CN102321146A

CN102321146A - Method for analyzing epitope of monoclonal antibody by using yeast surface display system and application of method in vaccine development

Info

Publication number: CN102321146A
Application number: CN201110206801A
Authority: CN
Inventors: 张林琦; 史宣玲; 左腾; 汪桦; 姚辰
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2011-07-22
Filing date: 2011-07-22
Publication date: 2012-01-18

Abstract

The invention discloses a method for analyzing epitope of monoclonal antibody by using a yeast surface display system and application of the method in vaccine development. The method comprises the following steps of: (1) constructing a DNA library of target protein; (2) displaying the DNA library on the surface of yeast to obtain a yeast display library; (3) mixing the monoclonal antibody of the target protein and a yeast display library A, and screening yeast combined with the monoclonal antibody; and (4) extracting plasmid of the screened yeast, sequencing, and analyzing the epitope of the target protein. Compared with the traditional method, the method has the advantages that: (1) ways of modifying the protein by yeast cells on are more, and high molecular weight and complicated protein can be displayed; (2) the yeast can be screened one by one by fluorescence-activated cell sorting (FACS), and the accuracy and screening flux are improved; (3) the yeast can independently complete self growing and reproducing process, the operation is simple and convenient and interference factors are a few; and (4) the yeast is an immunologic adjuvant, and can check whether the epitope can induce the generation of antibody with similar activity again.

Description

Utilize method and the application in vaccine development thereof of the epitope of yeast surface display systems analysis monoclonal antibody

Technical field

The present invention relates to a kind of method of the epitope that utilizes yeast surface display systems analysis monoclonal antibody and the application in vaccine development thereof.

Background technology

Host's antibody response plays an important role in the process of opposing pathogenic infection, and intravital polyclonal antibody reaction is by forming to the monoclonal antibody of synantigen or same antigenic different epitopes not.Deep understand this complicated process crucial information can be provided for the pathogenesis of study of disease, can also important theoretical foundation be provided for the research and development and the disease treatment of efficient vaccine.

In many research methods of epitope; Rise phage display peptide library technology (Scott in nineteen ninety; J.K.and G.P.Smith, Searching for peptide ligands with an epitope library.Science, 1990.249 (4967): p.386-90.); (general 6-12 amino acid, storage capacity reaches 10 promptly to use at random synthetic small peptide library ¹¹-10 ¹²) and phage display system carry out method for screening, fast, accurately, do not receive the advantage of amino acid sites restriction and become one of present application epitope screening platform wide, the most with fastest developing speed gradually with it.Yet also there is significant limitation in the phage display peptide library technology: the overwhelming majority that (1) small peptide library can produce is a linear epitope, can not simulate well to account for the conformation type epi-position of epitope more than 80%; (2) capsid protein of phage the exogenous molecules that can hold is little, simple in structure, foreign protein is lacked modify, and a lot of cytotoxicity molecules are with eukaryotic protein is beyond expression of words and displaying in this system; (3) the phage individuality is too small, adopts affinity chromatographic column or affinity XPS to screen more, and screening efficiency is low; (4) every take turns screening after, phage host cells infected again carries out next round screening so that obtain enough virus particle, the difference of infection ability can cause certain amplification advantage between the recombinant virus particle; (5) phage is prone to by airborne transmission, thereby causes crossed contamination easily.

The characteristics of monoclonal antibody are to combine with antigenic single-minded epi-position, judge the similarities and differences of the monoclonal antibody epi-position of different sources, and are all significant to the use value of understanding antibody and extensive Antibody Preparation etc.

Summary of the invention

The purpose of this invention is to provide a kind of method of the epitope that utilizes yeast surface display systems analysis monoclonal antibody and the application in vaccine development thereof.

The method of the proteic epitope of analysis purposes provided by the invention comprises the steps:

(1) the DNA library of the said target protein of structure; In the said DNA library, the dna fragmentation of the encoding sox of said target protein inserts the T carrier; Said T carrier contains the encoding sox of yeast a lectin Aga2p, and the C end of the N end of the peptide fragment of said dna fragmentation coding and said yeast a lectin Aga2p merges;

(2) said DNA library is illustrated in yeast surface, obtains yeast display libraries first;

(3) monoclonal antibody of said target protein is mixed with said yeast display libraries first, sub-elect and said monoclonal antibody bonded yeast;

(4) yeast that filters out is extracted plasmid and check order, according to the epitope (peptide fragment of the total dna fragmentation coding that inserts in each plasmid is candidate's identification epi-position) of the said target protein of nucleotide sequence analysis of the dna fragmentation that inserts said T carrier.

The pCTCON2-T plasmid that the sequence table 3 that said T carrier specifically can be insertion sequence table between the Nhe of pCTCON2 plasmid I and BamH I site obtains from the DNA shown in 5 ' terminal the 11st to 40 Nucleotide.

The concrete Xcm I restriction enzyme site that inserts said pCTCON2-T plasmid of the dna fragmentation of the encoding sox of said target protein.

But said yeast is a yeast saccharomyces cerevisiae, is preferably yeast saccharomyces cerevisiae EBY100.

In the said step (1), the method that makes up said DNA library can comprise the steps:

1. the encoding sox with said target protein digests with DNase I, reclaims the dna fragmentation of 50-100bp;

2. the dna fragmentation that 1. step is reclaimed carries out the random fragment reorganization;

3. the dna fragmentation after 2. step being recombinated adds the A tail;

4. add the Xcm I restriction enzyme site that dna fragmentation behind the A tail inserts said pCTCON2-T plasmid with what 3. step obtained, transform the host bacterium then, obtain said DNA library.

