CN102732974B - Method for constructing phage antibody libraries and CD6-resisting antibody obtained by screening by using phage antibody libraries - Google Patents
Method for constructing phage antibody libraries and CD6-resisting antibody obtained by screening by using phage antibody libraries Download PDFInfo
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Abstract
The invention discloses a method for constructing high-diversity high-capacity natural human Fab phage antibody libraries, comprising the following steps: obtaining genes of variable regions of a full set of human antibody light chain and a full set of human antibody heavy chain from the peripheral blood lymphocytes of 15 healthy volunteers by amplifying through DNA recombination technology, and inserting the genes into the corresponding positions of a phage carrier PFK-1 and a phage carrier PFL-6 respectively and constructing the high-diversity high-capacity natural human Fab phage antibody libraries. The kappa-chain Fab antibody library constructed by adopting the method has the capacity of 2.85*10<8>, and the lambda-chain Fab antibody library constructed by adopting the method has the capacity of 2.9*10<8>. The kappa-chain Fab antibody library and the lambda-chain Fab antibody library reach the standard of the high-capacitya antibody library, comprise the genes of the variable regions of nearly all the human antibody light chains and heavy chains, have high diversity and are mainly used for the high-throughout screening of the low-antigenicity high-affinity human antibody for clinical treatment. A CD6-resisting monoclonal antibody can be screened out by using the Fab antibody libraries.
Description
Technical field
The present invention relates to antibody engineering technical field, the particularly effect of a kind of construction process Ji Ci storehouse of phage antibody library in antibody screening, relates in particular to the anti-CD 6 antibody that this storehouse of application screens.
Background technology
Antibody library is the major progress in antibody engineering nineties field, it is the integrated of phage display and two kinds of technology of antibody library, and its appearance depends on the foundation of round pcr, antibody molecule in the development of colibacillary functional expression and display technique of bacteriophage.The principle of phage antibody library technique is exactly that the fragments such as the Fab of antibody molecule or ScFv and single stranded phage coat protein are formed to fusion rotein, is showed in phage particle surface.Phage antibody library technique has been simulated the selective action of antibody mediated immunity system, the antibody that is presented on phage surface can interact with immobilised target antigen in vitro, then repetitive scrubbing is removed non-specific binding antibody, the phage that wash-out collection are combined with antigen again, ehec infection, makes special phage obtain enrichment again.
The object of setting up phage antibody library is in order effectively to obtain the human antibody for various antigens.The screening method of antibody library can be divided into: the screening of (1) liquid phase antigen, can keep the native conformation of antigen preferably; (2) screening of intact cell, be applicable to be difficult to purifying or unknown antigen, but this method cell membrane component is complicated, and difficulty is larger; (3) tissue screening, the tissue that is difficult to obtain individual cells for some carries out the screening of specific antibody.Phage antibody library is unified in one by genotype and phenotype, and selective power and amplification ability is coupled, has powerful screening function, and the antibody generative process in analogue body, makes antibody engineering technology enter new epoch in vitro.
At present, phage antibody library is mainly used in expression screening Fab and single-chain antibody (ScFv).Fab is that Fab (fragment of antigen binding) is comprised of heavy chain Fd section and complete light chain, it is 1/3rd of complete antibody molecule, stability in vivo will be far above single-chain antibody, and have and be easy to carry out the advantages such as antibody fragment that expression product in structure of modification and bacterium mostly is function, therefore, Fab has incomparable advantage in functionally active and the clinical application of research antibody variable region.
The development experience of therapeutic antibodies several stages of heterologous antibody, humanized antibody and human antibody.At present, human antibody is the main direction of therapeutic antibodies development, and the preparation that appears as human antibody of phage antibody library technique provides good technology platform, and becomes gradually one of Main Means of current acquisition human antibody.
Anti-CD 6 monoclonal antibody is showing good result for the treatment of aspect the treatment of the diseases such as ox-hide aquatic foods and rheumatoid arthritis, so the screening of anti-CD 6 monoclonal antibody has become the study hotspot in antibody field.
