CN102295702B

CN102295702B - Preparation method of specific single-chain antibody aimed at T cell receptor variable range

Info

Publication number: CN102295702B
Application number: CN201110247320.3A
Authority: CN
Inventors: 高福; 施一
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2011-08-26
Filing date: 2011-08-26
Publication date: 2014-01-29
Anticipated expiration: 2031-08-26
Also published as: CN102295702A

Abstract

The invention discloses a preparation method of a specific single-chain antibody aimed at a T cell receptor variable range, the preparation method comprises the following steps: preparing soluble T cell receptor protein, then employing positive screening and reverse screening to obtain hybridoma capable of producing the specific variable range single-chain antibody. The specific single-chain antibody aimed at T cell receptor variable range can be rapidly obtained with high efficiency by using the method of the invention, and has an important meaning for designing and exploiting the correlative vaccine and immunotherapy means base on the T cell.

Description

A kind of preparation method for φt cell receptor variable region specific monoclonal antibody

Technical field

The present invention relates to a kind of preparation method of monoclonal antibody, especially a kind of method that obtains monoclonal antibody specific by positive and reverse screening method.

Background technology

T cell is by its surperficial φt cell receptor (T cell receptor, TCR) identification Major histocompatibility complex molecule (Major histocompatibility complex, MHC) polypeptide of presenting is exercised its cell function, so the recognition capability of TCR is the prerequisite of T cell functionating.The same with antibody molecule, also there is gene rearrangement phenomenon in the formation that TCR forms chain, and the encoding gene of every polypeptide chain is all to be combined by 3 or 4 gene fragments.Wherein, short chain α and γ are formed by connecting by variable region gene fragment (V gene segment), connexon gene fragment (J gene segment) and three kinds of fragments of constant region gene fragment (C gene segment), and long-chain β and δ are combined by 4 gene fragments, comprise 3 gene fragments and the extra diversity gene fragment (D gene segment) the same with short chain.There is missing at random or the increase of Nucleotide in the place of interconnecting of each gene fragment, thereby causes the height diversity of junction.For example, on human chromosome, contain altogether 42 V alpha gene fragments and 46 V beta gene fragments, and huge connexon gene fragment J α or the J β of number, the height diversity of gene fragment junction while adding gene rearrangement, just makes the number in α β TCR storehouse up to 10 ¹⁴individual.We know, for the cell receptor of other types, do not have a kind of cell receptor can equally with TCR have so many diversity.So we can imagine, the identification between TCR and its part will be all more complicated and marvellous than other any cell receptors.TCR mainly comes to interact with part by 6 complementarity determining regions (Complementarity determining region, CDR) of its variable region, so variable region determines the recognition capability of TCR.And in the research of the φt cell receptor storehouse of pathogen antigen specificity or specific for tumour antigen, we tend to find that some variable region gene fragment can preponderate, there is Preference, and monitor the have superiority changing conditions of φt cell receptor of variable region gene fragment of these bands, for relevant vaccine and the immunotherapy means of our design and developments based on T cell, have great importance.The monitoring method of ripe application is exactly low cytometric analysis at present, and want to apply low cytometric analysis, must need to use specific monoclonal antibody.But up to the present, still do not have a kind of general method to produce the preparation method for φt cell receptor variable region monoclonal antibody specific, this patent aims to provide a kind of method that can effectively produce such monoclonal antibody, and application protection.The present invention obtains soluble T cell receptor albumen by external renaturation technology, and then using monoclonal antibody hybridoma technology is produced monoclonal antibody, and adopts positive and reverse screening method to obtain the specific monoclonal antibody in certain variable region.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of preparation method for φt cell receptor variable region specific monoclonal antibody.

For solving the problems of the technologies described above, technical scheme of the present invention is: first prepare soluble T cell receptor albumen, then adopt positive and reverse screening to obtain the hybridoma cell strains that can produce specificity variable region monoclonal antibody.