The reaction system of said random fragment reorganization specifically can be: 90ng small pieces segment DNA, and 4 μ l, 5 * Phire buffer, 0.8 μ l dNTPs, 0.5 μ l Phire archaeal dna polymerase (1 unit) adds ddH ₂O to 20 μ l.The response procedures of said random fragment reorganization specifically can be 95 ℃ 2 minutes; 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ of N seconds, 10 circulations or 15 circulations; 72 ℃ 10 minutes; The 1st circulation time, N is 60 seconds, from the 2nd circulation beginning, the last circulation N of each recycle ratio increase 5s (i.e. the 2nd circulation time, N is 65 seconds, the 3rd circulation time N is 70 seconds, by that analogy).In the said response procedures, preferred 10 circulations or 15 circulations.

The also proteic DNA of method establishing target libraries such as available physical shearing method, random primer TRAP and enzyme digestion.

Said host bacterium can be intestinal bacteria, preferred escherichia coli DH5a.

Said step (1) is preceding carrying out, and can pass through the encoding sox of the said target protein of pcr amplification earlier.The used enzyme of said pcr amplification can be high-fidelity DNA polymerase, is preferably PyroBest DNA Polymerase.

In the said method, flow cytometry capable of using carries out the said sorting in the step (3).

In the said step (2), said DNA library is illustrated in said yeast surface can be comprised the steps:

1. extract the plasmid that contains said DNA library that obtains in the said step (1), transform said yeast, obtain the recombination yeast first;

2. collect said recombination yeast first inducing culture more than 36 hours, obtain yeast display libraries first; Said inducing is to realize through in the system of culturing yeast, adding semi-lactosi.

The parameter of said inducing culture specifically can be: adopt the SGCAA substratum, cultivated 36 hours in 20 ℃, 250rpm shaking table.The prescription of SGCAA substratum is specially: semi-lactosi 20g, yeast nitrogen 6.7g, acid hydrolysis casein 5g, NaHPO ₄12H ₂O 13.6g, NaH ₂PO ₄H ₂O 8.56g adds redistilled water to 1L.

Said method also can comprise the steps:

(5) make up the dna mutation library of the encoding sequence of the said epitope that said step (4) analyze to obtain; In the said dna mutation library, the mutant DNA fragment of the encoding sequence of said epitope is inserted said pCTCON2-T plasmid;

(6) said dna mutation library is illustrated in said yeast surface, obtains yeast display libraries second;

(7) monoclonal antibody and the C-myc monoclonal antibody with said target protein mixes with said yeast display libraries second together, sub-elect combine with the C-myc monoclonal antibody and with the uncombined yeast of the monoclonal antibody of said target protein;

(8) yeast that filters out being extracted plasmid checks order; The proteic epitope of variance analysis according to the encoding sequence of the nucleotide sequence of the dna fragmentation that inserts said pCTCON2-T plasmid and said epitope (is compared with the protogene sequence; The Nucleotide that changes is candidate's destruction polypeptide fragment and said monoclonal antibody bonded Nucleotide, the key amino acid residue during corresponding amino-acid residue is albumen and monoclonal antibody combines).

In the said step (5), the method that makes up said dna mutation library can comprise the steps:

1. pass through the encoding sox of the said epitope of pcr amplification; The used enzyme of said pcr amplification is a low-fidelity DNA polyase, is preferably the rTaq enzyme;

2. said pcr amplification product is inserted the Xcm I restriction enzyme site of said pCTCON2-T plasmid, transform the host bacterium then, obtain said dna mutation library;

In the said method, flow cytometry capable of using carries out the said sorting in the step (7);

In the said step (6), said DNA library is illustrated in said yeast surface can be comprised the steps:

1. extract the plasmid that contains said dna mutation library that obtains in the said step (5), transform said yeast, obtain recombination yeast second;

2. collect said recombination yeast second inducing culture more than 36 hours, obtain yeast display libraries second; Said inducing is to realize through in the system of culturing yeast, adding semi-lactosi.

More than arbitrary said method all can be applicable to study antigen and antibody and interact.

More than arbitrary said method all can be applicable to vaccine research and development.

Said target protein can be the albumen (the HA albumen of H5N1 bird flu virus) shown in the sequence 3 of sequence table.DNA shown in the sequence 4 of the encoding sox preferred sequence table of said target protein.

Said target protein can be the albumen (gp160 albumen) shown in the sequence 1 of sequence table.DNA shown in the sequence 2 of the encoding sox preferred sequence table of said target protein.

Said monoclonal antibody can be 2F5 monoclonal antibody, 4E10 monoclonal antibody, VRC01 monoclonal antibody or AVFluIgG03 monoclonal antibody.

The preparation method of said AVFluIgG03 monoclonal antibody is following:

1. the chain variable region gene shown in the sequence 6 of composition sequence table is cloned between the Xba I and Sac I restriction enzyme site of Pac-L-Fc carrier, obtains the recombinant plasmid first.

2. the heavy chain variable region gene shown in the sequence 7 of composition sequence table is cloned between the Xho I and Spe I restriction enzyme site of said recombinant plasmid first, obtains recombinant plasmid second;

3. said recombinant plasmid second is imported the Sf9 cell, collect culture supernatant, obtain the AVFluIgG03 monoclonal antibody.

The yeast random fragment library screening route of monoclonal antibody is referring to Fig. 1.A large amount of purpose antigen gene fragments that increase are through T-A ways of connecting and the fusion of the proteic C end of Aga2 are passed through in cutting and reorganization back at random; Import yeast then and under corresponding inductive condition, be illustrated in the yeast saccharomyces cerevisiae surface; Carry out the FACS screening through corresponding monoclonal antibody; The fragment of acquisition and monoclonal antibody reaction, the protein gene fragment of then total same monoclonal antibody being reacted is carried out the structure in random mutation library, through the screening to negative yeast clone; Analyze the gene locus of sudden change, the identification epi-position of monoclonal antibody is carried out accurate localization.When the epi-position of carrying out monoclonal antibody is confirmed; Be illustrated in yeast surface through making up with the specific purpose proteic reorganization library of this monoclonal antibody correspondence and with it; Mix monoclonal antibody with the yeast storehouse then, utilize airflow classification technology (FACS) to filter out the positive yeast clone that reacts with monoclonal antibody again.Through analyzing the Tumor-necrosis factor glycoproteins of a large amount of positive colonies, just can position research to single identified epitope (comprising line style epi-position and conformation type epi-position).Carry out random mutation to repeating region sequence then,, filter out the negative yeast clone of different monoclonal antibody reactions, analyze the site of sudden change, can accurately locate the recognition site of monoclonal antibody again with the antigen protein library of same monoclonal antibody screening sudden change.The yeast cell that utilizes the cell surface expression epitope that monoclonal antibody screening obtains obtains to have the immune antibody of similar characteristics with monoclonal antibody as immunogen.

