CN105884902B - Fusion protein of mycoplasma pneumoniae protein epitope and preparation and application thereof - Google Patents
Fusion protein of mycoplasma pneumoniae protein epitope and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses fusion protein of mycoplasma pneumoniae protein epitope and preparation and application thereof, and relates to the fields of genetic engineering technology, vaccines and diagnostic reagents. The amino acid sequences of the Mycoplasma pneumoniae P116 protein, the P1 protein and the P30 protein are analyzed by a computer, and a Mycoplasma pneumoniae P116 protein fragment containing a strong epitope, namely 261 th amino acid to 466 th amino acid, a Mycoplasma pneumoniae P1 protein fragment containing a strong epitope, namely 1125 th amino acid to 1395 th amino acid, and a P30 protein fragment containing a strong epitope, namely 107 th amino acid to 161 th amino acid are screened out. Chemically synthesizing a brand-new tandem gene sequence of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, connecting the three gene fragments in series, expressing the fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a genetic engineering technology, connecting the three protein fragments by using glycine-serine, and obtaining the fusion protein with the total length of 538 amino acids.
Description
Technical Field
The invention discloses fusion protein of mycoplasma pneumoniae protein antigen epitope and preparation and application thereof, and relates to the fields of genetic engineering technology, vaccines and diagnostic reagents, wherein the amino acid sequences of mycoplasma pneumoniae P116 protein, P1 protein and P30 protein are analyzed by a computer, and a P116 protein fragment of mycoplasma pneumoniae containing a strong antigen epitope, a P1 protein fragment of mycoplasma pneumoniae containing a strong antigen epitope and a P30 protein fragment of mycoplasma pneumoniae containing a strong antigen epitope are screened out. Selecting codons preferred by both eukaryote and prokaryote, chemically synthesizing a brand-new tandem gene sequence of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae, expressing a fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a genetic engineering technology, connecting the three protein fragments by using glycine-serine, and obtaining the fusion protein with 538 amino acids in the total length. The expressed fusion protein can be used for detecting vaccines and mycoplasma pneumoniae antibodies or antigens, and can be used for preparing mycoplasma pneumoniae monoclonal antibodies and polyclonal antibodies, and the like, and the invention relates to the fields of genetic engineering technology, vaccines and diagnostic reagents.
Background
Mycoplasma pneumoniae (A), (B), (CMycoplasma pneumoniaeMp) is a prokaryotic cell type microorganism which can grow in an inanimate culture medium, is a main pathogen causing primary atypical pneumonia, can also cause pharyngitis, rhinitis, otitis media, bronchitis, tracheitis and the like, and has close relation with the attack of infantile asthma. In addition, Mp can escape the clearance of host immune system in the organism, cause persistent infection, lead to the organism organ to take place chronic pathological damage, and is closely related with the occurrence and development of cardiovascular diseases such as atherosclerosis, coronary heart disease, etc. The research on Mp is receiving more and more attention.
Mp has no cell wall and binds to receptors on host cell membrane mainly through the structure on the cell membrane surface to take in amino acids, nucleic acid precursors, free fatty acids, etc., to synthesize its own structure and nutrients. Thus, adhesion to host cells and tissues is critical to its pathogenesis. However, mycoplasma also loses its ability to adhere due to genetic mutations, and its virulence is lost.
Mp infection is commonly seen in school-age children and teenagers, is the most main pathogen of community-acquired pneumonia of children, has the incidence rate which is increased year by year and accounts for 10-30 percent, has the epidemic period of 30-50 percent, and can cause regional and worldwide epidemics. Although antibiotics such as macrolides can control infection, the increasing number of drug-resistant strains presents difficulties for clinical treatment. Therefore, the development of vaccines using immunodominant antigen genes is critical for the prevention and control of infection. The whole gene sequence of the main surface membrane protein of the gene is determined and published at present, and a foundation is laid for researching diagnostic reagents and vaccines and screening antiviral drugs by using a gene engineering technology.
The mycoplasma pneumoniae can be cultured in vitro by using PPLO culture medium, so an immunofluorescence method and an enzyme-linked immunoassay method for detecting mycoplasma pneumoniae antibodies are established by using the cultured mycoplasma pneumoniae, but the cultured mycoplasma pneumoniae is used as an antigen detection antibody, so that the mass production is difficult, false positive can be generated, and the development of specific gene recombinant antigen is a development direction for establishing a reagent for detecting mycoplasma pneumoniae specific antibodies. The most ideal way for preventing Mp infection is to inject a vaccine, but no vaccine for preventing Mp is available at present, the development of the vaccine is actively carried out at home and abroad, particularly, the research progress of inactivated vaccines is fast, and genetic engineering vaccines are the final development direction.
Disclosure of Invention
The invention aims to provide a fusion protein of mycoplasma pneumoniae protein epitope and preparation and application thereof aiming at the defects.
The mycoplasma pneumoniae can stimulate the body to generate a plurality of antibodies after infecting the human body, but sometimes, only antibodies aiming at partial mycoplasma pneumoniae proteins in serum are possibly related to the time of generating various antibodies or the immune state of the body, so a plurality of antigens are used for detecting the mycoplasma pneumoniae antibodies in the serum to improve the detection rate of the antibodies.
The P1 adhesin is the most prominent adhesion protein and immunodominant antigen, where neutralizing antibodies prevent more than 80% of Mp adhesion to host cells. Thus, the P1 protein is the most potential vaccine candidate. The P1 protein and the P30 protein are mainly gathered in the apical structure of adhesion bodies and can be directly combined with neuraminic acid receptors on the epithelial cell membranes of respiratory mucosa, so that the Mp is adhered to the cell surface. The P116 protein and the like are mainly involved in or maintain the integrity and stability of the apical structure, constitute a scaffold of the apical structure of the cohesin aggregates such as P1 and P30, and play an auxiliary role in the process of Mp adsorption to host cells. Through computer analysis of amino acid sequences of mycoplasma pneumoniae P116 protein, P1 protein and P30 protein, P116 protein fragment, P1 protein fragment and P30 protein fragment of mycoplasma pneumoniae containing strong antigen epitope are screened out, codons preferred by both eukaryotes and prokaryotes are selected, gene sequences of brand-new P116 protein fragment, P1 protein fragment and P30 protein fragment are chemically synthesized, the three gene fragments are connected in series, fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment is expressed by using a genetic engineering technology, and the expressed fusion protein can be used for detection of vaccines, mycoplasma pneumoniae antibodies or antigens and the like.
The invention screens P116 protein fragment containing strong antigen epitope, namely 261 th amino acid to 466 th amino acid, and 206 amino acids in total, P1 protein fragment containing strong antigen epitope, namely 1125 th amino acid to 1395 th amino acid, and 271 amino acids in total, and P30 protein fragment containing strong antigen epitope, namely 107 th amino acid to 161 th amino acid, and 55 amino acids in total by analyzing amino acid sequences of P116 protein, P1 protein and P30 protein of Mycoplasma pneumoniae through a computer. Selecting codons preferred by both eukaryote and prokaryote, chemically synthesizing a brand-new tandem gene sequence of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae, expressing a fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a genetic engineering technology, connecting the three protein fragments by using glycine-serine, and obtaining the fusion protein with 538 amino acids in the total length. The expressed fusion protein can be used for detecting vaccines and mycoplasma pneumoniae antibodies or antigens, and can be used for preparing mycoplasma pneumoniae monoclonal antibodies and polyclonal antibodies and the like.
