CN105566492A

CN105566492A - Preparing method for anti-hepatitis B-surface-antigen fluorescent antibody

Info

Publication number: CN105566492A
Application number: CN201610029748.3A
Authority: CN
Inventors: 张贯京; 陈兴明; 张少鹏; 高伟明; 李慧玲
Original assignee: Shenzhen Beiwo Deke Biotechnology Research Institute Co Ltd
Current assignee: Shenzhen Beiwo Deke Biotechnology Research Institute Co Ltd
Priority date: 2016-01-16
Filing date: 2016-01-16
Publication date: 2016-05-11

Abstract

The invention discloses a preparing method for an anti-hepatitis B-surface-antigen fluorescent antibody. The DNA sequence of pyrrolysine aminoacyl-tRNA synthetase (PylRS) of M.Barkeri is synthesized and is connected to pPICZ plasmids; a mutation DNA sequence of the hepatitis B surface antigen (HBsAg) antibody is designed, and mutation DNA and plasmids pPIC9K are recombined to obtain pPIC9K-antiHBsAg; electroporation, screening and verification are conducted on the linearized plasmids pPICZ-CouKRS and pPIC9K-antiHBsAg to obtain a eucaryon expression system for preparing the anti-hepatitis B-surface-antigen fluorescent antibody, and consequently the anti-hepatitis B-surface-antigen fluorescent antibody is prepared. According to the preparing method for the anti-hepatitis B-surface-antigen fluorescent antibody, HBsAg detection sensitivity is improved, and the monitoring strength for hepatitis B paroxysm is enhanced.

Description

The preparation method of the fluorescence antibody of anti-hepatitis b surface antigen

Technical field

The present invention relates to biological technical field, particularly relate to a kind of preparation method of fluorescence antibody of anti-hepatitis b surface antigen.

Background technology

Hepatitis B (abbreviation hepatitis B) infects by hepatitis B virus (HepatitisBvirus, HBV) the global great public health problem caused, and is also the modal hepatopathy cause of disease.According to World Health Organization's statistics, the whole world has 2,000,000,000 people once to infect HBV, wherein about has 300,000,000 people to develop into HBV chronic infection.HBV chronic infection is the major cause causing liver cirrhosis and liver cancer, about has the liver cirrhosis patient of 1/3 and the liver cancer patient of more than 3/4 to be caused by hepatitis B chronic infection in the world.The disease such as liver cirrhosis or liver cancer having nearly 1,000,000 people to die from HBV chronic infection every year to cause.China is the hotspot of HBV infection, state-owned 9,300 ten thousand HBV chronic infection crowds at present.In recent years, along with the popularization of Hepatitis B virus vaccine, de novo hepatitis B infection quantity reduces, but China still has every year, and 100,000 examples are new sends out HBV infection person.The difficulty of hepatitis B prevention and control can not be ignored.Hepatitis B surface antigen (HBsAg) detection by quantitative evaluates the important means of chb result for the treatment of.Chinese Medical Association issue 2015 version " the guideline " point out: Quantitative Monitoring is carried out to the hepatitis B surface antigen in serum, can be used for predictive disease progress, Anti-viral Treatment and prognosis, obtain lasting HBsAg after drug withdrawal and disappear for the desirable terminal of chb treatment.Whether disappear for assessment chb HBsAg, then require quantitatively to reach accurate level to HBsAg.Limitting by technology, the detection of HBsAg is in manual qualitative or sxemiquantitative state always.Recently, external company has by the next quantitative HBsAg of the method for chemical colour reaction, but the sensitivity of chemical colour reaction method is dripped, and can't detect low-abundance HBsAg in blood, bring erroneous judgement can to the detection of HBsAg.Sensitiveer than chemical colour reaction more than 10000 times of the method that fluorescence radiation detects, can detect the detection of the target of single copy in sample.

Based on this, be necessary the preparation method of the fluorescence antibody designing a kind of anti-hepatitis b surface antigen, be used for increasing substantially the sensitivity that HBsAg detects, the accurate quantification realizing HBsAg detects, and assistant reinforcement is to the control and monitoring of the morbidity of hepatitis B.

Summary of the invention

The object of the present invention is to provide a kind of preparation method of fluorescence antibody of anti-hepatitis b surface antigen, be intended to the detection sensitivity of raising HBsAg, stable in properties and specificity, the accurate quantification being applicable to HBsAg detects.

For achieving the above object, the invention provides a kind of preparation method of fluorescence antibody of anti-hepatitis b surface antigen, the preparation method of the fluorescence antibody of described anti-hepatitis b surface antigen comprises the steps:

S1: obtain M.BarkeriPylRS mutation library;

S2: obtain tonka bean camphor Methionin;

S3: pyrrolysine aminoacyl-tRNA synthetase (CouKRS) mutant of screening specific recognition umbelliferone;

S4: recombinated by the encoding gene coukrs of the pyrrolysine aminoacyl-tRNA synthetase mutant in step S3 and Pichia anomala expression plasmid pPICZ, obtains expression plasmid of yeast pPICZ-CouKRS;

S5: the mutant DNA sequences of design AntiHBsAg antibody, obtains this mutant, the mutant DNA sequences of described AntiHBsAg antibody and plasmid pPIC9K are recombinated, and obtains expression plasmid of yeast pPIC9K-antiHBsAg;