Yeast cell still is an effective immunological adjuvant; The generation of can the direct immunization animal inducing antibodies specific uses yeast cell to carry out immunity as antigen, effectively activated dendritic cell (Dendritic cell; DC) function is strengthened the protectiveness cellullar immunologic response.Therefore, the yeast cell that utilizes the cell surface expression epitope that monoclonal antibody screening obtains produces humoral immunization and cellular immunization effect preferably at the body of accepting immunity probably as immunogen.

Characterization of antigenic epitopes method with traditional is compared; That utilizes that the yeast surface display technology carries out monoclonal antibody identification epi-position confirms to have original advantage: (1) yeast cell is more to proteic modification approach; Can show macromolecule and complicated albumen easily, the space conformation epi-position that therefore can be used for screening monoclonal antibody; (2) yeast can adopt FACS to screen one by one, improves accuracy and screening flux greatly; (3) yeast can independently be accomplished the growth reproduction process of self, and easy and simple to handle, interfering factors is few; Can (4) yeast itself be immunological adjuvant, can check epitope induce similar active antibody to produce again.

Description of drawings

Fig. 1 is the yeast random fragment library screening route of monoclonal antibody.

Fig. 2 is for making up 1% agarose gel electrophoresis figure in the process of the proteic DNA of gp160 library.

Fig. 3 is the sequential analysis of 2F5 monoclonal antibody identified epitope.

Fig. 4 is the sequential analysis of 4E10 monoclonal antibody identified epitope.

Fig. 5 is for using the separating effect evaluation of AVFluIgG03 monoclonal antibody.

Fig. 6 is for respectively organizing the detected result of tiring of mice serum among the embodiment 5.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

Yeast saccharomyces cerevisiae EBY100:invitrogen company, catalog number C839-00.The little extraction reagent kit of Biomed plasmid: Bo Maide company.Bacillus coli DH 5 alpha: day root company.Phire archaeal dna polymerase (Phire hot-start DNA Polymerase): New England Biolabs.Strand carrier DNA (salmon sperm dna): Merck company, catalog number CB262012.HA albumen: stick up Divine Land Bioisystech Co., Ltd available from Beijing justice, Catalog Number:11048-V08H1.2F5 monoclonal antibody: available from NIH AIDS Research&Reference Reagent Program, Catalog Number:1475.4E10 monoclonal antibody: available from NIH AIDS Research & Reference Reagent Program, Catalog Number:10091.VRC01 monoclonal antibody: available from NIH AIDS Research & Reference Reagent Program, Catalog Number:12033.2F5 monoclonal antibody, 4E10 monoclonal antibody and VRC01 monoclonal antibody are to have in the wide spectrum and active monoclonal antibody to HIV-1, and wherein the antigen recognition sequence of 2F5 and 4E10 is a linear epitope, is respectively ELDKWA and NWFDIT; VRC01 then is the wide spectrum neutralizing antibody to the CD4bs district, and the epi-position of its identification is a conformational epitope.

PCTCON2 plasmid (yeast display carrier): be so kind as to give by MIT Dane professor Wittrup, the public can obtain from Tsing-Hua University; Reference: Chao, G., et al., Isolating and engineering human antibodies using yeast surface display.NATURE PROTOCOLS, 2006.1 (2): p.755-768).

Pac-L-Fc carrier: reference: Yao LS, et al.Generation of human recombinant antibody Fab fragment and its IgG to adeno-associated virus type II from phage display library.Zhonghua Shi Yah He Lin Chuang Bing Du Xue Za Zhi.2003Sep; 17 (3): 240-3..

Sf9 cell: reference: Sun L; Et al.Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1viruses.PLoS One.2009; 4 (5): e5476.Epub 2009May 7..

The YPD substratum: glucose 2g, peptone 2g, yeast extract 1g adds redistilled water to 100ml.

SDCAA substratum: glucose 20g, yeast nitrogen 6.7g, acid hydrolysis casein 5g, NaHPO ₄12H ₂O 13.6g, NaH ₂PO ₄H ₂O 8.56g adds redistilled water to 1L.

SDCAA is dull and stereotyped: glucose 20g, yeast nitrogen 6.7g, acid hydrolysis casein 5g, agar powder 15g, NaHPO ₄12H ₂O13.6g, NaH ₂PO ₄H ₂O 8.56g adds redistilled water to 1L.

SGCAA substratum: semi-lactosi 20g, yeast nitrogen 6.7g, acid hydrolysis casein 5g, NaHPO ₄12H ₂O 13.6g, NaH ₂PO ₄H ₂O 8.56g adds redistilled water to 1L.

The structure of embodiment 1, pCTCON2-T plasmid (recombination yeast display carrier)

For the ease of building the storehouse, need the pCTCON2 plasmid be transformed into the T carrier, step is following:

1, the DNA (two strands) shown in the sequence 5 of composition sequence table.

5 '-AGTC GCTAGC

GGATCCGGGT-3 ' (sequence 5).

The underscore mark Nhe I recognition sequence on the left side, the underscore mark BamH I recognition sequence on the right, two Xcm I recognition sequences of runic mark.

2,, reclaim enzyme and cut product with restriction enzyme Nhe I and BamH I double digestion step 1 synthetic DNA.

3,, reclaim carrier framework with restriction enzyme Nhe I and BamH I double digestion pCTCON2 plasmid.