The fusion protein of the mycoplasma pneumoniae protein epitope and the preparation and the application thereof are realized by adopting the following technical scheme:
a fusion protein of a P116 protein fragment, a P1 protein fragment and a P30 protein fragment of Mycoplasma pneumoniae, namely a fusion protein of 261 amino acid fragment to 466 amino acid fragment of P116 protein of Mycoplasma pneumoniae, 1125 amino acid fragment to 1395 amino acid fragment of P1 protein and 107 amino acid fragment to 161 amino acid fragment of P30 protein of Mycoplasma pneumoniae, wherein the P116 protein fragment is at the N end of the fusion protein, the P1C protein fragment is in the middle of the fusion protein, the P30 protein fragment is at the C end of the fusion protein, the three protein fragments are connected by glycine-serine, the full-length 538 amino acids of the fusion protein have the following amino acid sequences:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val
1 5 10 15
Asn Ala Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu
20 25 30
Leu Met Gly Lys Ala Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln
35 40 45
Pro Leu Ser Phe Leu Lys Val MET Asp Lys Val Lys Glu Asp Phe Glu
50 55 60
Lys Leu Phe Asn Leu Val Arg Pro Gly Leu Gly Lys Phe Val Lys Asp
65 70 75 80
Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr Val Tyr Lys
85 90 95
Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu
100 105 110
Ser Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe
115 120 125
Asp Gly Ile Thr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe
130 135 140
Lys Asp Leu Ile Val Pro Ala Val Lys Thr Asn Glu Lys Thr Ala Ala
145 150 155 160
Leu Ser Pro Leu Ile Glu Glu Leu Leu Thr Gln Lys Asp Thr Tyr Val
165 170 175
Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr Asn Leu Leu Lys
180 185 190
Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala Gly Gly
195 200 205
Ser Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu
210 215 220
Thr GlyLeu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala
225 230 235 240
Pro Arg Pro Trp Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu Gly
245 250 255
Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln Ala
260 265 270
Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asn Ala Leu
275 280 285
Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu Ala
290 295 300
Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn
305 310 315 320
Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser
325 330 335
Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val
340 345 350
Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln Pro
355 360 365
Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser Ser
370 375 380
Gly Asn Leu SerSer Val Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser
385 390 395 400
Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val
405 410 415
Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr
420 425 430
Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val
435 440 445
Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp
450 455 460
Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly
465 470 475 480
Gly Gly Ser Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu
485 490 495
Gln Leu Gln Arg Ile Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu
500 505 510
Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala
515 520 525
Pro Gln Pro Val Pro Val Pro Pro Gln Pro
530 535。
the fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is prepared by recombinant expression and preparation by utilizing bacteria, yeast cells, insect cells, mammal cells and transgenic animals and plants through a gene recombination technology.
The fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is characterized in that the fusion protein is formed by connecting any combination of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, and expressing and preparing in a fusion protein mode.
The fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is used for developing vaccines, antibodies or antigen detection reagents and preparing mycoplasma pneumoniae monoclonal antibodies and polyclonal antibodies.
A fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment is expressed and prepared by using a genetic engineering technology, and the specific method comprises the following steps:
1. screening of antigen epitopes of mycoplasma pneumoniae P116 protein, P1 protein and P30 protein and chemical synthesis of gene fragments thereof:
by analyzing the amino acid sequences of Mycoplasma pneumoniae P116 protein, P1 protein and P30 protein by computer using ANTHEWIN et al software, it was found that the N-terminal 261 th to 466 th amino acids of P116 protein contain a strong epitope, the N-terminal 1125-1395 th amino acids of P1 protein contain a strong epitope, and the N-terminal 107 th to 161 th amino acids of P30 proteinAmino acids contain stronger antigenic determinants. Selecting codons preferred by both eukaryote and prokaryote, chemically synthesizing a brand-new tandem gene sequence of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the Mycoplasma pneumoniae, connecting the three gene fragments in series, expressing a fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a genetic engineering technology, sequentially connecting the P116 protein fragment, the P1 protein fragment and the P30 protein fragment from the N end to the C end of the fusion protein, connecting the three protein fragments by using glycine-serine, and connecting the fusion protein by 538 amino acids in total length. At the 5' end of the synthetic fusion protein gene segment is addedNdeI cleavage site, 3' end added with termination codon TAA andEcoRI cleavage site. Facilitating cloning of the synthetic fusion protein Gene fragment into plasmid pET28a (+)NdeI andEcowithin the RI cleavage site.
Epitope amino acid sequence (261 aa to 466 aa) in the screened mycoplasma pneumoniae P116 protein:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val AsnAla Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu Leu Met Gly LysAla Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln Pro Leu Ser Phe Leu Lys ValMet Asp Lys Val Lys Glu Asp Phe Glu Lys Leu Phe Asn Leu Val Arg Pro Gly LeuGly Lys Phe Val Lys Asp Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile ThrVal Tyr Lys Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu LeuSer Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe Asp Gly IleThr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe Lys Asp Leu Ile Val ProAla Val Lys Thr Asn Glu Lys Thr Ala Ala Leu Ser Pro Leu Ile Glu Glu Leu LeuThr Gln Lys Asp Thr Tyr Val Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu ThrAsn Leu Leu Lys Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala
epitope amino acid sequence (1125 aa to 1395 aa) in the selected Mycoplasma pneumoniae P1 protein:
Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu Thr GlyLeu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala Pro Arg Pro Trp AlaAla Phe Arg Gly Ser Trp Val Asn Arg Leu Gly Arg Val Glu Ser Val Trp Asp LeuLys Gly Val Trp Ala Asp Gln Ala Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr AlaThr Arg Asn Ala Leu Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val ValGlu Ala Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn SerSer Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser Asp Lys Leu AspAsp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val Arg Thr Lys Leu Arg Gln SerPhe Gly Thr Asp His Ser Thr Gln Pro Gln Pro Gln Ser Leu Lys Thr Thr Thr ProVal Phe Gly Thr Ser Ser Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala GlyGly Gly Ser Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys ValSer Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser ThrAsn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Arg LeuSer Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val Asp Gly Ile Val Arg Thr ProLeu Ala Glu Leu Leu Asp
Gly
epitope amino acid sequence (107 aa to 161 aa) in the selected mycoplasma pneumoniae P30 protein:
Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu Gln Leu Gln ArgIle Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu Gln Gln Ala Ala Ala Glu AlaHis Ala Glu Ala Glu Val Glu Pro Ala Pro Gln Pro Val Pro Val Pro Pro Gln Pro
tandem gene sequence (1629 bp) of chemically synthesized mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment:
NdeI
Cat atgAAA TAC CTG AAT ACC CAC GTT AAA GCG GAA GAT GTG AAA AAA GATGTG AAT GCT AAT ATC AAA AAC CAG TTC GAC ATT GCT AAA ATC ATC GCG GAA CTG ATGGGT AAA GCC CTG AAA GAA TTT GGT AAC CAG CAA GAA GGC CAG CCG CTG AGC TTC CTGAAA GTC ATG GAC AAA GTG AAA GAA GAT TTC GAA AAA CTG TTC AAC CTG GTC CGT CCGGGT CTG GGC AAA TTT GTG AAA GAC CTG ATT CAA AGC TCT AGT CAG GCG GAA AAC AAAATC ACC GTG TAC AAA CTG ATC TTC GAT AAC AAG AAA ACC ATT CTG AAT CTG CTG AAAGAA CTG TCC ATC CCG GAA CTG AAC TCC TCA CTG GGC CTG GTT GAC GTC CTG TTT GATGGT ATT ACC GAC TCC GAT GGC CTG TAT GAA CGC TTA CAG AGC TTT AAA GAC CTG ATCGTC CCG GCC GTG AAA ACC AAT GAA AAA ACG GCG GCC CTG AGC CCG CTG ATT GAA GAACTG CTG ACC CAA AAA GAC ACG TAC GTG TTT GAT CTG ATT CAG AAA CAT AAA GGT ATCCTG ACC AAC CTG CTG AAA AAT TTT CTG GCG GAC TTC CAG AAA TCT ACC CCG TTT ATGGCC GGC GGT AGT ACG GAT TTC GTT AAA CCG CGT GCG GGT TAC CTG GGT CTG CAA CTGACC GGC CTG GAC GCG AGC GAT GCA ACG CAG CGC GCG CTG ATC TGG GCA CCG CGT CCGTGG GCA GCT TTT CGC GGT TCG TGG GTC AAC CGT CTG GGC CGC GTT GAA AGC GTC TGGGAT CTG AAA GGT GTT TGG GCA GAC CAG GCT CAA TCG GAT AGC CAG GGC TCT ACC ACGACC GCA ACC CGT AAC GCT CTG CCG GAA CAT CCG AAT GCC CTG GCA TTC CAG GTG TCTGTG GTT GAA GCT AGT GCG TAT AAA CCG AAC ACC AGC AGC GGT CAG ACC CAG AGC ACCAAC AGC AGC CCG TAC CTG CAC CTG GTG AAA CCG AAA AAA GTT ACC CAG AGT GAC AAACTG GAT GAC GAT CTG AAA AAC CTG CTG GAC CCG AAT CAA GTT CGT ACG AAA CTG CGCCAG TCC TTT GGC ACC GAT CAT TCA ACG CAG CCG CAA CCG CAG TCT CTG AAA ACG ACCACG CCG GTT TTC GGC ACC AGC AGC GGC AAT CTG TCG AGC GTC CTG AGT GGC GGT GGCGCA GGT GGC GGT TCT AGT GGT TCG GGT CAG AGC GGC GTG GAT CTG TCG CCG GTG GAAAAA GTT AGC GGT TGG CTG GTT GGC CAG CTG CCG TCT ACC AGT GAC GGT AAC ACC AGCAGC ACC AAC AAT CTG GCC CCG AAC ACC AAT ACG GGC AAT GAT GTC GTG GGT GTG GGCCGT CTG TCC GAA TCA AAC GCG GCC AAA ATG AAT GAC GAT GTT GAC GGC ATC GT CCGCAC CCC GCT GGC AGA ACT GCT GGA TGG CGG TGG CTC CCT GCT GGA AGA AAA AGA ACGTCA AGA ACA GCT GGC TGA ACA ACT GCA ACG CAT TTC AGC ACA GCA AGA AGA ACA GCAAGC TCT GGA ACAG CAA GCA GCT GCG GAA GCC CAC GCA GAA GCT GAA GTG GAA CCG GCGCCG CAA CCG GTG CCG GTT CCG CCG CAG CCGTAA GAA TTC
EcoRI
2. construction of a fusion protein recombinant plasmid expressing a Mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment:
the plasmid pET28a (+) was extracted (Novagen, USA) and usedNdeI andEcor I double enzyme digestion, recovering the large plasmid fragment after electrophoresis, and dissolving in deionized water. By usingNdeI andBamand (3) carrying out electrophoretic recovery on the gene fragment of the mycoplasma pneumoniae fusion protein chemically synthesized by HI double enzyme digestion, and dissolving in deionized water. The three DNA fragments after enzyme digestion with equal molar concentration are connected by T4DNA ligase in the same centrifugal tube and inserted into the vector pET28a (+)NdeI andEcor I, a fusion protein of Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment is expressed.