S6: the pPICZ-CouKRS linearizing obtained by step S4, by the pPIC9K-antiHBsAg linearizing that step S5 obtains;

S7: the ratio mixing preset according to first by the pPICZ-CouKRS after step S6 linearizing and the pPIC9K-antiHBsAg after linearizing, through electroporated, imports in pichia spp X33, obtain strains A;

S8: the strains A that step S7 obtains is carried out resistance screening, obtains positive strain group B;

S9: the positive strain group B that step S8 obtains is carried out PCR Screening and Identification respectively, obtains several positive strains group C;

S10: the mono-clonal yeast in the positive strain group C that step S9 is obtained, coat in the YPDS solid medium containing G418 and cultivate, to verify the G418 resistance of described mono-clonal yeast, if be proved to be successful, then obtain and possess and identify and carry the mono-clonal yeast D that umbelliferone is inserted into 63 sites of anti-hepatitis b surface antigen antibody;

S11: the mono-clonal yeast D enlarged culturing obtained by step S10, centrifugal, ultrasonication and ni-sepharose purification obtain anti-hepatitis b surface antigen fluorescence antibody.

Preferably, described step S1 comprises the steps:

S101: the DNA sequence dna of synthetic M.BarkeriPylRS;

S102: be connected to by the DNA sequence dna of the M.BarkeriPylRS of above-mentioned synthesis on pBK plasmid, obtains recombinant plasmid A;

S103: design rite-directed mutagenesis primer, described rite-directed mutagenesis primer comprises primer f-Y349D, primer r-Y349D, primer f-L270I, primer r-L270I, primer f-N311F313NNK, primer r-N311F313NNK, primer f-Y349NNK and primer r-Y349NNK;

S104: with described recombinant plasmid A for template, with described primer f-Y349D and primer r-Y349D for primer, by polymerase chain reaction, obtains mutation library 1;

S105: with described mutation library 1 for template, with described primer f-L270I and primer r-L270I for primer, by polymerase chain reaction, obtains mutation library 2;

S106: with described mutation library 2 for template, with described primer f-N311F313NNK and primer r-N311F313NNK for primer, by polymerase chain reaction, obtains mutation library 3;

S107: with described mutation library 3 for template, with described primer f-Y349NNK and primer r-Y349NNK for primer, by polymerase chain reaction, obtains mutation library 4;

S108: described mutation library 1, mutation library 2, mutation library 3 and mutation library 4, according to the second predetermined ratio mixing, obtain the M.BarkeriPylRS mutation library being used for umbelliferone screening.

Preferably, described first predetermined ratio is 1:1.

Preferably, described step S2 comprises the steps:

S21: the Resorcinol getting the first predetermined amount, soluble in water, and be heated to 90 DEG C, drip the oxysuccinic acid of the second predetermined amount, react and can obtain umbelliferone A after 2 hours;

S22: get the above-mentioned umbelliferone A of the 3rd predetermined amount, Methionin and the LiCl aqueous solution in 60 DEG C of stirrings, through 12 hours; Be separated through liquid chromatography, membrane filtration is degerming, obtains umbelliferone mother solution C.

Preferably, described umbelliferone mother solution C is through 0.22 micron membrane filter filtration sterilization.

Preferably, the concentration of described umbelliferone mother solution C is set as 100mM.

Preferably, described step S8 comprises the steps:

S81: be coated on the YPDS flat board containing ZeocinTM and ammonia benzyl mycin after the strains A that step S7 obtains is cultivated two hours, cultivate 3 days, obtain multiple mono-clonal bacterial strain for 30 DEG C;

S82: the multiple mono-clonal bacterial strain pickings obtained by step S81 are in YPD liquid nutrient medium, and 30 DEG C of shaking tables are cultivated.Obtain positive strain group B

Preferably, described step S9 comprises the steps:

S91: it is centrifugal that the yeast liquid that the positive strain group B getting step S82 acquisition increases is placed in centrifuge tube respectively, obtains yeast sedimentation group 1;

S92: the yeast sedimentation group 1 that step S91 obtains is added sterilized water, whirlpool concussion is resuspended, obtains yeast sedimentation group 2;

S93: the refrigerator described yeast sedimentation group 2 being placed in respectively-80 DEG C, takes out after 30min, thaw at room temperature;

S94: repeating step S93 once, and centrifuging and taking supernatant liquor obtains yeast soln group 3 respectively;

S95: get yeast soln group 3, be placed in the enterprising performing PCR amplification of PCR amplification instrument respectively, the monoclonal exactness in verification step S82, obtains several positive strains group C.

Preferably, described step S4 inserts the encoding gene coukrs of pyrrolysine aminoacyl-tRNA synthetase mutant between the EcoRI and XbaI enzyme cutting site of Pichia anomala expression plasmid pPICZ, obtains expression plasmid of yeast pPICZ-CouKRS.

Preferably, described step S5 inserts the mutant DNA sequences of AntiHBsAg antibody between EcoRI and the NotI restriction enzyme site of plasmid pPIC9K, obtains expression plasmid of yeast pPIC9K-antiHBsAg.