4, the carrier framework of the enzyme of step 2 being cut product and step 3 is connected; Obtain pCTCON2-T plasmid (skeleton carrier is the pCTCON2 plasmid, between the Nhe of skeleton carrier I and BamH I site, has inserted sequence table 5 from the DNA shown in 5 ' terminal the 11st to 40 Nucleotide).The pCTCON2-T plasmid has been introduced two Xcm I restriction enzyme sites in the pCTCON2 plasmid, cut with restriction enzyme Xcm I enzyme and can form the T tail.

The preparation of embodiment 2, AVFluIgG03 monoclonal antibody

1, the chain variable region gene shown in the sequence 6 of composition sequence table is cloned between the Xba I and Sac I restriction enzyme site of Pac-L-Fc carrier (German PROGEN PR3003), obtains the recombinant plasmid first.

2, the heavy chain variable region gene shown in the sequence 7 of composition sequence table is cloned between the Xho I and SpeI restriction enzyme site of recombinant plasmid first, obtains recombinant plasmid second.

3, adopt the BaculoGold cotransfection test kit of U.S. Pharmogen company, recombinant plasmid second is imported the Sf9 cell, collect culture supernatant, obtain the AVFluIgG03 monoclonal antibody.

Embodiment 3, the proteic epitope of application methods analyst gp160 of the present invention

One, makes up gp160 proteic DNA library (cutting fragment library at random)

Make up the proteic DNA of gp160 library; At first the proteic encoding sox of gp160 is cut through DNase I at random; Obtain the small segment of about 50-100bp; Obtain having random fragment DNA library (Lin, Z., the S.Li and Y.Chen that length-specific distributes through recombinating at random then; Identification of Viral Peptide Fragments for Vaccine Development.Viral Applications of Green Fluorescent Protein:Methods and Protocols, 2009:p.261-274.; Chen; Y.; Et al., Random dissection to select for protein split sites and its application in protein fragment complementation.PROTEIN SCIENCE, 2009.18 (2): p.399-409.).Aforesaid method is similar to the program (Lorimer of DNA shuffling very much; I.and I.Pastan; Random recombination of antibody single-chain fv sequences after fragmentation with dnasei in the presence of Mn2+.Nucleic Acids Res; 1995.23 (15): p.3067-3608.); Utilize the fragment regrouping process to regulate the distribution in random fragment DNA library dexterously, can make up more rational library, the basis is provided for obtaining crucial conformation type epitope according to the screening needs.

1, a large amount of amplifying target genes

1. carry out the target gene PCR amplification

The proteic encoding sox of gp160 (the gp160 albumen shown in the sequence 1 of albumen coded sequence table shown in the sequence 2 shown in the sequence 2 of composition sequence table; Be the membranin of HIV-1B ' hypotype strain) as template; (used archaeal dna polymerase is the PyroBest DNA Polymerase of Takara company production to the primer of forming with gp160-F and gp160-R to carrying out pcr amplification; A kind of high-fidelity polysaccharase), obtain pcr amplification product (template DNA).

gp160-F：5’-ATGAGAGTGACGGGG-3’；

gp160-R：5’-TAGCAAAGCCCTTTC-3’。

2. adopt QIAquick PCR Purification Kit purifying and recovering pcr amplification product, carry out DNA quantitatively (concentration of DNA should greater than 130ng/ μ l).

2, DNase I digests at random

1. prepare DNase I substrate solution; 4 μ g template DNAs, 2.5 μ l 1M Tris-Cl (pH7.5), 10 μ l 50mMMnCl ₂, add ddH ₂O to 49 μ l.

2. add 1 μ l DNase I (0.075U) (available from Worthington company, catalog number LS006361), 15 ℃ of reaction 4min.

3. add 10 μ l 0.5M EDTA aqueous solution termination reactions.

4. placed 10 minutes for 90 ℃, further make DNase I inactivation.

3, purifying DNase I endonuclease bamhi product

1. use Pall ultrafiltration and concentration centrifuge tube (molecular weight cut-off is 30K) to filter the template DNA in the reaction system of removing step 2.

The filtrating of 2. 1. step being collected is through Pall ultrafiltration and concentration centrifuge tube (molecular weight cut-off is 3K), and reclaiming molecular weight is the small pieces segment DNA below the 3K.Adopt Nanodrop 2000 spectrophotometers (Thermo) to confirm DNA concentration.

The agarose gel electrophoresis figure of the small pieces segment DNA that 2. step obtains sees the swimming lane 1 of Fig. 2 A, and size is 50-100bp.

4, the reorganization of small pieces segment DNA

The small pieces segment DNA that step 3 is obtained carries out the random fragment reorganization, obtains random fragment.

The recombining reaction system: 90ng small pieces segment DNA, 4 μ l, 5 * Phire buffer, 0.8 μ l dNTPs, 0.5 μ l Phire archaeal dna polymerase (1 unit) adds ddH ₂O to 20 μ l.

The recombining reaction program: 95 ℃ 2 minutes; 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ of N seconds, 15 circulations; 72 ℃ 10 minutes; 4 ℃ of preservations.The 1st circulation time, N is 60 seconds, from the 2nd circulation beginning, the last circulation N of each recycle ratio increases 5s; I.e. the 2nd circulation time, N is 65 seconds, the 3rd circulation time N is 70 seconds, by that analogy).

The random fragment that obtains is with QIAquick PCR Purification Kit purifying and quantitative its concentration.

The agarose gel electrophoresis figure of random fragment sees the swimming lane 1 of Fig. 2 B, and size is 100-800bp.