The constructed recombinant plasmid expresses a fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment, the N end of the fusion protein is the P116 protein fragment with the length of 206 amino acids, the middle part of the fusion protein is the P1 protein fragment with the length of 271 amino acids, the C end of the fusion protein is the P30 protein fragment with the length of 55 amino acids, the P116 protein fragment, the P1 protein fragment, the P30 protein fragment and the P30 protein fragment are connected by three amino acids of amino acid-glycine-serine, the total length of the fusion protein is 538:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val
1 5 10 15
Asn Ala Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu
20 25 30
Leu Met Gly Lys Ala Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln
35 40 45
Pro Leu Ser Phe Leu Lys Val MET Asp Lys Val Lys Glu Asp Phe Glu
50 55 60
Lys Leu Phe Asn Leu Val Arg Pro Gly Leu Gly Lys Phe Val Lys Asp
65 70 75 80
Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr Val Tyr Lys
85 90 95
Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu
100 105 110
Ser Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe
115 120 125
Asp Gly Ile Thr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe
130 135 140
Lys Asp Leu Ile Val Pro Ala Val Lys Thr Asn Glu Lys Thr Ala Ala
145 150 155 160
Leu Ser Pro Leu Ile Glu Glu Leu Leu Thr Gln Lys Asp Thr Tyr Val
165 170 175
Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr Asn Leu Leu Lys
180 185 190
Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala Gly Gly
195 200 205
Ser Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu
210 215 220
Thr Gly Leu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala
225 230 235 240
Pro Arg Pro Trp Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu Gly
245 250 255
Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln Ala
260 265 270
Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asn Ala Leu
275 280 285
Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu Ala
290 295 300
Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn
305 310 315 320
Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser
325 330 335
Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val
340 345 350
Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln Pro
355 360 365
Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser Ser
370 375 380
Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser
385 390 395 400
Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val
405 410 415
Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr
420 425 430
Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val
435 440 445
Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp
450 455 460
Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly
465 470 475 480
Gly Gly Ser Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu
485 490 495
Gln Leu Gln Arg Ile Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu
500 505 510
Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala
515 520 525
Pro Gln Pro Val Pro Val Pro Pro Gln Pro
530 535
3. screening and identifying recombinant plasmids:
the recombinant plasmid was transformed into E.coli BL21(DE3), and LB plate containing kanamycin (60. mu.g/ml) was spread and left overnight at 37 ℃. The transformed colonies and the control bacteria (the plasmid pET28a transformed bacteria) are randomly picked the next day, plasmids are respectively extracted, the plasmids containing the tandem gene are subjected to DNA sequence analysis, and the sequence analysis proves that the recombinant plasmids contain the mycoplasma pneumoniae P116 protein gene fragment, the P1 protein gene fragment and the P30 protein gene fragment which are connected together, and the sequences are completely correct.
4. Screening and identifying the engineering bacteria expressing the fusion protein:
the positive transformant containing the recombinant plasmid is inoculated into a test tube containing 3ml of LB culture medium (containing 60 mu g/ml of the kanamycin), the shaking culture is carried out for 3h at 37 ℃, IPTG is added to the final concentration of 0.5mmol/L, the shaking culture is continued for inducing for 6h, thalli are collected by centrifugation and are subjected to SDS-PAGE detection, the transformant expresses fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment with the relative molecular weight of about 60 kD, and a control bacterium BL21(DE3) does not have the protein band.
5. Purification of fusion protein expressing Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment:
1) ultrasonic lysis of fusion protein engineering bacteria expressing mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
Engineering bacteria for inducing expression of the fusion protein are centrifuged (8000 rpm, 10min and 4 ℃) to collect bacteria, the bacteria are suspended in bacterial lysate (20 mmol/L PB pH7.4, 10 mmol/L EDTA, 1mmol/L DTT and 5% glycerol) with the volume of original culture solution 1/10, the bacteria are broken by ultrasonic treatment in ice bath, and supernatant is collected by centrifugation.
2) Nickel ion affinity chromatography column purification of fusion protein expressing mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
Washing a balanced nickel ion affinity chromatography column by using a balanced solution (20 mmol/L PB pH7.4), directly loading the supernatant obtained by the ultrasonic crushing and centrifugation step into the column, fully washing the column by using the balanced solution after the loading is finished, eluting proteins by using the balanced solution containing imidazole with gradient concentration in sequence, collecting proteins of each elution peak, detecting proteins of each peak by using SDS-PAGE electrophoresis, determining which elution peak contains the expressed fusion proteins of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, and obtaining a 200 mmol/L imidazole elution protein peak which is the fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment;
6. the fusion protein of the purified Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is used as an antigen to detect Mycoplasma pneumoniae antibodies:
the fusion protein of the renatured Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is diluted by 50mmol/L carbonate (pH 9.6) in a multiple ratio and then coated on an enzyme linked plate, and the prepared Mycoplasma pneumoniae, rabbit positive serum and blank control serum are detected by indirect enzyme linked immunization, wherein the fusion protein of the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment can react with the Mycoplasma pneumoniae positive serum and does not react with the blank control serum, so that the fusion protein of the expressed Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment has better antigenicity and specificity.
7. And (3) labeling a fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae by using horseradish peroxidase (HRP), and detecting anti-mycoplasma pneumoniae IgM or IgG antibodies by using a capture method or a sandwich method. Fusion proteins of the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment were used as antigens, and anti-Mycoplasma pneumoniae antibodies were detected by the gold-labeling method.
8. The expressed fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae is used for vaccine, detection of mycoplasma pneumoniae antibodies or antigens, immune preparation of monoclonal antibodies and polyclonal antibodies against mycoplasma pneumoniae and the like.
9. Connecting the gene fragment of the Mycoplasma pneumoniae P116 protein, the gene fragment of the P1 protein and the gene fragment of the P30 protein, and expressing and preparing the fusion protein.
10. Through gene recombination technology, bacteria, yeast cells, insect cells, mammal cells and transgenic animals and plants are used for carrying out recombination expression to prepare fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment.
Description of english abbreviations: mp (Mycoplasma pneumoniae) Mycoplasma pneumoniae, EDTA-EDTA; IPTG: isopropyl thiogalactoside; DTT: dithiothreitol; SDS (sodium dodecyl sulfate): sodium dodecyl sulfonate; PAGE: performing polyacrylamide gel electrophoresis; SKL: sodium inosinate; PB: phosphate buffer; DNA: deoxyribonucleic acid; RNA: ribonucleic acids; kD: kilodalton; triton: triton.
Compared with the prior art, the invention has the advantages of
The fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the Mycoplasma pneumoniae expressed by the fusion protein has more advantages:
1. the antigen used by the currently applied enzyme-linked immunoassay kit for the mycoplasma pneumoniae antibody is a whole virus antigen, and has the defects of dangerous production, high cost, cross reaction with other respiratory pathogens and the like. The expressed fusion protein of P116 protein fragment, P1 protein fragment and P30 protein fragment of Mycoplasma pneumoniae can be used as an antigen to overcome the defects.