Described DNA sequence dna by synthesizing the DNA sequence dna of the pyrrolysine aminoacyl-tRNA synthetase (PylRS) of M.Barkeri species, and is connected on pPICZ plasmid by the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen provided by the invention; By designing the mutant DNA sequences of AntiHBsAg antibody, obtaining this mutant, the mutant DNA of described AntiHBsAg antibody and plasmid pPIC9K are recombinated, obtain expression plasmid of yeast pPIC9K-antiHBsAg; By linearization plasmid pPICZ-CouKRS, pPIC9K-antiHBsAg are mixed according to certain ratio, electroporated, import in pichia spp X33, carry out screening and verifying, obtain the eukaryotic expression system preparing anti-hepatitis b surface antigen fluorescence antibody, this System-mediated umbelliferone inserts in 63 sites of the aminoacid sequence of anti-hepatitis b surface antigen antibody, thus achieves the preparation of anti-hepatitis b surface antigen fluorescence antibody; Increase substantially the sensitivity that HBsAg detects, the auxiliary accurate quantification realizing HBsAg detects, and strengthens the control and monitoring of the morbidity to hepatitis B.

Accompanying drawing explanation

Fig. 1 is the schematic flow sheet of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention;

Fig. 2 is the schematic flow sheet of the step S1 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention;

Fig. 3 is the schematic flow sheet of the step S2 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention;

Fig. 4 is the structural representation of tonka bean camphor Methionin of the present invention;

Fig. 5 is the schematic flow sheet of the step S8 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention;

Fig. 6 is the schematic flow sheet of the step S9 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail, and following examples are explanation of the invention, and the present invention is not limited to following examples.

The preparation method of the fluorescence antibody of anti-hepatitis b surface antigen provided by the invention, the preparation method of the fluorescence antibody of described anti-hepatitis b surface antigen comprises the steps:

S1: obtain M.BarkeriPylRS mutation library;

S2: obtain tonka bean camphor Methionin;

S10: the mono-clonal yeast in the positive strain group C obtain step S9 is coated in the YPDS solid medium containing G418 and cultivated, to verify the G418 resistance of described mono-clonal yeast, if be proved to be successful, then obtain and possess and identify and carry the mono-clonal yeast D that umbelliferone is inserted into 63 sites of anti-hepatitis b surface antigen antibody;

In the present embodiment, M.Barkeri represents a kind of methane phase anerobe; PylRS (pyrrolysineaminoacyl-tRNAsynthetase) Chinese is pyrrolysine aminoacyl-tRNA synthetase.Nucleotides sequence list 1 is the gene order of the pyrrolysine aminoacyl-tRNA synthetase (CouKRS) of specific recognition umbelliferone; Aminoacid sequence table is the aminoacid sequence of the pyrrolysine aminoacyl-tRNA synthetase (CouKRS) of specific recognition umbelliferone; Nucleotides sequence list 2 is the mutant gene sequence of AntiHBsAg antibody.

In an embodiment, as shown in Figure 1, Fig. 1 is the schematic flow sheet of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention.The PylRS (pyrrolysine aminoacyl-tRNA synthetase) of wild-type methane phase anerobe only identifies pyrrolysine (natural amino acid of the 22nd kind of genes encoding), and there is transhipment reaction in catalysis pyrrolysine and corresponding tRNA, thus pyrrolysine is connected on corresponding tRNA, elongation factor EF-Tu carries above-mentioned product to rrna, participates in protein synthesis.TRNA, also known as transfer RNA (transferribonucleicacidtRNA), is have the small molecule Yeast Nucleic Acid carrying and transport aminoacid functional.But the PylRS of the methane phase anerobe of wild-type (pyrrolysine aminoacyl-tRNA synthetase) nonrecognition tonka bean camphor Methionin, therefore can not determine that any PylRS mutant can identify tonka bean camphor Methionin, so the storehouse-screening (orthogenesis) that suddenlys change-build will be done, finishing screen is chosen can the PylRS mutant of specific recognition tonka bean camphor Methionin, i.e. called after CoukRS.

In the present embodiment, as shown in Figure 2, Fig. 2 is the schematic flow sheet of the step S1 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention.Step S1 is for obtaining M.BarkeriPylRS mutation library; In the process realizing step S1,8 steps can be divided again to realize, and are step S101, S102, S103, S104, S105, S106, S107 and S108 respectively.The DNA sequence dna of first synthetic M.BarkeriPylRS in step S101, then by step S102, the DNA of M.BarkeriPylRS is connected on pBK plasmid, both link can be cut by traditional enzyme, also substep PCR method can be adopted, utilize the method for saturation mutation, design primer, be that (N represents A to NNK the codon mutation of single amino acids, T, C, G tetra-kinds of codons, K represents T, G two kinds of codons) NNK combination is 4*4*2=32 kind an amino acid whose codon mutation, corresponding 20 seed amino acids.Reach the object of " creating groundless rumors ", final acquisition recombinant plasmid A.In addition, pBK plasmid is smaller, is conducive to the structure of the high-capacity library in downstream.When the coding DNA of the M.BarkeriPylRS of above-mentioned synthesis is connected in pBK plasmid, when being by step-by-step polymerization polymerase chain reaction (PCR) method, performing step is as follows: the structure realizing mutation library under specific 50 μ LPCR amplification systems and ad hoc approach.50 μ LPCR amplification systems comprise: the damping fluid 5 μ L of sterilized water 40 μ L, 10 times of concentration, plasmid DNA template 1 μ L, each 1 μ L, 10mMdNTPs1 μ L, the high-fidelity enzyme 1 μ L of the positive and negative chain of pcr amplification primer.Following steps PCR reaction system is all carried out according to this system.Above-described reaction system, PCR amplification instrument increases, and the method for PCR reaction amplification is: 94 DEG C, 4 minutes, denaturation; 94 DEG C, 1 minute, sex change; 58 DEG C, 30 seconds, annealing; 72 DEG C, 3 minutes, extend; After repeating the circulation of sex change-annealing-extension step 26,72 DEG C are incubated 5 minutes, then cool to 16 degree, complete reaction.Following steps mutation library, carries out all in this way.After the pcr amplification of each step, need to transform through electrocompetent intestinal bacteria, and increase in intestinal bacteria, obtain through plasmid extraction step the plasmid that can be used for next step pcr amplification reaction.The above-described competent preparation that can be used for shocking by electricity adopts standard step to carry out.

