CN111253478B - Mycoplasma pneumoniae antigen and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of in-vitro diagnosis immunodetection, and particularly provides a mycoplasma pneumoniae antigen as well as a preparation method and application thereof. The mycoplasma pneumoniae antigen provided by the invention is an antigenic protein sequence P1M: residues 1340-1518, which is specifically a protein composed of the amino acid sequence shown in SEQ ID NO.1 or a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.1 and having the same function. The antigen is verified by an immune serological detection technology, the antigen has strong specificity and high sensitivity, and the antigen is easy to culture and purify, is more beneficial to industrial production and saves cost. In addition, the antigen is suitable for preparing MP antibody detection products, can be products in any form in the field of in vitro immunodiagnosis, and has wide market prospect.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis immunodetection, and in particular relates to a mycoplasma pneumoniae antigen and a preparation method and application thereof.
Background
Mycoplasma Pneumoniae (MP) is a pathogenic mycoplasma, an important pathogen of community-acquired pneumonia, whose infection can cause primary atypical pneumonia; it can also cause human respiratory infectious diseases such as bronchitis, pharyngitis, etc., and serious patients can cause complications affecting nervous system, blood system, cardiovascular system, skin, muscle, joint, etc. In summary, respiratory tract injury and various extrapulmonary complications caused by MP infection have attracted widespread attention.
Because MP infection often has no specific clinical manifestation, the etiology is diagnosed in time, the disease deterioration is avoided, early detection and early treatment are achieved, and the early detection and early treatment become the urgent priority for treating MP infectious diseases, so the early diagnosis of MP infection is particularly important.
Mycoplasma pneumoniae antibodies are classified into IgG and IgM antibodies, the incubation period of MP infection is 2-3 weeks, IgM antibodies appear in about 7-10 days, and IgG antibodies appear in about 20 days. IgM antibody has reached a considerably high level when a patient is diagnosed with symptoms, and therefore, IgM antibody positivity can be used as a diagnostic index in the acute infection stage. However, if IgM antibody is negative, Mycoplasma pneumoniae infection cannot be denied, and IgG antibody needs to be detected. Furthermore, the detection of MP-specific IgM does not indicate that the patient is in the acute stage of infection, since specific IgM is still continuously elevated within one year after infection. Therefore, in the MP serological diagnosis method, dynamic observation is needed to detect IgM antibody and IgG antibody total antibody so as to be capable of better assisting the clinical diagnosis of MP.
In the in vitro diagnosis of mycoplasma pneumoniae infection, it is most important to select an antigen with strong antigenicity and good specificity. With the continuous development of molecular biotechnology, the main antigen genes of many MPs are cloned, expressed, purified and used for MP antibody detection. Schurwanz N et al divide the full length of the P1 protein into 16 segments to respectively construct recombinant proteins, find that HP14 segment has the strongest immunogenicity, construct the segment of protein and P30 protein into fusion protein HP14/30, and stimulate the production of corresponding specific antibodies by using the purified fusion protein. The HP14/30 immune guinea pig serum can inhibit the adhesion rate of MP to human lung bronchial epithelial cells to 5%, and the effect is close to that of MP whole-cell immune guinea pig serum. However, the preparation process of MP whole bacteria antigen is strict, the steps are complex, other proteins are often mixed, the price is high, the quality of products in batches is not uniform, and the risk of infecting human beings is existed in the production and use process.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a mycoplasma pneumoniae antigen.
It is a second object of the invention to provide a nucleic acid molecule encoding a mycoplasma pneumoniae antigen.
It is a third object of the present invention to provide a biological material related to a nucleic acid molecule.
The fourth purpose of the invention is to provide the application of the mycoplasma pneumoniae antigen or the nucleic acid molecule or the biological material in preparing a mycoplasma pneumoniae detection product.
The fifth object of the present invention is to provide a reagent for detecting Mycoplasma pneumoniae.
The sixth purpose of the invention is to provide a mycoplasma pneumoniae detection kit.