①
2. The expressed fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the Mycoplasma pneumoniae exists in a soluble form, is easy to purify, and does not need renaturation treatment.
3. The fusion expression of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae is more convenient and economical than the respective use of three separate protein fragments. When the two proteins are used as antigens to detect the anti-mycoplasma pneumoniae antibody, the three proteins do not need to be prepared respectively, and the use proportion of the three antigens does not need to be adjusted, so the application is convenient and the cost is low. When the capture method or the sandwich method is used for detecting the anti-mycoplasma pneumoniae antibody, only the fusion protein needs to be labeled by enzyme, and three proteins do not need to be labeled respectively.
4. There is currently no mycoplasma pneumoniae vaccine. The research of the inactivated vaccine has made great progress, but the production cost is high and the danger is large. The most important P1 protein of Mycoplasma pneumoniae is the main protein mediating the combination of pathogen and host cell, and blocking the combination site of the protein can prevent pathogen from infecting cell, so that P1 protein of Mycoplasma pneumoniae is the first choice protein for developing vaccine. The P116 protein and the P30 protein can stimulate the body to generate stronger cellular immunity. The fusion protein of P116 protein fragment, P1 protein fragment and P30 protein fragment of mycoplasma pneumoniae is prepared through gene engineering expression process, and this lays the foundation for developing gene engineering vaccine. The genetic engineering vaccine has the advantages of safety and low cost.
Drawings
The invention will be further explained with reference to the drawings, in which:
FIG. 1 shows the result of analyzing the epitope of Mycoplasma pneumoniae P116 protein by molecular biology software. The results showed that the P116 protein of Mycoplasma pneumoniae contains a strongly hydrophilic epitope from the 261 th amino acid to the 466 th amino acid at the N-terminus, i.e., the position indicated by the arrow in the figure.
FIG. 2 shows the result of analyzing the epitope of Mycoplasma pneumoniae P1 protein by molecular biology software. The results showed that the protein P1 of Mycoplasma pneumoniae contains a strong hydrophilic epitope from the 1125 th amino acid to the 1395 th amino acid at the N-terminus, i.e.at the position indicated by the arrow in the figure.
FIG. 3 shows the result of analyzing the epitope of Mycoplasma pneumoniae P30 protein by molecular biology software. The results showed that the P30 protein contains strong hydrophilic epitopes from amino acid 107 to amino acid 161 at the N-terminus of Mycoplasma pneumoniae, i.e.the positions indicated by the arrows in the figure.
FIG. 4 is a flow chart of recombinant plasmid construction for expressing fusion protein of P116 protein fragment, P1 protein fragment and P30 protein fragment of Mycoplasma pneumoniae.
FIG. 5 shows the transformants detected on a 1.2% Agarose gel. M1 high molecular weight DNA standard (TaKaRa, DL 10000); m2: low molecular weight DNA standards (TaKaRa, DL 5000); 1: a chemically synthesized fragment containing the gene of interest.
FIG. 6 shows the result of SDS-PAGE analysis of engineered bacteria expressing fusion proteins of P116 protein fragment, P1 protein fragment and P30 protein fragment of Mycoplasma pneumoniae. 4: low molecular weight protein standards (Pharmacia); 1.2, 3, 5, 6, 7, 8, 12, 13: the 9. sup. st recombinants 1, 2, 3, 4, 5, 6, 7, 11, 12 all expressed the fusion protein of interest with a relative molecular weight of about 60000, i.e.the position indicated by the arrow in the figure. 9. 10, 11: no. 8, 9, 10 transformants 3 did not express the target protein. 14. 15: 0.5% BSA, 1% BSA
FIG. 7 shows the results of SDS-PAGE analysis of the fusion protein before and after purification of the fusion protein expressing the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of Mycoplasma pneumoniae. Low molecular weight protein standards (Pharmacia); 1: a flow-through peak; 2: 20mM imidazole; 3: elution with 50mM imidazole; 4: elution with 100mM imidazole; 5: elution with 200mM imidazole; 6: fusion protein eluted with 500mM imidazole; 7: 1% BSA.
FIG. 8 is a graph showing the results of ELISA detection of rabbit immune antiserum.
Detailed Description
Detailed description of embodiments of the invention:
analysis, gene synthesis and expression of P116 protein, P1 protein and P30 protein epitope of mycoplasma pneumoniae
The amino acid sequences of the P116 protein, the P1 protein and the P30 protein of the Mycoplasma pneumoniae are analyzed by a computer, and a P116 protein fragment containing a strong epitope, namely the 261 th amino acid to the 466 th amino acid, a P1 protein fragment containing the strong epitope, namely the 1125 th amino acid to the 1395 th amino acid, and a P30 protein fragment containing the strong epitope, namely the 107 th amino acid to the 161 th amino acid, of the Mycoplasma pneumoniae are screened out.
Selecting codons preferred by eukaryote and prokaryote, chemically synthesizing novel tandem gene sequences of the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, and cloning the tandem gene fragments into a plasmid pET28a (+) by using a genetic engineering technologyNdeI／EcoAnd the RI site expresses a fusion protein of a P116 protein fragment, a P1 protein fragment and a P30 protein fragment, and the fusion protein sequentially comprises the P116 protein fragment, the P1 protein fragment and the P30 protein fragment from the N end to the C end and is 538 amino acids long.
The recombinant plasmid is transformed into escherichia coli BL21(DE3), engineering bacteria for efficiently expressing fusion proteins of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae are obtained through screening, and the expressed fusion proteins of the mycoplasma pneumoniae account for about 30% of the total mycoprotein amount.
Materials and methods
1. The strain and plasmid, host strain BL21(DE3) and expression vector pET28a (+) are products of Novagen company of America.
2. Molecular biological reagent restriction endonucleaseNdeI、EcoRI and T4DNA ligase are products of TaKaRa company. The plasmid purification kit and the kit for recovering DNA fragments from agarose gel are products of TaKaRa company. DTT and IPTG are products of TaKaRa company. Other reagents are imported or domestic analytical pure reagents.
3. Synthesis of Gene fragment Synthesis was aided by Nanjing Kingsry.
4. The gene cloning method comprises the steps of DNA enzyme digestion, connection and electrophoresis; extracting and transforming plasmids; general molecular cloning methods such as SDS-PAGE analysis of proteins are carried out by conventional methods. Other kits were run as described.
5. DNA sequence analysis: plasmid was purified using plasmid purification kit from Nanjing Kingsrey and sequenced using a DNA full-automatic sequencer.
Results
1. Screening of antigen epitopes of mycoplasma pneumoniae P116 protein, P1 protein and P30 protein and chemical synthesis of gene fragments thereof:
the amino acid sequences of Mycoplasma pneumoniae P116 protein, P1 protein and P30 protein (GeneBank, accession numbers: AGC041381, AAA883251 and AAB366031, respectively) were analyzed by computer using ANTHEWIN et al software, and it was found that the amino acids 261 to 466 at the N-terminus of P116 protein contained a strong epitope (FIG. 1), the amino acids 1125 to 1395 at the N-terminus of P1 protein contained a strong epitope (FIG. 2), and the amino acids 107 to 161 at the N-terminus of P30 protein contained a strong epitope (FIG. 3).
Epitope amino acid sequence (261 aa to 466 aa) in the screened mycoplasma pneumoniae P116 protein:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val AsnAla Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu Leu Met Gly LysAla Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln Pro Leu Ser Phe Leu Lys ValMet Asp Lys Val Lys Glu Asp Phe Glu Lys Leu Phe Asn Leu Val Arg Pro Gly LeuGly Lys Phe Val Lys Asp Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile ThrVal Tyr Lys Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu LeuSer Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe Asp Gly IleThr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe Lys Asp Leu Ile Val ProAla Val Lys Thr Asn Glu Lys Thr Ala Ala Leu Ser Pro Leu Ile Glu Glu Leu LeuThr Gln Lys Asp Thr Tyr Val Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu ThrAsn Leu Leu Lys Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala
epitope amino acid sequence (1125 aa to 1395 aa) in the selected Mycoplasma pneumoniae P1 protein:
Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu Thr GlyLeu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala Pro Arg Pro Trp AlaAla Phe Arg Gly Ser Trp ValAsn Arg Leu Gly Arg Val Glu Ser Val Trp Asp LeuLys Gly Val Trp Ala Asp Gln Ala Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr AlaThr Arg Asn Ala Leu Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val ValGlu Ala Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn SerSer Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser Asp Lys Leu AspAsp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val Arg Thr Lys Leu Arg Gln SerPhe Gly Thr Asp His Ser Thr Gln Pro Gln Pro Gln Ser Leu Lys Thr Thr Thr ProVal Phe Gly Thr Ser Ser Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala GlyGly Gly Ser Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys ValSer Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser ThrAsn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Arg LeuSer Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val Asp Gly Ile Val Arg Thr ProLeu Ala Glu Leu Leu Asp Gly
epitope amino acid sequence (107 aa to 161 aa) in the selected mycoplasma pneumoniae P30 protein:
Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu Gln Leu Gln ArgIle Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu Gln Gln Ala Ala Ala Glu AlaHis Ala Glu Ala Glu Val Glu Pro Ala Pro Gln Pro Val Pro Val Pro Pro Gln Pro
the codons preferred by both eukaryotes and prokaryotes were selected, and the tandem gene sequences of P116 protein, P1 protein and P30 protein containing the above-mentioned epitopes were chemically synthesized. At the 5' end of the synthetic fusion protein gene segment is addedNdeI enzyme cutting site, increased at the 3' endEcoRI enzyme cutting site. To facilitate tandem cloning of the synthetic tandem gene fragment into plasmid pET28a (+)NdeI andEcowithin the RI cleavage site.