5, random fragment adds the A tail

The random fragment that step 4 is obtained adopts A-tailing DNA Kit (Takara) to add the A tail by the specification sheets of test kit, and concrete steps are following:

In Eppendorf tube, prepare and add " A " reaction solution (50ul) as follows: 10 * A-Tailing Buffer, 5 μ l, dNTP Mixture 4 μ l, A-Tailing Enzyme 0.5 μ l, random fragment 2 μ g add ddH ₂O to 50 μ l; 72 ℃ were reacted 20 minutes, and ice bath is 4 minutes then; Use QIAquick PCR Purification Kit purifying then, and with the quantitative dna fragmentation concentration of Nanodrop2000 spectrophotometer.

6, the preparation of carrier framework

Cut the pCTCON2-T plasmid with restriction enzyme Xcm I enzyme, obtain carrier framework.

Enzyme is cut system: pCTCON2-T plasmid 24 μ g, and 10 * NEB buffer2,10 μ l, 10 * BSA, 1 μ l, restriction enzyme Xcm I 2 μ l add ddH ₂O to 100 μ l.

Reaction conditions: 37 ℃ of water-baths 4 hours (3-6 hour all can).

Adopt QIA quick Gel Extraction Kit to reclaim enzyme and cut product.The enzyme that reclaims is cut product with QIAquick PCR Purification Kit purifying, and with the quantitative dna fragmentation concentration of Nanodrop2000 spectrophotometer.

7, random fragment storehouse and carrier combines

The carrier framework thermostat metal that step 5 is added random fragment and step 6 after " A " is bathed 16 ℃ and is connected and spends the night (more than 12 hours all can).Linked system: 10 * T4 ligase buffer, 20 μ l, add the random fragment 1 μ g after " A ", skeleton carrier 4 μ g, T4 ligase (New England Biolabs) 10 μ l add ddH ₂O to 200 μ l.

8, electricity changes

1. use the connection mixture of QIAquick PCR Purification Kit purification step 7.

2. the fragment electricity behind the purifying is forwarded in the escherichia coli DH5a cell.

3. be coated with LB flat board (containing the 50mg/ml penbritin), incubated overnight in 37 ℃ of incubators is checked conversion results next day, the colony count (storage capacity) on the statistics plate.Storage capacity is about 500,000.10 single bacterium colonies of picking at random adopt the sequencing primer of pCTCON2 plasmid upstream and downstream to insert evaluation (Fig. 2 C of clip size through PCR.1 to 12 is different monoclonal pcr amplification products).

4. adopt the scraper plate device to scrape the bacterium colony on the clean LB flat board, put into 60ml LB substratum (containing the 100mg/ml penbritin), be the proteic DNA of gp160 library.

Two, yeast conversion, cultivate and induce

1, yeast conversion

Parallelly carry out 10 groups of yeast conversion, every group is all adopted following method:

(1) from fresh yeast saccharomyces cerevisiae EBY100 mono-clonal of picking on the YPD flat board to 5ml YPD liquid nutrient medium, 30 ℃ of 250rpm incubated overnight;

(2) overnight culture is diluted to the 0D600 value and is 0.4-0.5, in 30 ℃ of shaking tables, continue to be cultured to the 0D600 value and reach 2 (about 4 hours);

(3) room temperature 1500g is centrifugal 5 minutes, abandons supernatant, collects yeast cell; With the resuspended yeast cell of 25ml sterilized water, centrifugal 5 minutes of room temperature 1500g abandons supernatant, collects yeast cell;

(4) with the resuspended yeast cell of 1ml 0.1M Lithium Acetate solution, 30 ℃ of water-baths 10 minutes, the centrifugal 5s of 13000g abandons supernatant then, collects yeast cell;

(5) with the resuspended yeast cell of 0.5ml 0.1M Lithium Acetate solution, the every pipe packing of 50ul (competent cell);

(6) strand carrier DNA is put into the boiling water water-bath 5 minutes, take out ice bath then immediately;

(7) adopt the plasmid in the DNA library that the little extraction reagent kit of Biomed plasmid (Bo Maide) extraction step one obtains, 1 μ g plasmid is diluted to 50ul;

(8) get the competent cell that a pipe step (5) obtains, the centrifugal 5s of 13000g abandons supernatant; The plasmid that adds 240 μ l 50%PEG solution, 36 μ l 1M Lithium Acetate solution, 25 μ l strand carrier DNA, 50ul step (7) successively, fully mixing; 30 ℃ of water-baths 30 minutes, 42 ℃ of water-baths are 15 minutes then; The centrifugal 5s of 13000g abandons supernatant, collects yeast cell;

(9) with the resuspended yeast cell of 1ml sterilized water, take out 100ul and be coated with the SDCAA flat board, in 30 ℃ of thermostat containers, placed about 48 hours, can see tangible bacterium colony, the statistics colony count calculates yeast conversion efficient.The transformation efficiency of each group (colony count) is 50,000-100, and 000,10 group storage capacity is exactly 500,000-1000,000, can contain overwhelming majority clone through Theoretical Calculation.

2, induce

(1) every group of remaining 900ul bacterium liquid mixes after resuspended with 10ml SDCAA, 30 ℃, is cultured to OD600 in the 250rpm shaking table and is about 3 (24 hours).

(2) get 500 microlitre bacterium liquid, centrifugal 1 minute of room temperature 8000rpm abandons supernatant, with 1ml SGCAA substratum re-suspended cell, is diluted to OD600 then between 0.5-1;

Induced 36 hours in (3) 20 ℃ of 250rpm shaking tables, the bacterium liquid that obtains is the yeast display libraries.