The seventh object of the present invention is to provide a method for producing a mycoplasma pneumoniae antigen.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a Mycoplasma pneumoniae antigen which is (a) or (b) below:
(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO.1 and has the same function.
A nucleic acid molecule encoding a mycoplasma pneumoniae antigen which is (a) or (b) as follows:
(a) a DNA sequence encoding SEQ ID No. 1;
(b) a DNA sequence having a homology of 90% or more with the DNA sequence of (a) and encoding a protein having the same function.
Further, the nucleic acid molecule is a DNA sequence shown in SEQ ID NO. 2.
A biological material related to the nucleic acid molecule of the present invention, which is any one of:
(a) an expression cassette comprising a nucleic acid molecule of the invention;
(b) a recombinant vector comprising a nucleic acid molecule of the invention or an expression cassette of (a);
(c) a recombinant eukaryotic cell comprising a nucleic acid molecule of the invention, an expression cassette of (a) or a recombinant vector of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of the invention, the expression cassette of (a) or the recombinant vector of (b).
The mycoplasma pneumoniae antigen or the nucleic acid molecule or the biological material disclosed by the invention is applied to preparation of a mycoplasma pneumoniae detection product.
Further, the detection method adopted by the mycoplasma pneumoniae detection product comprises a chemiluminescence method, an ELISA method or a colloidal gold rapid detection method.
A Mycoplasma pneumoniae detection reagent, wherein the antigen used by the Mycoplasma pneumoniae detection reagent comprises the Mycoplasma pneumoniae antigen.
A Mycoplasma pneumoniae detection kit comprises the Mycoplasma pneumoniae antigen or detection reagent.
A preparation method of a mycoplasma pneumoniae antigen comprises the step of expressing a nucleic acid molecule to obtain the mycoplasma pneumoniae antigen.
Further, recombining the nucleic acid molecule with an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a host for expression to obtain a mycoplasma pneumoniae antigen;
preferably, the expression vector is a pET28a vector;
preferably, the host is escherichia coli, preferably e.coli Rosetta;
preferably, the recombinant vector is introduced into a host, expressed, purified and concentrated to obtain the mycoplasma pneumoniae antigen.
Compared with the prior art, the invention has the beneficial effects that:
the mycoplasma pneumoniae antigen provided by the invention is an antigenic protein sequence P1M: residues 1340-1518, which is specifically a protein composed of the amino acid sequence shown in SEQ ID NO.1 or a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.1 and having the same function. The antigen sequence is an MP specific antigen fragment obtained by technical methods such as bioinformatics, genetic engineering and the like, and the antigen is verified by an immune serology detection technology to have strong specificity and high sensitivity, the detection effect is higher than that of the MP antigen P1, and the antigen is easy to culture and purify, is more beneficial to industrial production and saves cost. In addition, the antigen is suitable for preparing MP antibody detection products, can be products in any form in the field of in vitro immunodiagnosis, and has wide market prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a SDS-PAGE protein electrophoresis chart of the induced expression of Mycoplasma pneumoniae antigen and Ni column purified samples in example 2 of the present invention, wherein M: protein marker; s, centrifuging supernatant after ultrasonic crushing; 50: 50mM imidazole eluent; 100: 100mM imidazole eluent; 500: 500mM imidazole eluent;
FIG. 2 is an SDS-PAGE protein electrophoresis chart of a Q column purified sample of Mycoplasma pneumoniae antigen in example 2 of the present invention, wherein M: protein marker; FT: the Q column flows through the liquid; 1: 100mM NaCl eluate; 2: 200mM NaCl eluate; 3: 1M NaCl eluate.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The invention protects a mycoplasma pneumoniae antigen which is (a) or (b) as follows:
(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO.1 and has the same function.