Tandem gene sequence (1629 bp) of chemically synthesized mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment:
NdeI
Cat atgAAA TAC CTG AAT ACC CAC GTT AAA GCG GAA GAT GTG AAA AAA GATGTG AAT GCT AAT ATC AAA AAC CAG TTC GAC ATT GCT AAA ATC ATC GCG GAA CTG ATGGGT AAA GCC CTG AAA GAA TTT GGT AAC CAG CAA GAA GGC CAG CCG CTG AGC TTC CTGAAA GTC ATG GAC AAA GTG AAA GAA GAT TTC GAA AAA CTG TTC AAC CTG GTC CGT CCGGGT CTG GGC AAA TTT GTG AAA GAC CTG ATT CAA AGC TCT AGT CAG GCG GAA AAC AAAATC ACC GTG TAC AAA CTG ATC TTC GAT AAC AAG AAA ACC ATT CTG AAT CTG CTG AAAGAA CTG TCC ATC CCG GAA CTG AAC TCC TCA CTG GGC CTG GTT GAC GTC CTG TTT GATGGT ATT ACC GAC TCC GAT GGC CTG TAT GAA CGC TTA CAG AGC TTT AAA GAC CTG ATCGTC CCG GCC GTG AAA ACC AAT GAA AAA ACG GCG GCC CTG AGC CCG CTG ATT GAA GAACTG CTG ACC CAA AAA GAC ACG TAC GTG TTT GAT CTG ATT CAG AAA CAT AAA GGT ATCCTG ACC AAC CTG CTG AAA AAT TTT CTG GCG GAC TTC CAG AAA TCT ACC CCG TTT ATGGCC GGC GGT AGT ACG GAT TTC GTT AAA CCG CGT GCG GGT TAC CTG GGT CTG CAA CTGACC GGC CTG GAC GCG AGC GAT GCA ACG CAG CGC GCG CTG ATC TGG GCA CCG CGT CCGTGG GCA GCT TTT CGC GGT TCG TGG GTC AAC CGT CTG GGC CGC GTT GAA AGC GTC TGGGAT CTG AAA GGT GTT TGG GCA GAC CAG GCT CAA TCG GAT AGC CAG GGC TCT ACC ACGACC GCA ACC CGT AAC GCT CTG CCG GAA CAT CCG AAT GCC CTG GCA TTC CAG GTG TCTGTG GTT GAA GCT AGT GCG TAT AAA CCG AAC ACC AGC AGC GGT CAG ACC CAG AGC ACCAAC AGC AGC CCG TAC CTG CAC CTG GTG AAA CCG AAA AAA GTT ACC CAG AGT GAC AAACTG GAT GAC GAT CTG AAA AAC CTG CTG GAC CCG AAT CAA GTT CGT ACG AAA CTG CGCCAG TCC TTT GGC ACC GAT CAT TCA ACG CAG CCG CAA CCG CAG TCT CTG AAA ACG ACCACG CCG GTT TTC GGC ACC AGC AGC GGC AAT CTG TCG AGC GTC CTG AGT GGC GGT GGCGCA GGT GGC GGT TCT AGT GGT TCG GGT CAG AGC GGC GTG GAT CTG TCG CCG GTG GAAAAA GTT AGC GGT TGG CTG GTT GGC CAG CTG CCG TCT ACC AGT GAC GGT AAC ACC AGCAGC ACC AAC AAT CTG GCC CCG AAC ACC AAT ACG GGC AAT GAT GTC GTG GGT GTG GGCCGT CTG TCC GAA TCA AAC GCG GCC AAA ATG AAT GAC GAT GTT GAC GGC ATC GT CCGCAC CCC GCT GGC AGA ACT GCT GGA TGG CGG TGG CTC CCT GCT GGA AGA AAA AGA ACGTCA AGA ACA GCT GGC TGA ACA ACT GCA ACG CAT TTC AGC ACA GCA AGA AGA ACA GCAAGC TCT GGA ACAG CAA GCA GCT GCG GAA GCC CAC GCA GAA GCT GAA GTG GAA CCG GCGCCG CAA CCG GTG CCG GTT CCG CCG CAG CCGTAA GAA TTC
EcoRI
2. construction of a fusion protein recombinant plasmid expressing a Mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment:
the plasmid pET28a (+) was extracted (Novagen, USA) and usedNdeI andEcor I double enzyme digestion, recovering the large plasmid fragment after electrophoresis, and dissolving in deionized water. By usingNdeI and EcoRI are subjected to double enzyme digestion and chemical synthesis to obtain the Mycoplasma pneumoniae fusion protein gene fragment, and the Mycoplasma pneumoniae fusion protein gene fragment is dissolved in deionized water after electrophoresis recovery.
Connecting the two enzyme-digested DNA fragments with equal molar concentrations in the same centrifugal tube by using T4DNA ligase to insert the P116 protein gene fragment, the P1 protein gene fragment and the P30 protein gene fragment of the mycoplasma pneumoniae into a vector pET28a (+)NdeI andEcor I, a fusion protein of Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment is expressed. The construction flow is shown in FIG. 4.
3. Screening and identifying recombinant plasmids:
the recombinant plasmid ligated in the above step was transformed into Escherichia coli BL21(DE3), and the transformed product was spread on a solid LB medium containing kanamycin (60. mu.g/ml) and cultured overnight at 37 ℃. The next day, colony of transformant was randomly selected, inoculated into a test tube containing 4ml of liquid LB medium (containing 60. mu.g/ml kanamycin), subjected to shake culture at 37 ℃ for 6 hours, and 1ml of bacterial liquid was collected and centrifuged. Respectively suspending the thallus by 50 mul of deionized water, boiling for 5min, centrifuging (4 ℃, 12000 rpm) for 5min, and determining the gene sequence of the mycoplasma pneumoniae P116 protein gene fragment, the tandem gene sequence of the P1 protein gene fragment and the P30 protein gene fragment in the plasmid, wherein DNA sequence analysis proves that the recombinant plasmid contains the mycoplasma pneumoniae P116 protein gene fragment, the P1 protein gene fragment and the P30 protein gene fragment which are connected in series, and the sequences are completely correct.