Three, analyze the epitope of gp160 albumen to the 2F5 monoclonal antibody

1, the FACS sorting of yeast display libraries

The yeast display libraries that step 2 is obtained carries out the FACS sorting, and step is following:

1. with yeast display libraries (10 ⁷-10 ⁸Individual cell) pack in the 1.5ml Ep pipe, centrifugal 1 minute of 8000rpm (or＞8000rpm also can) abandons supernatant;

2. add 1ml PBS, it is resuspended with yeast to flick tube wall, and centrifugal 1 minute of 8000rpm gets deposition; Repeat this operation once;

3. add and use the 2F5 monoclonal antibody of 50ul protein concentration, flick mixing as 10ug/ml, ice bath 1 hour, during flicked tube wall in every separated 15-20 minute;

4. add 1ml PBS, mixing, centrifugal 1 minute of 4 ℃ of 8000rpm get deposition; Repeat this operation once;

5. add fluorescently-labeled two anti-(PE mark sheep anti-mouse igg is available from Santa Cruz company, article No. sc-3738), flick mixing with 100 times of dilutions of 50ul PBS, ice bath 45 minutes, during flicked tube wall (attention lucifuge) in every separated 15-20 minute;

6. add 1ml PBS, mixing, centrifugal 1 minute of 4 ℃ of 8000rpm get deposition (yeast); Repeat this operation once;

7. use the resuspended yeast of PBS (3-4ml PBS) to transfer to flow cytometer and carry out the FACS sorting, collect and monoclonal antibody bonded yeast cell.

2, the FACS separating effect is estimated

The yeast cell (100,000-500,000) that step 1 sorting obtains is transferred in the test tube, and adding SDCAA substratum to TV is 2ml, and test tube is placed 30 ℃ of shaking tables, lets yeast recovery 2-3 hour.The bacterium liquid that takes out 200ul (approximately 5000-10000 yeast cell) recovery is coated with the SDCAA flat board, and flat board is upside down in 30 ℃ of thermostat containers, receives bacterium after 48 hours, obtains positive monoclonal.

Picking positive monoclonal list bacterium colony is to 5-6ml SDCAA substratum from flat board, in 30 ℃, 250rpm shaking table, grows, and OD600 is approximately 6-8 after 24 hours; Get the centrifugal supernatant of abandoning of part bacterium liquid, make its OD600 between 0.5-1, induce after 36 hours in 20 ℃, 250rpm shaking table and carry out facs analysis with SGCAA substratum re-suspended cell.The facs analysis method with step 31 in the method for FACS sorting, difference only is to get 10 during step is 1. ⁶-10 ⁷Individual cell, step are 7. middle with the resuspended yeast of 0.5-1ml PBS.24 mono-clonal facs analysis are positive.

3, order-checking and sequential analysis

1. for step 33 in the positive mono-clonal of facs analysis, after the enlarged culturing,, remain whole bacterium liquid and extract plasmid with the little extraction reagent kit of plasmid with the frozen bacterial classification of glycerine (glycerine final concentration 15%);

2. because the plasmid copy number that from yeast, extracts is very low, the plasmid that extracts is transformed into escherichia coli DH5a, check order then (detecting the segmental nucleotide sequence that inserts in the pCTCON2-T plasmid).

3. sequential analysis

The random fragment and the full length sequence of gp160 protein coding gene that adopt Sequencher software will be inserted in the pCTCON2 carrier are compared, and the result sees Fig. 3.Sequencher software is analyzed according to the Software Operation handbook.

Find that through sequence alignment the 2F5 monoclonal antibody is LELDKWA to each monoclonal total recognition sequence, all consistent with known recognition sequence, show that method provided by the invention is very accurate for the location of epitope.

Four, analyze the epitope of gp160 albumen to the 4E10 monoclonal antibody

Replace 2F5 monoclonal antibody, other same step 3 with the 4E10 monoclonal antibody.

25 mono-clonal facs analysis are positive.The positive monoclonal The sequencing results of facs analysis is seen Fig. 4.Find that through sequence alignment the 4E10 monoclonal antibody is WASLWNWFDIT to each monoclonal total recognition sequence, all consistent with known recognition sequence, show that method provided by the invention is very accurate for the location of epitope.

Five, analyze the epitope of gp160 albumen to the VRC01 monoclonal antibody

Replace 2F5 monoclonal antibody, other same step 3 with the VRC01 monoclonal antibody.

32 mono-clonal facs analysis are positive.The VRC01 monoclonal antibody from N-terminal the 257th to 471 amino acids residue, is a conformational epitope to each monoclonal total recognition sequence sequence 1 that is sequence table, comprised V3, V4 and the V5 section of membranin, and the CD4bs district of HIV is included promptly.

Embodiment 4, accurately confirm the antigen recognition epi-position of H5N1-HA albumen to the AVFluIgG03 monoclonal antibody

One, makes up H5N1-HA proteic DNA library (cutting fragment library at random)

1, a large amount of amplifying target genes

1. carry out the target gene PCR amplification

The proteic encoding sox of H5N1-HA shown in the sequence 4 of composition sequence table (the H5N1-HA albumen shown in the sequence 3 of dna encoding sequence table shown in the sequence 4 of sequence table); As template; The primer of forming with HA-F and HA-R obtains pcr amplification product (template DNA) to carrying out pcr amplification.

HA-F：5’-GATCAGATTTGCATTGGTTA-3’；

HA-R：5’-TATTTGGTAAGTTCCTATTG-3’。

Step 2 is to 8 with 2 to 8 of the step 1 of embodiment 3.

Two, yeast conversion, cultivate and induce

Step 2 with embodiment 3.

Three, tentatively confirm the epitope of H5N1-HA albumen to the AVFluIgG03 monoclonal antibody

The yeast display libraries that step 2 is obtained carries out the FACS sorting: replace the 2F5 monoclonal antibody with the AVFluIgG03 monoclonal antibody, other is with the step 3 of embodiment 3.

Use the screening of AVFluIgG03 monoclonal antibody, 7 mono-clonal facs analysis are positive.Find that through sequence alignment the AVFluIgG03 monoclonal antibody is that the sequence 3 of sequence table is from N-terminal the 51st to 260 amino acids residue (sequence 4 of corresponding sequence table is from 5 ' terminal the 241st to 774 Nucleotide) to the proteic recognition sequence of H5N1-HA.The recognition sequence of this monoclonal antibody is positioned at the HA1 section of H5N1, and this epi-position is a conformation property epitope.