CD6 molecule is I type transmembrane glycoprotein, and there are 3 functional zone tumor-necrosis factor glycoproteinss that are rich in halfcystine extracellular region, belongs to SRCRSF family, and CD6 has 105/110KD and two kinds of heterogeneous of 130KD.668 amino acid of CD6 molecular sequences total length, remove after 17 amino acid signal peptide sequences of N-end, 385 of CD6 molecule extracellular regions amino acid, and extracellular region has 3 epi-position: SRCR1, SRCR2 and SRCR3 at least.The more CD6 part of research is ALCAM (CD166) at present.CD6/CD6L ligand interaction may regulate T cell activation by following approach: 1. by PKC pathway activation T cell.2. CD6 and CD6L adhere to and promote and extend contacting of T cell and APC, thereby have extended the stimulation through TRC/CD3 or the generation of CD2 path; 3. CD6 is crosslinked can trigger cytoplasm Free Ca
2+concentration increases.
CD6 participates in the activation of T cell, and performance immunological effect shows that CD6 also participates in the function of immunological homeostasis simultaneously.At present, to CD6, the accurate mechanism in lymphocytic activation and differentiation is also not very clear.But increasing research shows that CD6 is the important accessory molecule of T lymphocyte activation, provides the costimulatory signal of lymphocyte activation.Vital role based on CD6 molecule in lymphocyte maturation, activation and propagation, the treatment that the function of blocking-up CD6 molecule can be the autoimmune disorders such as rheumatoid arthritis, psoriatic provides new treatment means and method.
Therefore, low antigenicity, the high-affinity antibody in order to meet high flux screening clinical treatment, used, build there is height diversity, versatility is good, without the large capacity natural human source Fab phage antibody library of antigen skewed popularity, have extremely important using value.And this antibody library has important effect aspect the screening of the humanized antibodies such as anti-CD 6 antibody.
Summary of the invention
The object of the present invention is to provide the method for the highly multifarious large capacity natural human of a kind of structure source Fab phage antibody library, and apply the monoclonal antibody that this storehouse filters out a strain anti-CD 6 antigen.This antibody library is mainly used in low antigenicity and the high-affinity human antibody that high flux screening clinical treatment is used.
Technical scheme of the present invention is as follows:
A construction process for natural human source Fab phage antibody library, comprises the steps:
(1) build the phasmid carrier PFK-1 of phage display Fab antibody, PFL-6;
(2) extraction of separated and total RNA of human peripheral lymphocyte, synthesizes cDNA by RT-PCR;
(3) take cDNA as template, adopt the Auele Specific Primer of one group of whole heavy chain of nearly cover people antibody or variable region of light chain encoding sequence to carry out PCR reaction, amplification light chain kappa, lambda and heavy chain variable region gene;
(4) by light chain kappa chain and lambda chain variable region gene respectively with phasmid carrier PFK-1, PFL-6 connects, and builds kappa chain, lambda chain light chain antibody library;
(5) heavy chain VH fragment gene is connected with phasmid carrier PFK-1, collects the DNA of all cloned plasmids, build heavy chain antibody storehouse;
(6) heavy chain variable region gene is cut out from the DNA of heavy chain antibody storehouse, be connected with light chain lambda storehouse DNA and be connected and obtain complete antibody library DNA with light chain kappa storehouse DNA respectively;
(7) carry out the diversity analysis of natural human source Fab phage antibody library.
In aforesaid method, the method that builds phasmid carrier described in step (1) is: heavy chain CH1 constant region gene carries out enzyme with restriction enzyme A scI and NotI respectively and cuts, after purifying, heavy chain CH1 constant region gene is connected with phage vector PCSm, transform in bacillus coli DH 5 alpha, built PCSm-CH1 carrier.Light chain Lambda chain and kappa chain constant region gene carry out enzyme with HindIII and AscI respectively and cut, after purifying, respectively lambda is connected with PCSm-CH1 carrier with kappa chain constant region gene, transform in bacillus coli DH 5 alpha the carrier called after PFK-1 of structure, PFL-6.