The constructed recombinant plasmid expresses a fusion protein of a mycoplasma pneumoniae P116 protein gene fragment, a P1 protein gene fragment and a P30 protein gene fragment, the total length is 538 amino acids, the N end of the fusion protein is the P116 protein fragment, the length of the fusion protein is 206 amino acids, the middle part of the fusion protein is the P1 protein fragment, the length of the fusion protein is 271 amino acids, the C end of the fusion protein is the P30 protein fragment, the length of the fusion protein is 55 amino acids, the three amino acids are connected through three amino acids of amino acid-glycine-serine, and the amino acid sequences are as follows:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val
1 5 10 15
Asn Ala Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu
20 25 30
Leu Met Gly Lys Ala Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln
35 40 45
Pro Leu Ser Phe Leu Lys Val MET Asp Lys Val Lys Glu Asp Phe Glu
50 55 60
Lys Leu Phe Asn Leu Val Arg Pro Gly Leu Gly Lys Phe Val Lys Asp
65 70 75 80
Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr Val Tyr Lys
85 90 95
Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu
100 105 110
Ser Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe
115 120 125
Asp Gly Ile Thr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe
130 135 140
Lys Asp Leu Ile Val Pro Ala Val Lys Thr Asn Glu Lys Thr Ala Ala
145 150 155 160
Leu Ser Pro Leu Ile Glu Glu Leu Leu Thr Gln Lys Asp Thr Tyr Val
165 170 175
Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr Asn Leu Leu Lys
180 185 190
Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala Gly Gly
195 200 205
Ser Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu
210 215 220
Thr Gly Leu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala
225 230 235 240
Pro Arg Pro Trp Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu Gly
245 250 255
Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln Ala
260 265 270
Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asn Ala Leu
275 280 285
Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu Ala
290 295 300
Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn
305 310 315 320
Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser
325 330 335
Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val
340 345 350
Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln Pro
355 360 365
Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser Ser
370 375 380
Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser
385 390 395 400
Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val
405 410 415
Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr
420 425 430
Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val
435 440 445
Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp
450 455 460
Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly
465 470 475 480
Gly Gly Ser Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu
485 490 495
Gln Leu Gln Arg Ile Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu
500 505 510
Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala
515 520 525
Pro Gln Pro Val Pro Val Pro Pro Gln Pro
530 535
4. screening and identifying the engineering bacteria expressing the fusion protein:
the selected 11 transformants were inoculated into a tube containing 3ml of LB medium (containing 60. mu.g/ml of kanamycin), cultured with shaking at 37 ℃ for 3 hours, IPTG was added to a final concentration of 0.5mmol/L, induction with shaking was continued for 6 hours, cells were collected by centrifugation and examined by SDS-PAGE, and the transformants No. 1, 2, 3, 4, 5, 6, 7, 11, and 12 expressed a fusion protein of Mycoplasma pneumoniae S protein and N protein having a relative molecular weight of about 60 kD, whereas the transformants No. 8, 9, and 10 had no band of this protein (FIG. 6).
Purification of fusion proteins expressing Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
Based on the amino acid sequence of the fusion protein expressing the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, the physicochemical characteristics of the fusion protein are analyzed, and a proper purification method is determined. The fusion proteins expressing the Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment contained His tags by computer analysis, and we therefore decided to purify them on a nickel ion affinity chromatography column in phosphate buffer at pH 7.4. The method comprises the following specific steps:
materials and methods
1. The main reagents are as follows:
the nickel ion gel is a product of Nanjing Kinshire company, and IPTG and DTT are products of TaTaRa company. Other reagents are domestic or imported analytical pure reagents.
2. Induced expression and ultrasonic lysis of engineering bacteria for expressing fusion protein of mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
Selecting a single colony from an LB flat plate inoculated with No. 5 engineering bacteria, inoculating the single colony into a triangular flask containing 200ml of LB liquid culture medium, adding kanamycin to the final concentration of 60 mug/ml, and culturing overnight in a shaking table at 37 ℃. The next day, the bacterial solution was inoculated into 4 Erlenmeyer flasks each containing 200ml of LB liquid medium, 50ml of the bacterial solution was inoculated into each flask, cultured in a shaker at 37 ℃ for 1 hour with shaking, and then IPTG was added to a final concentration of 0.1mmol/L for induction expression for 4 hours.
1000ml of engineering bacteria for inducing expression of the fusion protein are centrifuged (8000 rpm, 10min, 4 ℃) to collect the bacteria, the bacteria are suspended in 100ml of bacterial lysate (20 mmol/L PB pH7.4, 10 mmol/L EDTA, 1mmol/L DTT, 5% glycerol), the bacteria are broken by ultrasonic in an ice bath for 10min, and the supernatant is collected by centrifugation (12000 rpm, 30 min, 4 ℃).
3. Nickel ion affinity chromatography column purification of fusion protein expressing mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
The nickel ion affinity chromatography column is washed by balance liquid (20 mmol/L PB pH7.4), and then the supernatant obtained by the crushing and centrifugation in the previous step is directly loaded on the column at the loading flow rate of 1.5 ml/min. And after the sample loading is finished, fully washing the column by using the equilibrium solution, eluting proteins by using the equilibrium solution containing 20, 50, 100, 200 and 500 mmol/L of imidazole in sequence, collecting proteins of each elution peak, detecting the proteins of each peak by using 12% SDS-PAGE electrophoresis, and determining which elution peak contains the fusion protein of the expressed mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment.
Results
SDS-PAGE analysis is carried out on proteins eluted from a nickel column by imidazole with different concentrations, and the result shows (see figure 7), and the electrophoresis result shows that the imidazole elution protein peak of 200 mmol/L is the fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae, a very concentrated fusion protein (at about 60 kD) of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae is developed, and the fusion protein band of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment of the mycoplasma pneumoniae does not exist in other elution peaks.
Preparation and purification of multi-antiserum of fusion protein of mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment
The purified fusion protein of the recombinant mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is used as an antigen to prepare a multi-antiserum, the antigen is used for preparing and purifying a large amount of rabbit antiserum in a mode of immunizing white New Zealand big ear rabbits by combining with an adjuvant, and the antibody titer against mycoplasma pneumoniae is detected by an indirect ELISA method.
Materials and methods
1. The main reagents are as follows:
DEAE SepharoseTMfast Flow is a product of GE healthcare, and complete Freund's adjuvant and incomplete Freund's adjuvant are products of SIGMA. Other reagents are domestic or imported analytical pure reagents.
2. Immunogen and immunization procedure:
using the fusion protein as immunogen, 5 New Zealand big ear rabbits were immunized, each rabbit was immunized with 100. mu.g of antigen, and the immunization mode was back multipoint immunization. The first immunization uses complete Freund's adjuvant mixed with antigen, and the second and third immunizations use incomplete Freund's adjuvant.
3. The indirect ELISA method is used for measuring the antiserum titer:
2 weeks after each immunization, blood was taken from the rabbit ear vein and centrifuged at 4000rpm for 10min to obtain antiserum. Coating fusion protein, each hole is 1 mu g, keeping the temperature at 4 ℃ overnight, sealing 20% calf serum, keeping the temperature at 37 ℃ for 2h, diluting antiserum obtained by centrifugation in a gradient way to be used as a primary antibody, adding a secondary antibody after 2h at 37 ℃, diluting goat anti-rabbit IgG at a ratio of 1:5000, adding A and B solutions for developing and reading.
4. Obtaining antiserum:
measuring serum titer by indirect ELISA method, collecting blood from rabbit carotid artery, standing overnight at 4 deg.C, centrifuging at 3000rpm for 10min to obtain antiserum, filtering for sterilization, and freezing at-20 deg.C.
5. Purification of antiserum:
isovolumetric saturation (NH)4)2SO4Mixing the solution with serum, standing at 4 deg.C for 1 hr, centrifuging at 3000rpm for 10min, discarding supernatant, and adding 0.07M Na3PO4Dissolving the solution, precipitating, dialyzing, loading on column, and purifying with DEAE SepharoseTMThe multiple antibodies were purified by Fast Flow anion exchange chromatography.
Results
2 weeks after the last immunization, 60ml of blood was obtained by rabbit carotid artery blood sampling, and after standing overnight at 4 ℃, serum was separated and subjected to DEAE SepharoseTMFast Flow anion exchange chromatography purificationThe polyclonal antibody is used as a primary antibody in ELISA detection, and the result shows that 1:100000 is effective serum dilution gradient, namely serum titer, and as shown in FIG. 8, the prepared fusion protein of P116 protein fragment, P1 protein fragment and P30 protein fragment of Mycoplasma pneumoniae has better antigenicity and specificity.