In the present embodiment, the principle of design rite-directed mutagenesis primer is first the pylrs of wild-type is connected on pbk, then primer (having sudden change at specific site) is designed, after PCR, obtain background pylrs-pbk and sudden change pylrs-pbk, because the pylrs-pbk of background extracts from DH5a, adenosine can methylate, cut can remove background with the methylate DpnI enzyme of adenosine of specific recognition.The linear plasmid fragment (containing sudden change) obtained is transformed in competent cell, obtains the plasmid of cyclisation.Extract the plasmid of cyclisation, as masterplate, recycle another and mutant primer is suddenlyd change, equally with DpnI process, obtain mutation library 2, again repeat, obtain mutation library 3, obtain mutation library 4.Be that (N represents A to NNK, T, C, G tetra-kinds of codons, and K represents T, G two kinds of codons the codon mutation of single amino acids.) NNK combination is 4*4*2=32 kind an amino acid whose codon mutation, corresponding 20 seed amino acids.According to protein structures, the activity center of bound substrates can be obtained, this center is made up of multiple amino acid, calculate through InsightII molecular simulation, molecular simulation software InsightII calculates PylRS enzyme activity center, can predict the amino acid around substrate molecule, these amino acid are important evidence of evolution PylRS.In step s 103, by rite-directed mutagenesis principle design 4 to different primers, as shown in table 1, table 1 is the sequence of the primer adopting present pre-ferred embodiments.These 4 pairs of primers are primer f-Y349D, primer r-Y349D, primer f-L270I, primer r-L270I, primer f-N311F313NNK, primer r-N311F313NNK, primer f-Y349NNK and primer r-Y349NNK respectively.In step S104, utilize the primer f-Y349D in step S103, primer r-Y349D, by PCR (polymerase chain reaction), mutation library 1 can be obtained; In like manner, utilize the primer f-L270I in step S103 and primer r-L270I, primer f-N311F313NNK and primer r-N311F313NNK, primer f-Y349NNK and primer r-Y349NNK, respectively by PCR (polymerase chain reaction), mutation library 2, mutation library 3, mutation library 4 can be obtained respectively.By the mutation library 1, mutation library 2, mutation library 3 and the mutation library 4 that obtain according to the second predetermined ratio mixing, this ratio can be arranged to 1:1:32:32, finally can obtain the M.BarkeriPylRS mutation library for umbelliferone screening.Detailed step is as follows: in the above reaction system, add above-mentioned primer f-Y349D and primer r-Y349D, carry out pcr amplification, obtain PCR primer 1; PCR primer 1, increases in competence intestinal bacteria through electroporated, through plasmid extraction, obtains mutation library 1; With above-mentioned mutation library 1 for template, in reaction system, add above-mentioned primer f-L270I and primer r-L270I, through the step process that pcr amplification is identical with said mutation storehouse 1, obtain mutation library 2; With above-mentioned mutation library 2 for template, increase through primer f-N311F313NNK and primer r-N311F313NNK, obtain PCR primer 3, the preparation method that PCR primer 3 is identical through said mutation storehouse 1, obtain mutation library 3, mutation library 3 obtains mutation library 4 through primer Y349NNK-f and primer Y349NNK-r primer amplification, conversion, plasmid extraction step; The above mutation library 1-4 is mixed according to the ratio of 1:1:32:32, obtains finally for the M.BarkeriPylRS mutation library (Lib-CouK) of umbelliferone screening.

Table 1 adopts the sequence of the primer of present pre-ferred embodiments

As shown in Figure 3, Fig. 3 is the schematic flow sheet of the step S2 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention.In step s 2, relate generally to the synthesis of tonka bean camphor Methionin, as shown in Figure 4, Fig. 4 is the structural representation of tonka bean camphor Methionin of the present invention.Its synthesis step is as follows: in the step s 21, first gets Resorcinol 5.5 grams, soluble in water, and is heated to 90 DEG C, slowly in resorcinol solution, drips water-soluble oxysuccinic acid 6.7 grams, reacts 2 hours, obtains umbelliferone; In step S22, get umbelliferone 10 grams, Methionin 2.24 grams, water-soluble respectively, drip the LiCl aqueous solution 10 milliliters of 10 mmoles in the solution, spend the night in 60 degrees Celsius of stirrings, the time can be 12 hours.Be separated through liquid chromatography, obtain umbelliferone Methionin 9.5 grams for screening.The LiCl aqueous solution is also known as lithium chloride solution, and typical concentrations is 1mol/L.In the present embodiment, tonka bean camphor Methionin B is through 0.22 micron membrane filter filtration sterilization.