In aforesaid method, step (2) separated and total RNA extract and derive from the lymphocyte that 15 parts of age structures are the peripheral blood separation of the healthy volunteer between 30-40 year.
In aforesaid method, the Auele Specific Primer of the described one group of whole heavy chain of nearly cover people antibody of step (3) or variable region of light chain encoding sequence, can increase and obtain whole heavy chains and the chain variable region gene of nearly cover human normal immunoglobulin (IgG), described primer is as follows:
The upstream primer of amplification kappa chain variable region gene is:
5’VK-1：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGACATCCAGWTGACCCA
5’VK-2：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGATGTTGTGATGACTCAG
5’VK-3：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGAAATTGTGWTGACRCA
5’VK-4：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGATATTGTGATGACCCAG
The downstream primer of amplification kappa chain variable region gene is:
3’VK-1：
ACGTTTGAT
CTCGAGCTTGGTCCCYTGGCCRAA
3’VK-2：
ACGTTTGAT
CTCGAGTTTGGTCCCAGGGCCGAA
3’VK-3：
ACGTTTGAT
CTCGAGCTTGGTCCCTCCGCCGAA
The upstream primer of amplification lambda chain variable region gene is:
5’VL-1：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACAGTCTGTGYTGACKCAG
5’VL-2：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACARTCTGCCCTGACTCAG
5’VL-3：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCATCCTATGWGCTGACTCAG
5’VL-4：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCATCTTCTGAGCTGACTCAG
5’VL-5：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACAGGCTGTGCTGACTCAG
5’VL-6：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAAATTTTATGCTGACTCAGC
The downstream primer of amplification lambda chain variable region gene is:
3’VL-1：
CTTGGGCTGA
CCTAGGACGGTSACCTTGGTSCC
3’VL-2：
CTTGGGCTGA
CCTAGGACGGTCAGCTYSGTCCC
3’VL-3：
CTTGGGCTGA
CCTAGGATGATCAGCTGGGTTCC
The upstream primer of amplification heavy chain variable region gene is:
5’VH-1：
TTCTATTCTCACA
GTGCACAGRTGCAGCTGGTGCARTCTGG
5’VH-2：
TTCTATTCTCACA
GTGCACAGSAGGTCCAGCTGGTRCAGTCTGG
5’VH-3：
TTCTATTCTCACA
GTGCACAGRTCACCTTGAAGGAGTCTGG
5’VH-4：
TTCTATTCTCACA
GTGCACAGSAGGTGCAGCTGGTGGAGTCTGG
5’VH-5：
TTCTATTCTCACA
GTGCACAGGAGGTGCAGCTGGTGGAGWCYGG
5’VH-6：
TTCTATTCTCACA
GTGCACAGGTGCAGCTACAGCAGTGGGG
5’VH-7：
TTCTATTCTCACA
GTGCACAGSTGCAGCTGCAGGAGTCSGG
5’VH-8：
TTCTATTCTCACA
GTGCACAGGARGTGCAGCTGGTGCAGTCTGG
The downstream primer of amplification heavy chain variable region gene is:
3’VH-1：
GGCCCTTGGT
GCTAGCTGAGGAGACGGTGACCAGGGTKCC
3’VH-2：
GGCCCTTGGT
GCTAGCTGAAGAGACGGTGACCATTGTCCC
3’VH-3：
GGCCCTTGGT
GCTAGCTGAGGAGACGGTGACCGTGGTCCC。
In aforesaid method, the method that builds light chain storehouse in step (4) is: light chain kappa chain and lambda chain variable region gene are used respectively to restriction enzyme SfiI and XhoI, SfiI and AvrII double digestion, after purifying Vkappa and Vlambda gene respectively with phasmid carrier PFK-1, PFL-6 connects; After kappa is partly connected to product purification, be dissolved in 100uL sterilized water, electricity is evenly coated transformed bacteria in the 2YTAG flat board containing 100ug/ml penbritin after proceeding to competent cell TG1; The DNA that collects all cloned plasmids is kappa chain light chain antibody library; By identical method, obtain lambda chain light chain antibody library.