Claims (4)
1. A fusion protein of a P116 protein fragment, a P1 protein fragment and a P30 protein fragment of Mycoplasma pneumoniae, namely a fusion protein of 261 amino acid fragment to 466 amino acid fragment of P116 protein of Mycoplasma pneumoniae, 1125 amino acid fragment to 1395 amino acid fragment of P1 protein and 107 amino acid fragment to 161 amino acid fragment of P30 protein of Mycoplasma pneumoniae, wherein the P116 protein fragment is at the N end of the fusion protein, the P1C protein fragment is in the middle of the fusion protein, the P30 protein fragment is at the C end of the fusion protein, the three protein fragments are connected by glycine-serine, the full-length 538 amino acids of the fusion protein have the following amino acid sequences:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val
1 5 10 15
Asn Ala Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu
20 25 30
Leu Met Gly Lys Ala Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln
35 40 45
Pro Leu Ser Phe Leu Lys Val MET Asp Lys Val Lys Glu Asp Phe Glu
50 55 60
Lys Leu Phe Asn Leu Val Arg Pro Gly Leu Gly Lys Phe Val Lys Asp
65 70 75 80
Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr Val Tyr Lys
85 90 95
Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu
100 105 110
Ser Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe
115 120 125
Asp Gly Ile Thr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe
130 135 140
Lys Asp Leu Ile Val Pro Ala Val Lys Thr Asn Glu Lys Thr Ala Ala
145 150 155 160
Leu Ser Pro Leu Ile Glu Glu Leu Leu Thr Gln Lys Asp Thr Tyr Val
165 170 175
Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr Asn Leu Leu Lys
180 185 190
Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala Gly Gly
195 200 205
Ser Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu
210 215 220
Thr Gly Leu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala
225 230 235 240
Pro Arg Pro Trp Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu Gly
245 250 255
Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln Ala
260 265 270
Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asn Ala Leu
275 280 285
Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu Ala
290 295 300
Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn
305 310 315 320
Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser
325 330 335
Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val
340 345 350
Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln Pro
355 360 365
Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser Ser
370 375 380
Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser
385 390 395 400
Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val
405 410 415
Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr
420 425 430
Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val
435 440 445
Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp
450 455 460
Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly
465 470 475 480
Gly Gly Ser Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu
485 490 495
Gln Leu Gln Arg Ile Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu
500 505 510
Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala
515 520 525
Pro Gln Pro Val Pro Val Pro Pro Gln Pro
530 535。
2. the method for preparing the fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment according to claim 1, wherein the protein is expressed and prepared by using a genetic engineering technology, and the specific method comprises the following steps:
screening of antigen epitopes of mycoplasma pneumoniae P116 protein, P1 protein and P30 protein and chemical synthesis of gene fragments thereof:
by using software such as ANTHEWIN and the like, the amino acid sequences of the P116 protein, the P1 protein and the P30 protein of the Mycoplasma pneumoniae are analyzed by a computer, and the N-terminal 261 th to 466 th amino acids of the P116 protein are found to contain a stronger epitope, the N-terminal 1125-th to 1395 th amino acids of the P1 protein are found to contain a stronger epitope, and the N-terminal 107 th to 161 th amino acids of the P30 protein are found to contain a stronger epitope; selecting codons preferred by both eukaryote and prokaryote, chemically synthesizing a brand-new tandem gene sequence of a P116 protein fragment, a P1 protein fragment and a P30 protein fragment of the mycoplasma pneumoniae, connecting the three gene fragments in series, expressing a fusion protein of the P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a genetic engineering technology, sequentially connecting the P116 protein fragment, the P1 protein fragment and the P30 protein fragment from the N end to the C end of the fusion protein, connecting the three protein fragments by using glycine-serine, and connecting the fusion protein by 538 amino acids in total length; at the 5' end of the synthetic fusion protein gene segment is addedNdeI cleavage site, 3' end added with termination codon TAA andEcoRI enzyme cutting sites; facilitating cloning of the synthetic fusion protein Gene fragment into plasmid pET28a (+)NdeI andEcowithin the RI enzyme cutting site;
the epitope amino acid sequence in the screened mycoplasma pneumoniae P116 protein is characterized in that the amino acid sequence of the epitope from 261 th aa to 466 th aa:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val Asn AlaAsn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu Leu Met Gly Lys AlaLeu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln Pro Leu Ser Phe Leu Lys Val MetAsp Lys Val Lys Glu Asp Phe Glu Lys Leu Phe Asn Leu Val Arg Pro Gly Leu GlyLys Phe Val Lys Asp Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr ValTyr Lys Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu SerIle Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe Asp Gly Ile ThrAsp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe Lys Asp Leu Ile Val Pro AlaVal Lys Thr Asn Glu Lys Thr Ala Ala Leu Ser Pro Leu Ile Glu Glu Leu Leu ThrGln Lys Asp Thr Tyr Val Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr AsnLeu Leu Lys Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala
the screened amino acid sequence of the antigen epitope in the mycoplasma pneumoniae P1 protein is that the amino acid sequence of 1125 aa to 1395 aa:
Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu Thr Gly LeuAsp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala Pro Arg Pro Trp Ala AlaPhe Arg Gly Ser Trp Val Asn Arg Leu Gly Arg Val Glu Ser Val Trp Asp Leu LysGly Val Trp Ala Asp Gln Ala Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala ThrArg Asn Ala Leu Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val GluAla Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn Ser SerPro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser Asp Lys Leu Asp AspAsp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val Arg Thr Lys Leu Arg Gln Ser PheGly Thr Asp His Ser Thr Gln Pro Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro ValPhe Gly Thr Ser Ser Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly GlyGly Ser Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val SerGly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser Thr AsnAsn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Arg Leu SerGlu Ser Asn Ala Ala Lys Met Asn Asp Asp Val Asp Gly Ile Val Arg Thr Pro LeuAla Glu Leu Leu Asp
Gly
the epitope amino acid sequence in the screened mycoplasma pneumoniae P30 protein is shown in the sequence from 107 aa to 161 aa:
Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu Gln Leu Gln Arg IleSer Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu Gln Gln Ala Ala Ala Glu Ala HisAla Glu Ala Glu Val Glu Pro Ala Pro Gln Pro Val Pro Val Pro Pro Gln Pro
the tandem gene sequence of the chemically synthesized Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment is 1629 bp:
NdeI
Cat atgAAA TAC CTG AAT ACC CAC GTT AAA GCG GAA GAT GTG AAA AAA GAT GTGAAT GCT AAT ATC AAA AAC CAG TTC GAC ATT GCT AAA ATC ATC GCG GAA CTG ATG GGTAAA GCC CTG AAA GAA TTT GGT AAC CAG CAA GAA GGC CAG CCG CTG AGC TTC CTG AAAGTC ATG GAC AAA GTG AAA GAA GAT TTC GAA AAA CTG TTC AAC CTG GTC CGT CCG GGTCTG GGC AAA TTT GTG AAA GAC CTG ATT CAA AGC TCT AGT CAG GCG GAA AAC AAA ATCACC GTG TAC AAA CTG ATC TTC GAT AAC AAG AAA ACC ATT CTG AAT CTG CTG AAA GAACTG TCC ATC CCG GAA CTG AAC TCC TCA CTG GGC CTG GTT GAC GTC CTG TTT GAT GGTATT ACC GAC TCC GAT GGC CTG TAT GAA CGC TTA CAG AGC TTT AAA GAC CTG ATC GTCCCG GCC GTG AAA ACC AAT GAA AAA ACG GCG GCC CTG AGC CCG CTG ATT GAA GAA CTGCTG ACC CAAAAA GAC ACG TAC GTG TTT GAT CTG ATT CAG AAA CAT AAA GGT ATC CTGACC AAC CTG CTG AAA AAT TTT CTG GCG GAC TTC CAG AAA TCT ACC CCG TTT ATG GCCGGC GGT AGT ACG GAT TTC GTT AAA CCG CGT GCG GGT TAC CTG GGT CTG CAA CTG ACCGGC CTG GAC GCG AGC GAT GCA ACG CAG CGC GCG CTG ATC TGG GCA CCG CGT CCG TGGGCA GCT TTT CGC GGT TCG TGG GTC AAC CGT CTG GGC CGC GTT GAA AGC GTC TGG GATCTG AAA GGT GTT TGG GCA GAC CAG GCT CAA TCG GAT AGC CAG GGC TCT ACC ACG ACCGCA ACC CGT AAC GCT CTG CCG GAA CAT CCG AAT GCC CTG GCA TTC CAG GTG TCT GTGGTT GAA GCT AGT GCG TAT AAA CCG AAC ACC AGC AGC GGT CAG ACC CAG AGC ACC AACAGC AGC CCG TAC CTG CAC CTG GTG AAA CCG AAA AAA GTT ACC CAG AGT GAC AAA CTGGAT GAC GAT CTG AAA AAC CTG CTG GAC CCG AAT CAA GTT CGT ACG AAA CTG CGC CAGTCC TTT GGC ACC GAT CAT TCA ACG CAG CCG CAA CCG CAG TCT CTG AAA ACG ACC ACGCCG GTT TTC GGC ACC AGC AGC GGC AAT CTG TCG AGC GTC CTG AGT GGC GGT GGC GCAGGT GGC GGT TCT AGT GGT TCG GGT CAG AGC GGC GTG GAT CTG TCG CCG GTG GAA AAAGTT AGC GGT TGG CTG GTT GGC CAG CTG CCG TCT ACC AGT GAC GGT AAC ACC AGC AGCACC AAC AAT CTG GCC CCG AAC ACC AAT ACG GGC AAT GAT GTC GTG GGT GTG GGC CGTCTG TCC GAA TCA AAC GCG GCC AAA ATG AAT GAC GAT GTT GAC GGC ATC GT CCG CACCCC GCT GGC AGA ACT GCT GGA TGG CGG TGG CTC CCT GCT GGA AGA AAA AGA ACG TCAAGA ACA GCT GGC TGA ACA ACT GCA ACG CAT TTC AGC ACA GCA AGA AGA ACA GCA AGCTCT GGA ACAG CAA GCA GCT GCG GAA GCC CAC GCA GAA GCT GAA GTG GAA CCG GCG CCGCAA CCG GTG CCG GTT CCG CCG CAG CCGTAA GAA TTC