Four, accurately confirm the antigen recognition epi-position of H5N1-HA albumen to the AVFluIgG03 monoclonal antibody

(1) makes up the proteic mutant DNA of H5N1-HA library

1, a large amount of amplifications are to the mutant DNA fragment of the epitope of step 3 discovery

1. the proteic encoding sox of H5N1-HA shown in the sequence 4 of composition sequence table; As template; (used archaeal dna polymerase is the rTaq enzyme of Takara company to the primer of forming with HA-F and HA-R to carrying out pcr amplification; The fidelity of this enzyme is lower, is easy to generate sudden change), obtain pcr amplification product (mutant DNA fragment).

F1:5 '-GAGC GCTAGCATTTTAAGAGATTGTA-3 ' (underscore mark NheI recognition sequence);

R1:5 '-TAGT GGATCCCCCTTTCTTGACAATTTT-3 ' (underscore mark BamHI recognition sequence).

2,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme NheI and BamHI double digestion step 1.

3, the preparation of carrier framework

With restriction enzyme NheI and BamHI double digestion pCTCON2-T plasmid, obtain carrier framework.

Enzyme is cut system: pCTCON2-T plasmid 24 μ g, and 10 * NEB buffer2,60 μ l, 100 * BSA, 6 μ l, each 12 μ l of restriction enzyme NheI and BamHI add ddH ₂O to 600 μ l.

Reaction conditions: 37 ℃ of water-baths 4 hours (3-6 hour all can).

Adopt QIA quick Gel Extraction Kit to reclaim enzyme and cut product.The enzyme that reclaims is cut product with QIAquickPCR Purification Kit purifying, and with the quantitative dna fragmentation concentration of Nanodrop2000 spectrophotometer.

4, random mutation storehouse and carrier combines

The carrier framework thermostat metal that the enzyme of step 2 is cut product and step 3 is bathed 16 ℃ and is connected and spends the night (more than 12 hours all can).Linked system: 10 * T4 ligase buffer, 20 μ l, enzyme cut product 1 μ g, skeleton carrier 4 μ g, and T4 ligase (New England Biolabs) 10 μ l add ddH ₂O to 200 μ l.

5, electricity changes

1. use the connection mixture of QIAquick PCR Purification Kit purification step 4.

3. be coated with LB flat board (containing the 50mg/ml penbritin), incubated overnight in 37 ℃ of incubators is checked conversion results next day, the colony count (storage capacity) on the statistics plate.Storage capacity is about 500,000.

4. adopt the scraper plate device to scrape the bacterium colony on the clean LB flat board, put into 60ml LB substratum (containing the 100mg/ml penbritin), be the random mutation storehouse.

(2) yeast conversion, cultivate and induce

Step 2 with embodiment 3.

(3) epitope of Accurate Analysis monoclonal antibody AVFluIgG03

1, FACS sorting

The yeast display libraries that step 2 is obtained carries out the FACS sorting first time: replace the 2F5 monoclonal antibody with C-myc monoclonal antibody and AVFluIgG03 monoclonal antibody; Add two kind two anti-(PE mark sheep anti-mouse igg and FITC mark goat-anti chicken IgG) simultaneously, other is with 1 of the step 3 of embodiment 3; Collection combines with the C-myc monoclonal antibody and carries out the FACS sorting second time with the uncombined yeast cell of AVFluIgG03 monoclonal antibody: replace the 2F5 monoclonal antibody with C-myc monoclonal antibody and AVFluIgG03 monoclonal antibody; Add two kind two anti-(PE mark sheep anti-mouse igg and FITC mark goat-anti chicken IgG) simultaneously, other is with 1 of the step 3 of embodiment 3; Collection combines with the C-myc monoclonal antibody and carries out FACS sorting for the third time with the uncombined yeast cell of AVFluIgG03 monoclonal antibody: replace the 2F5 monoclonal antibody with C-myc monoclonal antibody and AVFluIgG03 monoclonal antibody; Add two kind two anti-(PE mark sheep anti-mouse igg and FITC mark goat-anti chicken IgG) simultaneously, other is with 1 of the step 3 of embodiment 3; Collect combine with the C-myc monoclonal antibody and with the uncombined yeast cell of AVFluIgG03 monoclonal antibody.

2, the FACS separating effect is estimated

Replace the 2F5 monoclonal antibody with C-myc monoclonal antibody and AVFluIgG03 monoclonal antibody, add two kind two anti-(PE mark sheep anti-mouse igg and FITC mark goat-anti chicken IgG) simultaneously, other is with 2 of the step 3 of embodiment 3.

The FACS collection of illustrative plates of the yeast cell of collecting after yeast cell of collecting after the yeast cell of collecting after the yeast display libraries (A) before the sorting, the sorting for the first time (B), the sorting for the second time (C) and the sorting for the third time (D) is seen Fig. 5.For the third time after the sorting, obtain altogether 41 combine with the C-myc monoclonal antibody and with the uncombined mono-clonal of AVFluIgG03 monoclonal antibody.

3, order-checking and sequential analysis

With 3 of the step 3 of embodiment 3.

In 41 mono-clonals, 27 is the clone of simple point mutation.With the proteic encoding sox comparison of the H5N1-HA shown in the sequence 4 of sequence table; Simple point mutation among the clone of 27 simple point mutations (repeated cloning is disregarded) sees that (numeral of occurrence positions is all represented the figure place of the sequence 4 of sequence table from 5 ' end to table 1; The numeral front gets Nucleotide for sudden change is preceding, the numeral back is the Nucleotide after suddenling change).