In step s3, pyrrolysine aminoacyl-tRNA synthetase (CouKRS) mutant of main screening specific recognition umbelliferone: the alpha-non-natural amino acid umbelliferone synthesized in step s 2, be dissolved in water, through 0.22 micron membrane filter filtration sterilization, obtain the umbelliferone mother liquor that final concentration is 100mM.Before screening, build and just screening pylT-pREP plasmid, and for expressing the pylT-pBAD plasmid of checking.The screening step of described CouKRS is as follows: first cotransformation Lib-CouK and pylT-pREP plasmid are in Top10 E. coli competent, and Top10 competent escherichia coli cell can be used for the chemical conversion of DNA.Through 37 DEG C, after within 1 hour, hatching, be coated on positive sifting motion cultivation plate.Just screening and object clone is presented, corresponding negative screening is then that non-object clone is rejected.The compound method of positive sifting motion cultivation plate is as follows: add 0.015g agar powder in 100 milliliters of LB substratum, autoclaving process, and cool to 60 DEG C; Add the umbelliferone mother liquor that 1 milliliter of above-described concentration is 100mM wherein, the paraxin liquid of 100 μ L80 mg/ml, concentration is the tsiklomitsin 100 μ L of 50mM, concentration is the kantlex 100 μ L of 50mM, after mixing, pour in sterile culture plate, for subsequent use after cooling.At 37 DEG C, the cultivation of 48 hours, selects and carries out cultivation 12 hours just screening the mono-clonal bacterium that plank is survived, and the mono-clonal bacterium obtained to be imprinted on two kinds of substratum on 1 and substratum 2 respectively.The formula of substratum 1 is: add 0.015g agar powder in 100 milliliters of LB substratum, autoclaving process, and cool to 60 DEG C; Add the umbelliferone mother liquor that 1 milliliter of above-described concentration is 100mM wherein, the paraxin liquid of 150 μ L80 mg/ml, concentration is the tsiklomitsin 100 μ L of 50mM, concentration is the kantlex 100 μ L of 50mM, after mixing, pour in sterile culture plate, for subsequent use after cooling.The formula of substratum 2 is: add 0.015g agar powder in 100 milliliters of LB substratum, autoclaving process, and cool to 60 DEG C; Add the paraxin liquid of 50 μ L80 mg/ml wherein, concentration is the tsiklomitsin 100 μ L of 50mM, and concentration is the kantlex 100 μ L of 50mM, after mixing, pours in sterile culture plate, for subsequent use after cooling.The bacterium be imprinted on substratum 1 and 2 is cultivated 48 hours at 37 DEG C, and the mono-clonal can not survived on substratum 2 while of can surviving on substratum 1 is the bacterial strain containing CouKRS.This bacterial strain specific recognition umbelliferone Methionin.The mutational site of CouKRS is that L270I, Y349D, N311A, N313G, L270I, Y349D, N311A, N313G4 mutational site is not overlapping.

In the present embodiment, umbelliferone is object substrate, therefore adds on screening flat board when screening.Preparation mother liquor is routine or the general knowledge of Biochemical Research.Suppose that they are directly mixed, and volume can increase, and is only made into mother liquor, when guarantee concentration, does not affect volume containing some compositions in 100 milliliters of liquid.With reference to 10 times of buffer in PCR, it is exactly mother liquor.Transform successful bacterial strain to survive on chlorampenicol resistant flat board.The effect of pylT-pREP plasmid is exactly allow identify that amino acid whose enzyme survives.PylT genes encoding tRNA is tonka bean camphor aminoacyl-tRNA synthetase; Be that the design of L270I, Y349D, N311A, N313G and screening finally can obtain only identification tonka bean camphor Methionin by the mutational site of CouKRS, improve specificity.

The encoding gene coukrs and the Pichia anomala expression plasmid pPICZ that relate generally to pyrrolysine aminoacyl-tRNA synthetase mutant in step s 4 which recombinate, and obtain expression plasmid of yeast pPICZ-CouKRS.Detailed process can be expressed as: will screen the encoding gene coukrs (gene order of coukr sees the sequence 1 in sequence table, and protein sequence sees the sequence 2 in sequence table) of the pyrrolysine aminoacyl-tRNA synthetase mutant obtained in above-mentioned steps S3.Be building up to Pichia anomala expression plasmid pPICZ, this pPICZ plasmid is from Invitrogen company, the multiple clone site of this pPICZ plasmid is selected between EcoRI and XbaI enzyme cutting site, carry out enzyme and is cut connection, reaches the object of gene recombination, finally obtains expression plasmid of yeast pPICZ-CouKRS.