EcoRI
construction of a fusion protein recombinant plasmid expressing a Mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment:
extracting plasmid pET28a (+) byNdeI andEcor I double enzyme digestion, recovering the enzyme digested plasmid large fragment after electrophoresis, and dissolving in deionized water; by usingNdeI andBamthe gene fragment of the mycoplasma pneumoniae fusion protein chemically synthesized by HI double enzyme digestion is dissolved in deionized water after electrophoretic recovery; the three digested DNA fragments with equal molar concentration are connected by T4DNA ligase in the same centrifugal tube and inserted into the vector pET28a (+)NdeI andEcor I, expressing a fusion protein of Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment;
the constructed recombinant plasmid expresses a fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment, the N end of the fusion protein is the P116 protein fragment with the length of 206 amino acids, the middle part of the fusion protein is the P1 protein fragment with the length of 271 amino acids, the C end of the fusion protein is the P30 protein fragment with the length of 55 amino acids, the P116 protein fragment, the P1 protein fragment, the P30 protein fragment and the P30 protein fragment are connected by three amino acids of amino acid-glycine-serine, the total length of the fusion protein is 538:
Lys Tyr Leu Asn Thr His Val Lys Ala Glu Asp Val Lys Lys Asp Val
1 5 10 15
Asn Ala Asn Ile Lys Asn Gln Phe Asp Ile Ala Lys Ile Ile Ala Glu
20 25 30
Leu Met Gly Lys Ala Leu Lys Glu Phe Gly Asn Gln Gln Glu Gly Gln
35 40 45
Pro Leu Ser Phe Leu Lys Val MET Asp Lys Val Lys Glu Asp Phe Glu
50 55 60
Lys Leu Phe Asn Leu Val Arg Pro Gly Leu Gly Lys Phe Val Lys Asp
65 70 75 80
Leu Ile Gln Ser Ser Ser Gln Ala Glu Asn Lys Ile Thr Val Tyr Lys
8590 95
Leu Ile Phe Asp Asn Lys Lys Thr Ile Leu Asn Leu Leu Lys Glu Leu
100 105 110
Ser Ile Pro Glu Leu Asn Ser Ser Leu Gly Leu Val Asp Val Leu Phe
115 120 125
Asp Gly Ile Thr Asp Ser Asp Gly Leu Tyr Glu Arg Leu Gln Ser Phe
130 135 140
Lys Asp Leu Ile Val Pro Ala Val Lys Thr Asn Glu Lys Thr Ala Ala
145 150 155 160
Leu Ser Pro Leu Ile Glu Glu Leu Leu Thr Gln Lys Asp Thr Tyr Val
165 170 175
Phe Asp Leu Ile Gln Lys His Lys Gly Ile Leu Thr Asn Leu Leu Lys
180 185 190
Asn Phe Leu Ala Asp Phe Gln Lys Ser Thr Pro Phe Met Ala Gly Gly
195 200 205
Ser Thr Asp Phe Val Lys Pro Arg Ala Gly Tyr Leu Gly Leu Gln Leu
210 215 220
Thr Gly Leu Asp Ala Ser Asp Ala Thr Gln Arg Ala Leu Ile Trp Ala
225 230 235 240
Pro Arg Pro Trp Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu Gly
245 250 255
Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln Ala
260 265 270
Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asn Ala Leu
275 280 285
Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu Ala
290 295 300
Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn
305 310 315 320
Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Thr Gln Ser
325 330 335
Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln Val
340 345 350
Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln Pro
355 360 365
Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser Ser
370 375 380
Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser
385 390 395 400
Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys Val
405 410 415
Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr
420 425 430
Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val
435 440 445
Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp
450 455 460
Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly
465 470 475 480
Gly Gly Ser Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu
485 490 495
Gln Leu Gln Arg Ile Ser Ala Gln Gln Glu Glu Gln Gln Ala Leu Glu
500 505 510
Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala
515 520 525
Pro Gln Pro Val Pro Val Pro Pro Gln Pro
530 535
screening and identifying recombinant plasmids:
the recombinant plasmid was transformed into E.coli BL21(DE3), spread on LB plates containing 60. mu.g/ml kanamycin, and left overnight at 37 ℃;
randomly selecting a transformed colony and a control bacterium containing a plasmid pET28a transformed bacterium on the next day, respectively extracting plasmids, carrying out DNA sequence analysis on the plasmids containing the tandem genes, and confirming that the recombinant plasmids contain a mycoplasma pneumoniae P116 protein gene fragment, a P1 protein gene fragment and a P30 protein gene fragment which are connected together and the sequences are completely correct by the sequence analysis;
screening and identifying the engineering bacteria expressing the fusion protein:
inoculating a positive transformant containing a recombinant plasmid into a test tube containing 3ml of LB culture medium containing 60 mu g/ml of kanamycin, carrying out shake culture at 37 ℃ for 3h, adding IPTG (isopropyl-beta-thiogalactoside) to a final concentration of 0.5mmol/L, continuing to carry out shake culture induction for 6h, centrifuging and collecting thalli, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection, wherein the transformant expresses a fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment with a relative molecular weight of about 60 kD, and a control bacterium BL21(DE3) does not have the protein band;
purification of fusion protein expressing Mycoplasma pneumoniae P116 protein fragment, P1 protein fragment and P30 protein fragment:
ultrasonic cracking of fusion protein engineering bacteria for expressing mycoplasma pneumoniae P116 protein fragments, P1 protein fragments and P30 protein fragments, centrifuging the engineering bacteria for inducing expression of the fusion proteins, centrifuging at 8000 rpm, 10min and 4 ℃, collecting bacteria, suspending the bacteria in bacterial lysate with the volume of an original culture solution 1/10, performing ultrasonic bacteria breaking in ice bath, centrifuging and collecting supernatant, wherein the bacterial lysate is 20 mmol/L PB (phosphate buffer solution) pH7.4, 10 mmol/LEDTA, 1mmol/L DTT and 5% glycerol;
purifying the fusion protein expressing the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment by using a nickel ion affinity chromatography column;
washing a balanced nickel ion affinity chromatography column by using a balanced liquid containing 20 mmol/L PB pH7.4, then directly loading the supernatant obtained by the ultrasonic crushing and centrifugation step into the column, fully washing the column by using the balanced liquid after the loading is finished, eluting proteins by using the balanced liquid containing imidazole with gradient concentration in sequence, collecting proteins of each elution peak, detecting proteins of each peak by using SDS-PAGE electrophoresis, determining which elution peak contains the fusion proteins of the expressed mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment, and obtaining a 200 mmol/L imidazole elution protein peak which is the fusion protein of the mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment;
the fusion protein of the purified Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is used as an antigen to detect Mycoplasma pneumoniae antibodies:
the fusion protein of the renatured Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment is diluted by 50mmol/L carbonate with the pH value of 9.6, and then coated on an enzyme linked plate, and the prepared Mycoplasma pneumoniae, rabbit positive serum and blank control serum are detected by an indirect enzyme linked immunosorbent assay, wherein the fusion protein of the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment can react with the Mycoplasma pneumoniae positive serum and does not react with the blank control serum, so that the expressed fusion protein of the Mycoplasma pneumoniae P116 protein fragment, the P1 protein fragment and the P30 protein fragment has better antigenicity and specificity.
3. The method for producing a fusion protein of a Mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment, and a P30 protein fragment according to claim 1, wherein the fusion protein is produced by recombinant expression using bacteria, yeast cells, insect cells, mammalian cells, or transgenic animals and plants by a gene recombination technique.
4. The use of a fusion protein of a mycoplasma pneumoniae P116 protein fragment, a P1 protein fragment and a P30 protein fragment according to claim 1, for the preparation of a vaccine, an antibody or an antigen detection reagent, or for the preparation of a mycoplasma pneumoniae monoclonal antibody or polyclonal antibody.
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| AAA88325.1;无;《Genebank》;19960215;序列 * |
| AAB36603.1;无;《Genebank》;19961225;序列 * |
| Identification of an N-terminal 27 kDa fragment of Mycoplasma pneumoniae P116 protein as specific immunogen in M. pneumoniae infections;Irum Tabassum1等;《BMC Infectious Diseases》;20101231;第350-358页 * |
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