In described soluble T cell receptor albumen preparation process, by introducing artificial disulfide linkage, stablize tcr protein, described artificial disulfide linkage is comprised of the T48 – C of α chain constant region and the S57 – C of β chain constant region.

In order better to stablize tcr protein, can the free cysteine mutation in β chain be become to L-Ala simultaneously.

The principle of described positive and reverse screening is as follows: φt cell receptor extracellular fragment is comprised of 4 immunoglobulin domains, comprises two variable regions and two constant regions.So when we adopt tcr protein to go immune mouse to produce monoclonal antibody, the monoclonal antibody that we obtain is other three structural domains outside combining target variable region likely.For the efficient specific monoclonal antibody in target variable region that obtains, we also will adopt anti-sieve method to reject those not monoclonal antibodies of combining target variable region.

Its concrete steps are as follows: first utilize tcr protein 1 immune mouse to produce monoclonal antibody, can stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 1; Then use φt cell receptor 2 screening by the 1st) monoclonal antibody hybridoma cell strains that step obtains, can not stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 2, obtain producing the hybridoma cell strains of specificity variable region 1 monoclonal antibody; Described φt cell receptor 1 is just the different of target variable region from the difference of φt cell receptor 2, and other three structural domains are all identical.

Application present method can obtain φt cell receptor variable region specific monoclonal antibody efficiently, fast, and relevant vaccine and immunotherapy means to design and development based on T cell have great importance.

Accompanying drawing explanation

Fig. 1 monoclonal antibody detects.

Embodiment

embodiment 1 soluble T cell receptor albumen transformation

With screening V δ 1 variable region, (be also TRDV1, TCR variable region information please refer to website: specific monoclonal antibody http://www.imgt.org) is example, and we illustrate concrete embodiment.Wherein, we are S19-2 for the φt cell receptor 1 just sieving, and derive from patient HIV, and the part of identification is the restrictive nef138-10 epi-position of HLA-A*2402 (deriving from HIV virus nef albumen).The synthetic corresponding gene of all protein material Ke You gene Synesis Company is expressed.

The sequence information of HLA-A*2402 is as shown in SEQ ID NO.1, specific as follows:

GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRW

Nef138-10 epitope polypeptide Ke You company is synthetic, and its aminoacid sequence is as shown in SEQ ID NO.2, specific as follows:

For RYPLTFGWCF.

The genomic constitution information of two chains of S19-2 TCR is: α chain, TRDV1+TRAC; β chain, TRBV30+TRBC.

α catenin sequence is as shown in SEQ ID NO.3, specific as follows:

MLFSSLLCVFVAFSYSGSSV AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGELARSGGYQKVTFGTGTKLQVIP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS CDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

Wherein, with the part of underscore, be variable region sequences, with underscore and italics body, be divided into constant region sequence, is the joining region sequence of alterable height between variable region and constant region.T48 in constant region sequence is mutated into C, for the renaturation process in later stage, can introduce the conformation that a pair of artificial disulfide linkage is stablized TCR albumen.

β catenin sequence is as shown in SEQ ID NO.4, specific as follows:

MLRSLLALLLGTFFGVR SQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSVSVGAGVPTIYFGEGSWLTVV EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD CGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Wherein, with the part of underscore, be variable region sequences, with underscore and italics body, be divided into constant region sequence, is the joining region sequence of alterable height between variable region and constant region, after constant region is cross-film district and intracellular region sequence.S57 in constant region sequence need to be mutated into C, for the renaturation process in later stage, can introduce the conformation that a pair of artificial disulfide linkage is stablized TCR albumen, free halfcystine C96 is mutated into A simultaneously, reduces polymeric formation in renaturation process.