The mycoplasma pneumoniae antigen provided by the invention is an antigenic protein sequence P1M: senses 1340-1518. The antigen sequence is an MP specific antigen fragment obtained by technical methods such as bioinformatics, genetic engineering and the like, and the immune serology detection technology verifies that the antigen has strong specificity and high sensitivity, the detection effect is higher than that of the MP antigen P1, and the antigen is easy to culture and purify, is more beneficial to industrial production and saves cost. In addition, the antigen is suitable for preparing MP antibody detection products, can be products in any form in the field of in vitro immunodiagnosis, and has wide market prospect.
WLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQT(SEQ ID NO.1)。
The invention also protects a nucleic acid molecule encoding a mycoplasma pneumoniae antigen, which is (a) or (b) as follows:
(a) a DNA sequence encoding SEQ ID No. 1;
(b) a DNA sequence having a homology of 90% or more with the DNA sequence of (a) and encoding a protein having the same function.
The mycoplasma pneumoniae adopts a unique partial codon system, and the universal stop codon UGA codes tryptophan in the mycoplasma pneumoniae, so that translation is interrupted if a wild gene of the mycoplasma pneumoniae is directly adopted, and the preparation of an antigen is limited. In order to overcome the difficulty, the invention adopts the escherichia coli preference codon to reversely translate the antigen amino acid sequence of the invention, and obtains the recombinant antigen gene nucleic acid molecule consisting of the escherichia coli preference codon. The nucleic acid molecule can completely and accurately express the mycoplasma pneumoniae antigen. The nucleic acid molecule is preferably the DNA sequence shown in SEQ ID NO. 2.
TGGCTGGTTGGCCAGCTGCCGAGCACCAGCGATGGTAACACCAGCAGCACCAACAACCTGGCGCCGAACACCAACACCGGCAACGACGTGGTTGGTGTGGGCCGTCTGAGCGAAAGCAACGCGGCGAAAATGAACGATGACGTGGACGGTATCGTTCGTACCCCGCTGGCGGAGCTGCTGGATGGCGAGGGTCAGACCGCGGACACCGGTCCGCAGAGCGTGAAGTTTAAAAGCCCGGATCAAATCGACTTCAACCGTCTGTTTACCCACCCGGTTACCGACCTGTTCGACCCGGTGACCATGCTGGTTTACGATCAGTATATTCCGCTGTTTATCGACATTCCGGCGAGCGTTAACCCGAAGATGGTGCGTCTGAAAGTTCTGAGCTTCGATACCAACGAGCAAAGCCTGGGTCTGCGTCTGGAGTTCTTCAAACCGGATCAAGACACCCAGCCGAACAACAACGTGCAGGTTAACCCGAACAACGGTGACTTTCTGCCGCTGCTGACCGCGAGCAGCCAAGGTCCGCAGACCTAA(SEQ ID NO.2)。
The invention also protects biological materials related to nucleic acid molecules, wherein the biological materials are any one of the following materials:
(a) an expression cassette comprising a nucleic acid molecule of the invention;
(b) a recombinant vector comprising a nucleic acid molecule of the invention or an expression cassette of (a);
(c) a recombinant eukaryotic cell comprising a nucleic acid molecule of the invention, an expression cassette of (a) or a recombinant vector of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of the invention, the expression cassette of (a) or the recombinant vector of (b).
The invention also protects the application of the mycoplasma pneumoniae antigen or nucleic acid molecule or biological material in the preparation of mycoplasma pneumoniae detection products. The detection method adopted by the mycoplasma pneumoniae detection product can be a chemiluminescence method, an ELISA or a colloidal gold rapid detection method and the like, and the mycoplasma pneumoniae antigen provided by the invention is suitable for products in any forms in the field of in vitro immunodiagnosis.
The invention also provides a mycoplasma pneumoniae detection reagent, and the antigen used by the reagent comprises the mycoplasma pneumoniae antigen. Meanwhile, the invention provides a mycoplasma pneumoniae detection kit, which comprises the mycoplasma pneumoniae antigen or detection reagent. The detection reagent may be, for example, an immunochromatographic test strip.
The invention finally provides a preparation method of the mycoplasma pneumoniae antigen, which expresses the nucleic acid molecule to obtain the mycoplasma pneumoniae antigen.