In aforesaid method, the method that builds heavy chain storehouse in step (5) is: heavy chain VH fragment gene is carried out to double digestion with ApaLI and NheI respectively, after purifying, be connected with phasmid carrier PFK-1; After heavy chain is connected to product purification, be dissolved in 100uL sterilized water, minutes four times electric shocks transform after TG1 competent cells by transformed bacteria evenly coating with containing in the 2YTAG flat board of 100ug/mL penbritin; The DNA that collects all cloned plasmids is heavy chain antibody storehouse.
In aforesaid method, the method that obtains complete antibody storehouse described in step (6) is: adopt ApaLI and NheI to carry out double digestion, heavy chain variable region gene is cut out from the DNA of heavy chain antibody storehouse, be connected with light chain lambda storehouse DNA and be connected with light chain kappa storehouse DNA respectively.With method electric shock, transform the plasmid DNA that obtains all clones, be antibody library DNA.
The monoclonal antibody that the present invention provides the construction process by this phage antibody library to screen simultaneously.
The anti-CD 6 monoclonal antibody that the present invention provides the construction process by this phage antibody library to screen simultaneously, the aminoacid sequence of its Fab antibody VH-CH1 is as shown in SEQ ID NO:1, and the aminoacid sequence of Vkappa-Ckappa is as shown in SEQ ID NO:3.
The biological screening process of anti-CD 6 Fab antibody of the present invention is: enrichment screening kappa chain phage antibody library → phage library ELISA → mono-clonal Phage-ELISA evaluation → PCR finger printing authentication → mono-clonal bacterium colony order-checking → Phage-ELISA result verification.
Accompanying drawing explanation
Fig. 1 is PFK-1/PFL-6 plasmid vector figure.
Fig. 2 is heavy chain variable region gene and chain variable region gene pcr amplification product figure.Wherein, M is 100bp Ladder DNA Marker.
Fig. 3 is phage antibody library ELISA detected result after anti-CD 6 screening.
Fig. 4 is mono-clonal phage display ELISA result.
Fig. 5 is that BstNI enzyme is cut PCR fragment finger printing.
Fig. 6 is anti-CD 6 antibody checking ELISA detected result.
Embodiment
Embodiment of the present invention illustrate by the following example.Yet embodiment of the present invention are not limited to the specific detail of these embodiment, because for the person of ordinary skill of the art, other variation is known, or is apparent according to direct disclosed content and appended claims.Therefore, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.The reference of quoting is herein incorporated to herein in full by reference with it.
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
Adopt the synthetic method of gene, external synthetic heavy chain CH1 constant region gene, simultaneously synthetic light chain Lambda chain and kappa chain constant region gene.Heavy chain CH1 constant region gene carries out enzyme with restriction enzyme A scI and NotI respectively and cuts, after purifying heavy chain CH1 constant region gene with phage vector PCSm(purchased from company limited of Cuba Centro De Inmunologia Molecular) be connected, transform in bacillus coli DH 5 alpha, built PCSm-CH1 carrier.Light chain Lambda chain and kappa chain constant region gene are carried out to enzyme with Hind III and AscI respectively to be cut, after purifying, respectively lambda is connected with PCSm-CH1 carrier with kappa chain constant region gene, transform in bacillus coli DH 5 alpha, build PCSm-CH1-Ckappa carrier and PCSm-CH1-Clambda carrier.By these two kinds of carrier called after PFK-1, PFL-6, result is as shown in Figure 1.