For the φt cell receptor 2 of anti-sieve method, be the basis at S19-2, the variable region TRDV1 of α chain is replaced to TRAV2-1, and β chain remain unchanged, our called after S19-2-TRAV2-1 of this φt cell receptor.The α catenin sequence newly combining as shown in SEQ ID NO.5, (only comprising variable region, joining region and constant region sequence) specific as follows:

KEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVTLARSGGYQKVTFGTGTKLQVIP NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS

These three polypeptide chains are building up to expression vector pET21a upper, < < molecule clone technology handbook > > is asked for an interview in specific experiment operation.Application intestinal bacteria E.coli expression system is expressed inclusion body protein purifying, and concrete steps are as follows:

1) 1-2 μ L expression plasmid is transformed into BL21(DE3) in competent cell, spend the night, mono-clonal colony inoculation, in 40 mL LB substratum, is cultivated to 6-8 hour.

2) every 20 mL are transferred in the large shaking flask that contains 2 L LB substratum according to 1% inoculum size respectively, and 37 ℃ are cultured to OD600=0.4-0.6, and adding IPTG is 1 mM to final concentration, and 37 ℃ are continued to cultivate 5-6 hour.

3) receive bacterium (following steps are all carried out under low temperature or ice-water bath), with 60 mL left and right 1 * PBS, suspend, Φ 6 Probe Ultrasonic Searching cracking (ultrasonic 6 s, interval 12 s, 99 times, 250 W, 2 circulations).

4) 17500 rpm, centrifugal 15 min.The centrifuge tube bottom compact block that is white in color is inclusion body.

5) with pipettor or careful crisp piping and druming of one layer of cells above inclusion body is fallen of glass stick, abandon supernatant.With Washing buffer, suspend.

6) ultrasonic, 4 S, 10 S, 40 times, 250 W.

7) repeating step 4-6, with Washing buffer washing three times.

8) Resuspension buffer suspends, 17500 rpm, centrifugal 15 min.

9) remove cell debris, abandon supernatant, weigh.In the ratio of 30mg/mL, with solubilization of inclusion bodies liquid (Disolution buffer), dissolve, 4 ℃ of stirrings are spent the night.

10) 17500 rpm, 20 min, take out supernatant or be distributed into 1mL/ pipe be stored in-20 ℃ or-80 ℃ standby.

inclusion body scavenging solution (Washing buffer): 0.5% Triton-100,50 mM Tris pH 8.0,300 mM NaCl, 10 mM EDTA, 10 mM DTT (existing with now adding);

the resuspended liquid of inclusion body (Rsuspension buffer): 50 mM Tris pH 8.0,100 mM NaCl, 10 mM EDTA, 10 mM DTT (existing with now adding);

solubilization of inclusion bodies liquid (Dissolution buffer): 6 M Gua-HCl(or 8 M Urea), 10% glycerine, 50mM Tris pH8.0,100 mM NaCl, 10 mM EDTA, 10 mM DTT (existing with now adding);

the renaturation of embodiment 2 soluble T cell receptor albumen

concrete steps are as follows:

1) preparation TCR renaturation solution.Be generally 2 L, on ice or Cool Room 4℃ precooling, renaturation process carries out in Cool Room 4℃.

2) beaker that fills renaturation solution is placed in to magnetic stirring apparatus, adds rotor, suitable stirring velocity is set.Here with 1sec rotor, turn around as good.Look for 5 mL syringes to change the syringe needle of 1 mL, be fixed on beaker.

3) inclusion body of α chain and β chain adds syringe (mass ratio can be adjusted according to actual renaturation situation) according to the mass ratio of 2:1, once adds 2 mL α chains and 1 mL β chain, and mixes.Make it slowly splashing in renaturation solution drop by drop, slowly stir 8 hours.

4) every 8 hours, repeat step 4 one time, add altogether inclusion body three times, add altogether α chain and the 3 mL β chains of 6 mL.

5) add for the third time after inclusion body 8 hours, use tangential flow instrument (TFF) to concentrate, last solution system is about 300-400 mL, and the protein renaturation solution after concentrated is put into dialysis tubing.

6), in renaturation solution concentration process, prepare 4 L deionized waters and 4L 10 mM Tris damping fluids, Cool Room 4℃ precooling.