In a preferred embodiment, the nucleic acid molecule is recombined with an expression vector to obtain a recombinant vector, which is introduced into a host and expressed to obtain the mycoplasma pneumoniae antigen. Wherein, the expression vector can be pET28a vector; the host can be escherichia coli, preferably e.coli Rosetta; the recombinant vector is introduced into a host to be expressed, and then the mycoplasma pneumoniae antigen is obtained after purification and concentration.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1: synthesis of target Gene and construction of recombinant vector of the invention
1. The whole amino acid sequence (MP FH strain) of Mycoplasma pneumoniae antigen P1 was analyzed by ProtScale et al software to select a protein sequence P1M: residues 1340-1518 with antigenicity, the amino acid sequence of which is shown in SEQ ID NO: 1.
2. The invention adopts the escherichia coli preference codon to reversely translate the amino acid sequence to obtain the recombinant antigen gene consisting of the escherichia coli preference codon. The gene is connected to pET28a carrier by gene synthesis method, and the gene sequence of coded antigen is shown in SEQ ID NO. 2.
Example 2: induced expression purification and application of mycoplasma pneumoniae antigen
1. The recombinant vector P1M-pET28a in example 1 was transformed into E.coli Rosetta (DE3) competent cells, cultured on LB plate containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, and cultured at 37 ℃ for 14-16 hours; positive recombinant bacteria were selected, and a single colony was inoculated into 5ml of LB medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, and cultured overnight.
2. The overnight-cultured bacterial suspension was inoculated at 1% inoculum size to LB medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, and cultured at 37 ℃ and 250rpm until the OD of the bacterial suspension6001.0-1.3, and inducing with IPTG with final concentration of 0.5-1.0 mM at 25 deg.C and 250rpm for 3-4 h.
3. Coli e.coli Rosetta cells obtained in 2 were collected by centrifugation, and 20ml of lysine buffer (ph7.5) was added to ice to resuspend the solid until no clumpy cells were visible to the naked eye.
4. Ultrasonic crushing: and (4) breaking the bacteria according to the conditions of ultrasonic power of 400W, ultrasonic for 4s and stopping for 3s and circulation for 99 times until the bacteria liquid is clear and transparent. After the sonication was completed, the supernatant and the precipitate were separated by centrifugation at 12000rpm for 30min at 4 ℃.
5. The supernatant separated in step 4 was added with balanced 1ml of Ni resin and placed in an ice box and incubated with shaking for 1 hour.
6. Pouring the incubated mixture into a gravity column, collecting the flow-through liquid by using a clean EP tube, and adding 15mL of lysine buffer to wash out the residual flow-through liquid in the pipeline after the flow-through liquid completely flows out.
7. And (3) elution: the Ni column was eluted with 50mM, 100mM and 500mM imidazole, and the eluates were collected by peak separation tube and 20. mu.l was left.
8. The samples retained in the above steps were subjected to SDS-PAGE in the order of elution, and the results are shown in FIG. 1, wherein M: protein marker; s, centrifuging supernatant after ultrasonic crushing; 50: 50mM imidazole eluent; 100: 100mM imidazole eluent; 500: 500mM imidazole eluate, Colony-3 and Colony-4 are the results of the detection of the two strains.
9. Purification on Q column
Preparation of a Q column: wash 10CV with Q column high salt buffer, then balance 10CV with low salt buffer.
Loading: the sample purified by the Ni column was diluted to 10ml with BufferA, and subjected to column chromatography at a flow rate Q of 1ml/min, and the flow-through was collected in a clean bottle.
Cleaning: after loading, the column was washed with buffer A over 15 CV. And (3) elution: the elution was carried out with a gradient of 100mM NaCl, 200mM NaCl, 1M NaCl, and the eluate was collected by peak separation tube and 20. mu.l of the sample was retained. Electrophoresis: SDS-PAGE (4% -20% gradient gel) was performed in order of elution, and the results are shown in FIG. 2, in which M: protein marker; FT: the Q column flows through the liquid; 1: 100mM NaCl eluate; 2: 200mM NaCl eluate; 3: 1M NaCl eluate.