Design the mutant DNA sequences of AntiHBsAg antibody in step s 5, obtain this mutant, the mutant DNA of described AntiHBsAg antibody and plasmid pPIC9K are recombinated, obtain expression plasmid of yeast pPIC9K-antiHBsAg, plasmid pPIC9K also comes from Invitrogen company.Detailed process can be expressed as: the mutant DNA sequences (sequence 3 see in sequence table) of design AntiHBsAg antibody also can close this mutant by genome company, and it is cloned into plasmid pPIC9K, the multiple clone site of this pPIC9K plasmid is selected between EcoRI and NotI restriction enzyme site, carry out enzyme and is cut connection, reach the object of gene recombination, finally obtain expression plasmid of yeast pPIC9K-antiHBsAg.

The pPICZ-CouKRS plasmid linearization will built in step S4 respectively in step s 6; By the pPIC9K-antiHBsAg plasmid linearization built in step S5.In the process of this plasmid linearization, pPICZ-CouKRS plasmid EcoRI enzyme is cut, reaches pPICZ-CouKRS plasmid linearization; PPIC9K-antiHBsAg plasmid NotI enzyme is cut, reaches pPIC9K-antiHBsAg plasmid linearization.Linearization plasmid is to make plasmid be easily integrated into host genome, and in the present embodiment, host genome refers to the genome of pichia spp X33; Reason is: after plasmid transfection enters host cell, if not linearizing, then plasmid is when being integrated into karyomit(e), and its breaking point is random, in this case, some broken site may be arranged in goal gene, after such integration occurs, because screening-gene is normal, so when screening by screening of medicaments, cell can not be killed, but ruptures due to goal gene, thus can not express target protein.

In the step s 7 linearization plasmid pPICZ-CouKRS, pPIC9K-antiHBsAg of obtaining in step S6 are mixed according to certain ratio, through electroporated, import in pichia spp X33, obtain strains A.Linearization plasmid pPICZ-CouKRS and pPIC9K-antiHBsAg mixes according to the ratio of 1:1, through electroporated, imports to pichia spp X33, and pichia spp X33 bacterial strain comes from Invitrogen company.Electroporated, voltage is 3.8 kilovolts, and the electric shock time is 3.6 seconds, in electroporated process, also can distribute and carry out: first by electroporated for linearization plasmid pPICZ-CouKRS, then by electroporated for linearization plasmid pPIC9K-antiHBsAg, be advisable with the hobby of user.

With reference to the schematic flow sheet that Fig. 5, Fig. 5 are the step S8 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention; In step s 8 strains A in step S7 is carried out resistance screening, obtain positive strain group B.Step S8 specifically can be divided into S81 and S82 two steps.S81: the strains A of cultivating two hours in step S7 is coated on containing ZeocinTM and ammonia benzyl mycin YPDS flat board on, 30 DEG C cultivate 3 days, obtain multiple mono-clonal bacterial strain; ZeocinTM, also known as bleomycin, is used for screening the yeast strain of resistance; Ammonia benzyl mycin and bleomycin use simultaneously, screen the positive yeast bacterial strain of resistance; The concentration of ZeocinTM and ammonia benzyl mycin is 100 μ g/mL.S82: the multiple mono-clonal bacterial strain pickings obtained by step S81 are in YPD liquid nutrient medium, and 30 DEG C of shaking tables are cultivated, and obtain positive strain group B.YPD is also known as YEPD, and English is YeastExtractPeptoneDextroseMedium (1L), is again yeast extract powder peptone dextrose culture-medium, and this nutrient solution is used for the bacterial strain that culturing yeast belongs to, and this bacterial strain can be X33 pichia spp.In step s 8, adding 1 milliliter of YPD substratum to having transformed in the X33 pichia spp of linearizing pPICZ-CouKRS plasmid and pPIC9K-antiHBsAg plasmid, in 30 degrees Celsius, cultivating 2 hours under 200rpm, obtaining strains A.Strains A is coated on the YPDS flat board containing the ammonia benzyl mycin of 100 μ g/mLZeocinTM and 100 μ g/mL, cultivates 3 days for 30 DEG C.By the mono-clonal picking on the YPDS flat board in above-mentioned steps S81 in YPD liquid nutrient medium, in 30 degrees Celsius, cultivate 2 hours under 200rpm, final acquisition positive strain group B, the bacterial strain quantity in positive strain group B is at least 2.

With reference to the schematic flow sheet that Fig. 6, Fig. 6 are the step S9 of the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen of the present invention.In step s 9 positives for step S8 bacterial strain group B is carried out PCR Screening and Identification respectively, obtain several positive strains group C.Step S9 comprises 5 steps, is specially following steps: S91: it is centrifugal that the yeast liquid that the positive strain group B getting step S82 acquisition increases is placed in centrifuge tube respectively, obtains yeast sedimentation group 1; In S91, the yeast 1mL of amplification is placed in 1.5mL centrifuge tube, 10000rpm, and centrifugal 1 minute, now yeast sedimentation was in the bottom of centrifuge tube.S92: the yeast sedimentation group 1 that step S91 obtains is added sterilized water, whirlpool concussion is resuspended, obtains yeast sedimentation group 2; Containing in the centrifuge tube of yeast sedimentation in above-mentioned S91, add 0.3mL sterilized water, and resuspended with whirlpool concussion.S93: the refrigerator described yeast sedimentation group 2 being placed in respectively-80 DEG C, takes out after 30min, thaw at room temperature; S94: repeating step S93 is once, centrifugal, gets supernatant liquor respectively and obtains yeast soln group 3; S95: get yeast soln group 3, be placed in the enterprising performing PCR amplification of PCR amplification instrument respectively, the monoclonal exactness in verification step S82, obtains several positive strains group C.In step S95, get the yeast soln 3 i.e. supernatant liquor 10 μ L obtained in S94 be placed in PCR pipe, this PCR pipe is purchased from Qiagen, and in PCR pipe, add 31 μ LH2O2,1 μ L forward primer f1 and 1 μ L reverse primer r1, f1 and r1 primer sequence is as shown in table 2, concentration is the dNTPs of 10mM, and 1 μ L concentration is the Taq DNA polymerase of 100 units, and this Taq DNA polymerase purchases the damping fluid in Takara company and the supporting Taq DNA polymerase of 5 μ L.By adding primer, the PCR pipe of Taq DNA polymerase is placed in PCR amplification instrument, and this PCR amplification instrument is purchased in Biorad company, increases, the monoclonal exactness in checking S82.