The extraction of separated and total RNA of embodiment 2, human peripheral lymphocyte
Collecting respectively 15 parts of age structures is each 2mL of healthy volunteer's peripheral blood between 30 ~ 40 years old, adopts respectively the separated peripheral blood lymphocyte that obtains of lymphocyte separation medium, and every part of lymphocytic amount is about 5 * 106.Adopt Trizol method from lymphocyte, to extract total RNA.
The amplification of embodiment 3, human antibody heavy chain and chain variable region gene
Adopt omniscript RT kit (purchased from Qiagen company limited) to carry out RT-PCR reaction, the total RNA reverse transcription of lymphocyte is become to cDNA.Take cDNA as template, then adopt the Auele Specific Primer (summary of the invention partly has detailed description) of one group of whole heavy chain of nearly cover people antibody or variable region of light chain encoding sequence to carry out PCR reaction, amplification light chain kappa, lambda and heavy chain variable region gene.Amplification condition is: 98 ℃, and 3min, then 98 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 30sec, totally 30 circulations, are finally extended for 72 ℃, 10min.PCR product is identified through 1.5% gel electrophoresis, shows that heavy chain variable region gene length is 390bp left and right.Light chain kappa, lambda variable region gene length is 350bp left and right.Result as shown in Figure 2.
The structure of embodiment 4, natural human source Fab phage antibody library
Light chain kappa chain and lambda chain variable region gene are used respectively to restriction enzyme SfiI and XhoI, SfiI and AvrII double digestion, after purifying Vkappa and Vlambda gene respectively with phasmid carrier PFK-1, PFL-6 connects.After kappa is partly connected to product purification, be dissolved in 100uL sterilized water, minutes four times electric shocks transform after TG1 competent cells by transformed bacteria evenly coating with containing in the 2YTAG flat board of 100ug/mL penbritin.The DNA that collects all cloned plasmids is kappa chain light chain antibody library.By identical method, obtain lambda chain light chain antibody library.
Heavy chain VH fragment gene is carried out to double digestion with ApaLI and NheI respectively, after purifying, be connected with phasmid carrier PFK-1.After heavy chain is connected to product purification, be dissolved in 100uL sterilized water, minutes four times electric shocks transform after TG1 competent cells by transformed bacteria evenly coating with containing in the 2YTAG flat board of 100ug/mL penbritin.The DNA that collects all cloned plasmids is heavy chain antibody storehouse.
Adopt ApaLI and NheI to carry out double digestion, heavy chain variable region gene is cut out from the DNA of heavy chain antibody storehouse, be connected with light chain lambda storehouse DNA and be connected with light chain kappa storehouse DNA respectively.With method electric shock, transform the plasmid DNA that obtains all clones, be antibody library DNA.By transformed bacteria gradient dilution, after coated plate, calculate bacterium colony number simultaneously.Obtaining kappa chain Fab antibody library capacity is 2.85 * 10
8, lambda chain Fab antibody library capacity is 2.9 * 10
8, meet the requirement of Large phage library.
The diversity analysis of embodiment 5, natural human source Fab phage antibody library
Twice random choose clone is obtained to lambda chain and heavy chain gene and insert 28 correct clones and carry out sequential analysis, variable region of heavy chain V fragment belongs to VH1 ~ 7 gene family.Wherein, VH1 gene family has 11, accounts for 39%.8 of VH4 gene families (29%), 4 of VH5 gene families (14%), 3 of VH3 gene families (11%), 1 of VH2 gene family (3.5%), 1 of VH6 gene family (3.5%).Variable region of heavy chain J fragment belongs to 1 ~ 6 ，Yi JH4 family of family and accounts at most 43%.Article 28, Lambda variable region of light chain V fragment belongs to V λ 1 ~ 10 family.13 of 3 gene families of V λ (46%) wherein, 4 of 1 gene families of V λ (14%), 4 of 2 gene families of V λ (14%), 3 of 9 gene families of V λ (11%), 2 of 6 gene families of V λ (7%), 1 of 5 gene family of V λ (3.5%), 1 of 10 gene family of V λ (3.5%).Lambda chain variable region of light chain J fragment belongs to J λ 1 ~ 3 family.Illustrate that thus natural human source lambda chain IGg Fab phage antibody library has the diversity of height.