Example 3: specificity and sensitivity detection
Based on the principle of antigen-antibody specific binding, the binding reactivity of the mycoplasma pneumoniae antigen and the IgM antibody is detected by using an enzyme-labeled IgM antibody and sulfonated magnetic beads coated with the mycoplasma pneumoniae antigen and adopting a chemiluminescence method. The application effects of the recombinant P1M antigen and the recombinant P1 antigen (as a comparative example) provided by the invention are detected by adopting an immune serological detection technology, and specifically, the method comprises the following steps:
coating sulfonated magnetic beads: mixing the mycoplasma pneumoniae antigen and the sulfonated magnetic beads according to a certain proportion, and washing, coating and cleaning the mixture by using Buffer independently developed by Zhuhai pearl reagent GmbH to prepare magnetic bead working solution;
according to the process flow independently developed by Zhuhai Lizhu reagent GmbH, preparing enzyme-labeled antibody IgM working solution;
the reactivity of the mycoplasma pneumoniae antigen was detected by chemiluminescence, luminescence values were recorded, and statistical data analysis was performed, with the results shown in table 1 below.
TABLE 1 immune serological test results for P1M antigen and P1 antigen
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
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<120> mycoplasma pneumoniae antigen and preparation method and application thereof
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Claims (14)
1. A mycoplasma pneumoniae antigen which is a protein consisting of an amino acid sequence shown in SEQ ID No. 1.
2. A nucleic acid molecule encoding a mycoplasma pneumoniae antigen, characterised in that it is a DNA sequence encoding SEQ ID No. 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is the DNA sequence of SEQ ID No. 2.
4. Biological material associated with the nucleic acid molecule of claim 2 or 3, wherein the biological material is any one of:
(a) an expression cassette comprising the nucleic acid molecule of claim 2 or 3;
(b) a recombinant vector comprising the nucleic acid molecule of claim 2 or 3 or the expression cassette of (a);
(c) a recombinant eukaryotic cell comprising the nucleic acid molecule of claim 2 or 3, the expression cassette of (a), or the recombinant vector of (b);
(d) a recombinant prokaryotic cell comprising the nucleic acid molecule of claim 2 or 3, the expression cassette of (a), or the recombinant vector of (b).
5. Use of a mycoplasma pneumoniae antigen according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a biological material according to claim 4 for the preparation of a mycoplasma pneumoniae detection product.
6. The use according to claim 5, wherein the detection method used in the detection product for Mycoplasma pneumoniae comprises chemiluminescence, ELISA or rapid detection with colloidal gold.
7. A Mycoplasma pneumoniae detection reagent characterized in that the antigen used in the Mycoplasma pneumoniae detection reagent comprises the Mycoplasma pneumoniae antigen according to claim 1.
8. A Mycoplasma pneumoniae detection kit comprising the Mycoplasma pneumoniae antigen of claim 1 or the detection reagent of claim 7.
9. A method for producing a Mycoplasma pneumoniae antigen, comprising expressing the nucleic acid molecule according to claim 2 or 3 to obtain the Mycoplasma pneumoniae antigen.
10. The method according to claim 9, wherein the mycoplasma pneumoniae antigen is obtained by recombining the nucleic acid molecule with an expression vector to obtain a recombinant vector, and expressing the recombinant vector after introduction into a host.
11. The method according to claim 10, wherein the expression vector is pET28a vector.
12. The method according to claim 10, wherein the host is Escherichia coli.
13. The method of claim 12, wherein the E.coli is E.
14. The method according to claim 10, wherein the recombinant vector is introduced into a host, expressed, purified, and concentrated to obtain the mycoplasma pneumoniae antigen.
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| CN109342724A (en) * | 2018-10-31 | 2019-02-15 | 廖朝晖 | A kind of kit detecting mycoplasma pneumoniae |
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