7) first the albumen that packs dialysis tubing into is dialysed 24 hours in water, then with 10 mM Tris dialysis 24 hours, in dialysis procedure, need to stir with rotor.

8) protein liquid after dialysis is put into concentrated cup and be concentrated into 100 mL.

9) take out protein liquid, 4 ℃, centrifugal 10 minutes of 16,000 rpm.Get supernatant.

10) with ion exchange column Source 15Q, purify, first loading is by protein binding to ion exchange column several times, and each 5ml, generally above expires 50 mL albumen with regard to wash-out once, uses salt concn gradient elution, and gradient is generally in 90 minutes and moves 50% to from 0%.

11) run non-reduced and reduction albumin glue and determine object TCR protein peak, after this by Superdex 200 pillars purifying again for collected albumen.

renaturation solution formula is as follows:

5M Urea

100mM Tris pH8.0

400mM L-Arg HCl (mother liquor can be made into 2M)

2mM EDTA

5mM GSH (adding again after renaturation solution precooling)

0.5mM GSSG(is the same)

0.5mM PMSF (mother liquor 100mM can not add).

embodiment 3 positive and reverse screenings are prepared monoclonal antibody specific

φt cell receptor extracellular fragment is comprised of 4 immunoglobulin domains, comprises two variable regions and two constant regions.So when we adopt tcr protein to go immune mouse to produce monoclonal antibody, the monoclonal antibody that we obtain is other three structural domains outside combining target variable region likely.For the efficient specific monoclonal antibody in target variable region that obtains, we also will adopt anti-sieve method to reject those not monoclonal antibodies of combining target variable region.

So-called anti-sieve method, first needs us to apply a kind of tcr protein 2 for anti-sieve of external renaturation technique construction, and simultaneously for convenient statement, we are by the tcr protein called after φt cell receptor 1 of immune mouse before.φt cell receptor 1 is just the different of target variable region from the difference of φt cell receptor 2, and other three structural domains are all identical.

Preparation flow is as follows:

First we utilize φt cell receptor 1 immune mouse to produce monoclonal antibody, adopt afterwards ELISA(enzyme-linked immunosorbent assay) technology carrys out desired certain the variable region specific monoclonal antibody of positive and reverse screening.The first step, forward screening, can stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 1; Second step, in the monoclonal antibody hybridoma cell strains that the first step obtains, further oppositely screens with φt cell receptor 2, soon can not stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 2, reject those can with the monoclonal antibody hybridoma cell strains of φt cell receptor 2 combinations.The monoclonal antibody hybridoma cell strains obtaining through this two steps positive and reverse screening is exactly our last desired certain variable region specific monoclonal antibody.

Or screening V δ 1 specific monoclonal antibody of take is example, after our the method acquisition soluble T cell receptor 1 and φt cell receptor 2 by 2 li of embodiment, carry out above-mentioned preparation flow.We obtain 11 strain monoclonal antibody hybridoma cell strainses altogether result, adopt ELISA to verify.

operation steps:

1) coated

With coated damping fluid, albumen is diluted to 10ug/mL, in every hole, adds 0.1mL, first put 37 ℃ and hatch and within 2 hours, proceed to again 4 ℃ and spend the night.Also elisa plate directly can be put to 4 ℃ spends the night.

2) washing

After coated end, discard coating buffer, with PBST, wash, method is that PBST fills it up with every hole, and standing 5-10min, discards washing lotion, again fills it up with, and repeated washing 3-5 time, finally pats dry elisa plate and wait for next step sealing.

3) sealing

The elisa plate of getting the 2nd step adds 10% calf serum to the sealing of every hole, and volume is at least the twice of added coating buffer.Sealing condition also has two kinds of selections: be directly placed in 4 ℃ and spend the night, or be placed in 37 ℃ of incubation 2-4h.

4) washing

The elisa plate that sealing finishes washs, and method is with step 2.The primary antibodie to be added (monoclonal antibody hybridoma cell strains culturing cell supernatant or mouse ascites purified monoclonal antibody) such as finally pat dry.