Twice random choose clone is obtained to kappa chain and heavy chain gene and insert 20 correct clones and carry out sequential analysis, variable region of heavy chain V fragment belongs to VH1 ~ 7 gene family.Wherein, VH1 gene family has 7, accounts for 35%.4 of VH4 gene families (20%), 4 of VH3 gene families (20%), 3 of VH5 gene families (15%), 2 of VH2 gene families (10%).Variable region of heavy chain J fragment belongs to 1 ~ 6 ，Yi JH4 family of family and accounts at most 35%.Article 20, kappa variable region of light chain V fragment belongs to V κ 1 ~ 3 family.9 of 3 gene families of V κ (45%) wherein, 8 of 2 gene families of V κ (40%), 3 of 1 gene families of V κ (15%).Kappa chain variable region of light chain J fragment belongs to J κ 1 ~ 5 family.Illustrate that thus natural human source Kappa chain IGg Fab phage antibody library has the diversity of height.
The biopanning of embodiment 6, anti-CD 6 Fab antibody
1, the enrichment of kappa chain phage antibody library screening
Take CD6 as antigen coated immunity pipe, adopt 4% PBSM sealing.In immune pipe, add kappa phage library to carry out antibody antigen combination, PBS(polysorbas20) wash away unconjugated phage.100mM/L TEA wash-out antibody, by the antibody re-infection TG1 cell eluting, the dull and stereotyped overnight incubation of 2YTAG.Intestinal bacteria are scraped from flat board, again shake bacterium and add M13 helper phage to carry out phage amplification, PEG/Nacl deposition and purification phage is for next round screening.Carry out altogether four-wheel phage library enrichment screening.
2, phage library ELISA
With the coated elisa plate of CD6, BSA(bovine serum albumin), anti-Myc monoclonal antibody does respectively feminine gender and positive control.By every, take turns phage library after enrichment respectively through row 1:10,1:100,1:1000 dilution, then adds elisa plate to hatch.Wash after plate, add the anti-M13 monoclonal antibody/horseradish peroxidase of anti-M13 monoclonal antibody/HRP() hatch TMB(tetramethyl benzidine) colour developing.Result demonstration, CD6 takes turns enrichment since second and obtains positive findings, as shown in Figure 3.
3, mono-clonal Phage-ELISA is identified
Take turns second, third round, the phage-infect TG1 cell that fourth round elutes, is coated with that 2YTAG is dull and stereotyped obtains single bacterium colony.84 single bacterium colonies of picking are cultivated in 96 hole depth orifice plates, add helper phage M13 to carry out phage amplification.The phage drawing after amplification carries out ELISA evaluation.Result shows that each mono-clonal phage shows positive findings, as shown in Figure 4.
4, the authentication of PCR finger printing and the order-checking of mono-clonal bacterium colony
The mono-clonal of choosing after third round enrichment carries out the authentication of PCR finger printing.Adopt the method for the bacterium colony PCR Fab full length DNA that increases from 42 samples.Then adopting BstNI to carry out enzyme to PCR fragment cuts.4% agarose gel electrophoresis detects enzyme and cuts result.Result shows: 42 samples contain identical DNA fragmentation pattern, as shown in Figure 5.Choosing immediately 6 samples checks order.Result shows that 6 samples have aminoacid sequence and the DNA sequence dna of identical Fab antibody, and the aminoacid sequence of VH-CH1 is as shown in SEQ ID NO:1, and DNA sequence dna is as shown in SEQ ID NO:2; The aminoacid sequence of Vkappa-Ckappa is as shown in SEQ IDNO:3, and DNA sequence dna is as shown in SEQ ID NO:4.