Table 2 primer f1 and primer r1 gene order

Primer	Primer sequence
		f1	cagatgcagc tggtgcagtc
r1	ctatggtccctccgccgaatac

In step slo by the mono-clonal in positives for step S9 bacterial strain group C, coat in the YPDS solid medium containing G418 and cultivate, the G418 resistance of checking mono-clonal yeast, G418 (Geneticin, Geneticin) be a kind of aminoglycoside antibiotics, in molecular genetic test, be the most frequently used resistance screening reagent of stable transfection.It disturbs rrna function and blocking protein synthesizes by suppressing transposon Tn601, the gene of Tn5, produces toxin, comprise bacterium, yeast, plant and mammalian cell to the cell such as protokaryon and eucaryon.After neo gene is integrated into eukaryotic cell dna, the sequence transcribes that then can start neo genes encoding is mRNA, thus obtain the high expression of resistance product aminoglycoside phosphotransferase, make cell obtain resistance and can grow in containing the selective medium of G418.This selectivity characteristic of G418, is able to widespread use in transgenosis, gene knockout, resistance screening and transgenic animal etc.

Particularly, the mono-clonal of S95 step of learning from else's experience checking, coats in the YPDS solid medium containing 400ng/ μ LG418 and cultivates 3 days under 30 degrees Celsius, the G418 resistance of checking mono-clonal yeast.If after G418 resistance is proved to be successful, can obtains to possess and identify and carry the mono-clonal yeast D that umbelliferone is inserted into 63 sites of anti-hepatitis b surface antigen antibody.

In step s 11 the yeast D mono-clonal enlarged culturing of step S10, centrifugal, ultrasonication, ni-sepharose purification step are obtained anti-hepatitis b surface antigen fluorescence antibody.Particularly, the yeast mono-clonal be proved to be successful in step S10 is inoculated in the YPD substratum of 100mL, in 30 DEG C, cultivate under 200rpm, when A600 reaches 0.3, add the ZeocinTM of 100 μ L100 μ g/mL and the ammonia benzyl mycin of 100 μ g/mL in the medium respectively, after 0.5 hour, add the umbelliferone mother liquor 1mL in above-mentioned steps S22 in the medium, the IPTG1mL of 100mM, in 30 degrees Celsius, cultivate 4 hours under 200rpm.IPTG, full name Isopropyl β-D-1-thiogalactopyranoside, Chinese isopropyl-β-D-thiogalactoside(IPTG) by name; A kind ofly act on extremely strong inductor, very unstable by bacterial metabolism, therefore by laboratory widespread use.Continue to add umbelliferone mother liquor 1mL, in 30 degrees Celsius, continue cultivation and obtain yeast thalline in 4 hours under 200rpm, centrifugal through 5000rpm, 30min, ultrasonication, ni-sepharose purification step obtains anti-hepatitis b surface antigen fluorescence antibody.

For detecting efficiency and the specificity of the identification hepatitis B surface antigen of anti-hepatitis b surface antigen fluorescence antibody obtained above, we take following instance to make an explanation to the present invention, and the present invention is not limited to following examples.

Example 1:

Utilize the hepatitis B surface antigen of lower concentration as sample, the sensitivity of fluorescence antibody prepared by test the present invention, concrete steps are as follows.

S120: get hepatitis B surface antigen, dilute hepatitis B surface antigen with phosphoric acid buffer (PBS), being mixed with concentration is that the hepatitis B surface antigen liquid of 0ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 2.5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL10 gradient is for subsequent use.

S130: by described anti-hepatitis b surface antigen fluorescence antibody 10 μ L, phosphoric acid buffer 90 μ L, join in 11 holes of enzyme plate respectively, add in the 11st hole and do not have fluorescently-labeled hepatitis B surface antigen fluorescence antibody 10 μ L, phosphoric acid buffer 90 μ L, called after 1,2,3 respectively, 4,5,6,7,8,9,10,11 incubated at room 12h.

S140: inhaled by the liquid in the enzyme plate in described S130 and abandon, rejoin 100 μ L phosphoric acid buffers in enzyme plate, be placed on shaking table by described enzyme plate, per minute 30rpm, room temperature cleans 5 minutes.