5) add primary antibodie

Primary antibodie sample joins in respective aperture after being first diluted to desired concn with coating buffer.Reaction conditions is 37 ℃ and hatches 2h.

6) washing

Specifically with step 4.

7) add the commercialization two of corresponding HRP-mark anti-

Add the two anti-of corresponding commercialization HRP-mark, with reference to two anti-specification sheetss, by two, anti-ly with confining liquid, be diluted to suitable multiple.By two anti-each holes that add of having diluted, 37 ℃ of reaction 1-1.5h.

8) washing

Specifically with step 4.

9) colour developing

After step 8 completes, getting 100ulTMB substrate nitrite ion divides and adds in each hole of ELISA.Then elisa plate is placed in to 37 ℃ of about 10min of reaction.

10) stop

Elisa plate is taken out, and every hole adds stop buffer (2mol/L sulphuric acid soln) termination reaction, and to reading in microplate reader, when TMB is substrate, wavelength is 450nm immediately.

reagent preparation:

1) coating buffer (carbonate buffer solution of PH6.3)

Anhydrous Na2CO3 0.1696g

NaHCO3 0.2856g

Sterilized water 100ml

Be dissolved to 4 ℃ completely, in one week, use

2) PBST formula

NaCl 8.0g

Na2HPO4.12H2O 2.9g

KCl 0.2g

KH2PO4 0.24g

TW-20 0.5ml

Sterilized water 1000ml

3) stop buffer

The vitriol oil: sterilized water=1:8

The vitriol oil slowly joins in sterilized water, and stirs heat radiation.

Finally we obtain ELISA data as shown in Figure 1.Result shows that wherein 10 strains can be specifically in conjunction with V δ 1 variable region, and has strain combination specifically.So applying our method can high efficiency acquisition variable region specific monoclonal antibody, and guarantees the activity conformation epi-position of identification albumen, can be used for flow cytometer and detect.

Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.

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Claims

1. for a preparation method for φt cell receptor variable region specific monoclonal antibody, it is characterized in that first preparing soluble T cell receptor albumen, then adopt positive and reverse screening to obtain the hybridoma cell strains that can produce specificity variable region monoclonal antibody; Shown in positive and reverse screening comprise the steps:

1) utilize φt cell receptor 1 immune mouse to produce monoclonal antibody, can stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 1; Described φt cell receptor 1 is S19-2, and the part of identification is the restrictive nef138-10 epi-position of HLA-A*2402; The sequence information of HLA-A*2402 is as shown in SEQ ID NO.1; Nef138-10 aminoacid sequence is as shown in SEQ ID NO.2; The genomic constitution information of two chains of S19-2TCR is: α chain, TRDV1+TRAC; β chain, TRBV30+TRBC, α catenin sequence is as shown in SEQ ID NO.3, and β catenin sequence is as shown in SEQ ID NO.4;

2) use φt cell receptor 2 screenings by the 1st) monoclonal antibody hybridoma cell strains that step obtains, can not stay in conjunction with the monoclonal antibody hybridoma cell strains of φt cell receptor 2, obtain the hybridoma cell strains that specificity is produced φt cell receptor variable region 1 monoclonal antibody; Described φt cell receptor 2 is on the basis of S19-2, the variable region TRDV1 of α chain is replaced to TRAV2-1, and β chain remains unchanged; α catenin sequence is as shown in SEQ ID NO.5.

2. method claimed in claim 1, is characterized in that by introducing artificial disulfide linkage, stablizing tcr protein in described soluble T cell receptor albumen.

3. method claimed in claim 2, is characterized in that the 57th mutant serine that described artificial disulfide linkage sports halfcystine and β chain constant region by the 48th Threonine of α chain constant region is that halfcystine forms.

4. the method described in claim 2 or 3, characterized by further comprising simultaneously the free cysteine mutation of the 96th in β chain is become to L-Ala.