Table 1 sequencing result

Clone's numbering

Occurrence positions

Sudden change

Clone's numbering

Occurrence positions

Sudden change

1

T185A

I62H

15

A436G

N146D

2

T185C

I62P

16

T449C

L150P

3

G187A

G63R

17

A454G

K152E

4

T268A

C90S

18

T465G

N155K

5

T268C

C90R

19

A536G

H179R

6

C275T

P92L

20

A539G

H180R

7

T302A

L101Q

21

T549G

D183E

8

A374G

H125R

22

A560T

Q187L

9

T392A

V131E

23

T569A

L190H

10

T403C

C135R

24

T571A

Y190C

11

T424C

S142P

25

A572G

Y190N

12

T427C

F143L

26

T682C

F228L

13

T428C

F143S

27

A736G

N246D

14

T431C

F144S

The site that the amino-acid residue sudden change takes place is the critical sites of epitope, and this site mutation has caused polypeptide and can not combine with the AVFluIgG03 monoclonal antibody by combining to have become with the AVFluIgG03 monoclonal antibody.

The evaluation of the monoclonal antibody identification epi-position immunological competence of embodiment 5, yeast surface display

1, construction of recombinant plasmid

The sequence 4 of sequence table is inserted between the Nhe I and BamH I site of pCTCON2 plasmid from 5 ' terminal the 241st to 774 Nucleotide (sequence 3 of code sequence tabulation is from N-terminal the 81st to 258 amino acids residue), obtained recombinant plasmid.

2, the acquisition of recombination yeast

With recombinant plasmid transformed yeast strain E BY100, obtain recombination yeast.

With pCTCON2 plasmid transformed yeast bacterial strain EBY100, obtain contrasting yeast.

3, the abduction delivering of foreign protein

(1) recombination yeast (or contrast yeast) is mixed after resuspended with 10ml SDCAA, 30 ℃, be cultured to OD600 in the 250rpm shaking table and be about 3 (24 hours).

Induced 36 hours centrifugal collection thalline in (3) 20 ℃ of 250rpm shaking tables.

4, grouping immunity

To be divided into five groups available from the BALB/C mice of Beijing Experimental Animal Center, 5 every group, carry out following immunity respectively:

First group: with the recombination yeast thalline (4 * 10 of step 3 collection ⁷Individual) with every mouse of the resuspended pneumoretroperitoneum injection of 400ul PBS; Carry out identical immunity after two weeks at interval;

Second group: with the water intraperitoneal injection of mice, the immune volume of every mouse is 400ul; Carry out identical immunity after two weeks at interval;

The 3rd group: with the PBS intraperitoneal injection of mice, the immune volume of every mouse is 400ul; Carry out identical immunity after two weeks at interval;

The 4th group: the yeast strain that step 3 is collected contrasts yeast (4 * 10 ⁷Individual) with every mouse of the resuspended pneumoretroperitoneum injection of 400ul PBS; Carry out identical immunity after two weeks at interval.

In getting blood, separation of serum after immune 1 week for the second time.

Encapsulate elisa plate with the HA albumen of H5N1 with the concentration of 1ug/ul, with the dilution of serum continuous gradient, the antibody that detects in the serum is active with the combination of corresponding epitope.The result sees Fig. 6.It is thus clear that immune group (AVFluIgG03) and control group (second group, the 3rd group with the 4th group) are compared, and have produced higher titer antibody.The encoding sox that utilizes yeast cell surface antigen expressed epi-position is described, can be induced production of antibodies effectively.

Claims

1. the method for the proteic epitope of analysis purposes comprises the steps:

(4) yeast that filters out is extracted plasmid and check order, according to the epitope of the said target protein of nucleotide sequence analysis of the dna fragmentation that inserts said T carrier.

2. method as claimed in claim 2 is characterized in that: said step (1) is preceding carrying out, and passes through the encoding sox of the said target protein of pcr amplification earlier; The used enzyme of said pcr amplification is a high-fidelity DNA polymerase; Be preferably PyroBest DNA Polymerase.

3. according to claim 1 or claim 2 method is characterized in that: the pCTCON2-T plasmid that said T carrier obtains from the DNA shown in 5 ' terminal the 11st to 40 Nucleotide for the sequence table 5 of insertion sequence table between the Nhe of pCTCON2 plasmid I and BamH I site; The dna fragmentation of the encoding sox of said target protein inserts the Xcm I restriction enzyme site of said pCTCON2-T plasmid; Said yeast is a yeast saccharomyces cerevisiae, is preferably yeast saccharomyces cerevisiae EBY100.

4. method as claimed in claim 3 is characterized in that: in the step (1), the method that makes up said DNA library comprises the steps:

3. the dna fragmentation after 2. step being recombinated adds the A tail;

5. like arbitrary described method in the claim 1 to 4, it is characterized in that: in the said method, utilize flow cytometry to carry out the said sorting in the step (3).

6. like arbitrary described method in the claim 1 to 5, it is characterized in that: in the step (2), said DNA library is illustrated in said yeast surface comprises the steps:

7. like arbitrary described method in the claim 3 to 6, it is characterized in that: said method also comprises the steps:

(8) yeast that filters out is extracted plasmid and check order, according to the proteic epitope of variance analysis of the encoding sequence of the nucleotide sequence of the dna fragmentation that inserts said pCTCON2-T plasmid and said epitope.

8. method as claimed in claim 7 is characterized in that:

In the said step (5), the method that makes up said dna mutation library comprises the steps: 1. to pass through the encoding sox of the said epitope of pcr amplification; The used enzyme of said pcr amplification is a low-fidelity DNA polyase; Be preferably the rTaq enzyme; 2. said pcr amplification product is inserted the Xcm I restriction enzyme site of said pCTCON2-T plasmid, transform the host bacterium then, obtain said dna mutation library;

In the said method, utilize flow cytometry to carry out the said sorting in the step (7);

In the said step (6), said DNA library is illustrated in said yeast surface comprises the steps: 1. to extract the plasmid that contains said dna mutation library that obtains in the said step (5), transform said yeast, obtain recombination yeast second; 2. collect said recombination yeast second inducing culture more than 36 hours, obtain yeast display libraries second; Said inducing is to realize through in the system of culturing yeast, adding semi-lactosi.

9. the application of arbitrary said method in research antigen and antibody interaction in the claim 1 to 8.

10. the application of arbitrary said method in the vaccine research and development in the claim 1 to 8.