S150: repeating step S140 twice, and inhale the phosphoric acid buffer abandoned in enzyme plate in 1-11 plate hole.

S160: add 100 μ L phosphoric acid buffers in described enzyme plate plate hole 1, the hepatitis B surface antigen liquid that 10 μ L concentration are 0ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 2.5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL10 gradient is added respectively in plate hole 1-11, wherein 1 is positive control, 11 is negative control, and 2-10 is sample group.

S170: add 90 μ L phosphoric acid buffers in plate hole 1-11 respectively.Enzyme plate is placed on shaking table, according to the rpm speed incubated at room 2h of per minute 30rpm.

S180: inhaled by the liquid in enzyme plate 1-11 plate hole described in S170 and abandon, and add 100 μ LPBS in the 1-11 plate hole of enzyme plate, according to per minute 30rpm, room temperature cleans 5 minutes.

S190: repeat S180 twice.

S200: be placed in microplate reader by the enzyme plate after described cleaning and detect, 488nm laser excitation, gather the fluorescence under 525nm, result is as shown in table 3.

The fluorescence intensity level of fluorescence antibody prepared by table 3 the present invention

Plate hole is numbered	Fluorescence intensity (a.u)	Antigen final concentration (ng/mL)
			1	10000	0
2	8000	0.02
			3	7590	0.04
4	7100	0.06
			5	5520	0.13
6	4000	0.2
			7	3800	0.31
8	2600	0.89
			9	1700	1.34
10	1000	2.0
			11	0.21	0.02

Find from the test result of table 3: be 0.21a.u for negative control group 11 (i.e. unlabelled anti-hepatitis b surface antigen antibody) fluorescence intensity; For the hepatitis B surface antigen identification that positive controls 1 (fluorescently-labeled anti-hepatitis b surface antigen antibody) is not corresponding with it, fluorescence intensity is 10000 to the maximum, when the hepatitis B surface antigen of different concns and fluorescently-labeled anti-hepatitis b surface antigen antibody in conjunction with time, fluorescent signal is different, and increase gradually along with the concentration of hepatitis B surface antigen, fluorescent signal weakens gradually; In addition, can also show that this fluorescence antibody can detect the conclusion of the antigen of 0.02ng/L level by this test.

Described DNA sequence dna by synthesizing the DNA sequence dna of the pyrrolysine aminoacyl-tRNA synthetase (PylRS) of M.Barkeri species, and is connected on pPICZ plasmid by the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen provided by the invention; By designing the mutant DNA sequences of AntiHBsAg antibody, obtaining this mutant, the mutant DNA of described AntiHBsAg antibody and plasmid pPIC9K are recombinated, obtain expression plasmid of yeast pPIC9K-antiHBsAg; By linearization plasmid pPICZ-CouKRS, pPIC9K-antiHBsAg are mixed according to certain ratio, electroporated, import in pichia spp X33, carry out screening and verifying, obtain the eukaryotic expression system preparing anti-hepatitis b surface antigen fluorescence antibody, this System-mediated umbelliferone inserts in 63 sites of the aminoacid sequence of anti-hepatitis b surface antigen antibody, thus achieves the preparation of anti-hepatitis b surface antigen fluorescence antibody; Increase substantially the sensitivity that HBsAg detects, the accurate quantification realizing HBsAg detects, the control and monitoring of assistant reinforcement to the morbidity of hepatitis B.

These are only the preferred embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.

Claims

1. a preparation method for the fluorescence antibody of anti-hepatitis b surface antigen, is characterized in that, the preparation method of the fluorescence antibody of described anti-hepatitis b surface antigen comprises the steps:

S1: obtain M.BarkeriPylRS mutation library;

S2: obtain tonka bean camphor Methionin;

2. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 1, it is characterized in that, described step S1 comprises the steps:

S101: the DNA sequence dna of synthetic M.BarkeriPylRS;

3. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 2, is characterized in that, described first predetermined ratio is 1:1.

4. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 1, it is characterized in that, described step S2 comprises the steps:

5. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 4, it is characterized in that, described umbelliferone mother solution C is through 0.22 micron membrane filter filtration sterilization.

6. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 5, it is characterized in that, the concentration of described umbelliferone mother solution C is set as 100mM.

7. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 1, it is characterized in that, described step S8 comprises the steps:

S82: the multiple mono-clonal bacterial strain pickings obtained by step S81 are in YPD liquid nutrient medium, and 30 DEG C of shaking tables are cultivated, and obtain positive strain group B.

8. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 7, it is characterized in that, described step S9 comprises the steps:

S94: repeating step S93 is once, centrifugal, gets supernatant liquor respectively and obtains yeast soln group 3;

9. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 1, it is characterized in that, described step S4 inserts the encoding gene coukrs of pyrrolysine aminoacyl-tRNA synthetase mutant between the EcoRI and XbaI enzyme cutting site of Pichia anomala expression plasmid pPICZ, obtains expression plasmid of yeast pPICZ-CouKRS.

10. the preparation method of the fluorescence antibody of anti-hepatitis b surface antigen as claimed in claim 1, it is characterized in that, between EcoRI and the NotI restriction enzyme site of plasmid pPIC9K, insert the mutant DNA sequences of AntiHBsAg antibody in described step S5, obtain expression plasmid of yeast pPIC9K-antiHBsAg.