SEQ?ID?NO：1：
MKKIWLALAGLVLAFSASAQVQLQQWGAGLLKPSETLSLTCAVYGASFGDDYWSWIRQAPGRGLEWIGEINRAGRTFYDPSVKSRVTISMDTSKNQFSLRLTSVTAADTAVYYCARGRIIQRSSLHFDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
SEQ?ID?NO：2：
ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCAAGTGCACAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGCGTCCTTCGGTGATGACTACTGGAGCTGGATCCGCCAGGCCCCAGGGAGGGGGCTTGAGTGGATTGGTGAAATCAATCGTGCTGGACGCACCTTCTACGACCCGTCCGTCAAGAGTCGAGTCACCATATCAATGGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGACCTCTGTGACCGCCGCGGACACGGCTGTCTATTACTGTGCGAGAGGCAGAATAATCCAGAGGAGTAGCCTTCACTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTCCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
SEQ?ID?NO：3：
MKKIWLALAGLVLAFSASADIVMTQTPSPLSASVGDRVTITCRASQSISTYVNWYQQKPGKAPRLLIYDGSRLESGVPSRFSASGSGTDFTLTISGLQPEDCATYYCQQSYSASRGSFGPGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ?ID?NO：4：
ATGAAGAAAATCTGGCTGGCACTGGCTGGCTTAGTCTTGGCCTTCTCGGCCAGCGCAGATATTGTGATGACCCAGACTCCATCTCCCTTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGTACCTATGTAAATTGGTATCAACAAAAACCAGGGAAAGCCCCTAGGCTTCTGATTTACGATGGATCCAGATTGGAGAGTGGGGTCCCATCGAGGTTCAGTGCCAGTGGGTCTGGAACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTGTGCAACTTACTACTGTCAACAGAGTTACAGTGCCTCCCGAGGAAGTTTCGGCCCTGGGACCAAACTCGAGATCAAACGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCACCGGTGACAAAGAGCTTCAACAGGGGAGAGTGT
5, Phage-ELISA carries out result verification
From the identical sample of 6 Fab DNA sequence dnas, choose 1 sample (1A), carry out amplification and the purifying of phage.With coated elisa plate of CD6, and carry out ELISA detection.Result shows: at 1:10, two extent of dilution of 1:100 obtain strong positives, prove the Fab antibody fragment that obtains after enrichment can with CD6 specific binding, result is as shown in Figure 6.
Claims (4)
1. a construction process for natural human source Fab phage antibody library, is characterized in that, comprises the steps:
(1) build the phasmid carrier of phage display Fab antibody, the method of described carrier construction is: heavy chain CH1 constant region gene carries out enzyme with restriction enzyme A scI and NotI respectively and cuts, after purifying, heavy chain CH1 constant region gene is connected with phage vector PCSm, transform in bacillus coli DH 5 alpha, built PCSm-CH1 carrier, light chain Lambda chain and kappa chain constant region gene carry out enzyme with HindIII and AscI respectively and cut, after purifying, respectively lambda is connected with PCSm-CH1 carrier with kappa chain constant region gene, transform in bacillus coli DH 5 alpha, the carrier called after PFK-1 building, PFL-6,
(2) extraction of separated and total RNA of human peripheral lymphocyte, synthesizes cDNA by RT-PCR;
(3) take cDNA as template, then adopt the Auele Specific Primer of one group of whole heavy chain of nearly cover people antibody or variable region of light chain encoding sequence to carry out PCR reaction, amplification light chain kappa, lambda and heavy chain variable region gene;
The Auele Specific Primer of the described one group of whole variable region of light chain of nearly cover people antibody encoding sequence is as follows:
The upstream primer of amplification kappa chain variable region gene is:
5’VK-1：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGACATCCAGWTGACCCA
5’VK-2：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGATGTTGTGATGACTCAG
5’VK-3：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGAAATTGTGWTGACRCA
5’VK-4：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAGATATTGTGATGACCCAG
The downstream primer of amplification kappa chain variable region gene is:
3’VK-1：
ACGTTTGAT
CTCGAGCTTGGTCCCYTGGCCRAA
3’VK-2：
ACGTTTGAT
CTCGAGTTTGGTCCCAGGGCCGAA
3’VK-3：
ACGTTTGAT
CTCGAGCTTGGTCCCTCCGCCGAA
The upstream primer of amplification lambda chain variable region gene is:
5’VL-1：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACAGTCTGTGYTGACKCAG
5’VL-2：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACARTCTGCCCTGACTCAG
5’VL-3：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCATCCTATGWGCTGACTCAG
5’VL-4：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCATCTTCTGAGCTGACTCAG
5’VL-5：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCACAGGCTGTGCTGACTCAG
5’VL-6：
GCTGGCTTAGTCTT
GGCCTTCTCGGCCAGCGCAAATTTTATGCTGACTCAGC
The downstream primer of amplification lambda chain variable region gene is:
3’VL-1：
CTTGGGCTGA
CCTAGGACGGTSACCTTGGTSCC
3’VL-2：
CTTGGGCTGA
CCTAGGACGGTCAGCTYSGTCCC
3’VL-3：
CTTGGGCTGA
CCTAGGATGATCAGCTGGGTTCC
The Auele Specific Primer of the described one group of whole variable region of heavy chain of nearly cover people antibody encoding sequence is as follows:
The upstream primer of amplification heavy chain variable region gene is:
5’VH-1：
TTCTATTCTCACAGTGCACAGRTGCAGCTGGTGCARTCTGG
5’VH-2：
TTCTATTCTCACAGTGCACAGSAGGTCCAGCTGGTRCAGTCTGG
5’VH-3：
TTCTATTCTCACAGTGCACAGRTCACCTTGAAGGAGTCTGG
5’VH-4：
TTCTATTCTCACAGTGCACAGSAGGTGCAGCTGGTGGAGTCTGG
5’VH-5：
TTCTATTCTCACAGTGCACAGGAGGTGCAGCTGGTGGAGWCYGG
5’VH-6：
TTCTATTCTCACAGTGCACAGGTGCAGCTACAGCAGTGGGG
5’VH-7：
TTCTATTCTCACAGTGCACAGSTGCAGCTGCAGGAGTCSGG
5’VH-8：
TTCTATTCTCACAGTGCACAGGARGTGCAGCTGGTGCAGTCTGG
The downstream primer of amplification heavy chain variable region gene is:
3’VH-1：
GGCCCTTGGTGCTAGCTGAGGAGACGGTGACCAGGGTKCC
3’VH-2：
GGCCCTTGGTGCTAGCTGAAGAGACGGTGACCATTGTCCC
3’VH-3：
GGCCCTTGGTGCTAGCTGAGGAGACGGTGACCGTGGTCCC；
(4) light chain kappa chain and lambda chain variable region gene are used respectively to restriction enzyme SfiI and XhoI, SfiI and AvrII double digestion, after purifying Vkappa and Vlambda gene respectively with phasmid carrier PFK-1, PFL-6 connects; After kappa is partly connected to product purification, be dissolved in 100uL sterilized water, electricity is evenly coated transformed bacteria in the 2YTAG flat board containing 100ug/mL penbritin after proceeding to competent cell TG1; The DNA that collects all cloned plasmids is kappa chain light chain antibody library; By identical method, obtain lambda chain light chain antibody library;
(5) heavy chain VH fragment gene is carried out to double digestion with ApaLI and NheI respectively, after purifying, be connected with phasmid carrier PFK-1; After heavy chain is connected to product purification, be dissolved in 100uL sterilized water, minutes four times electric shocks transform after TG1 competent cells by transformed bacteria evenly coating with containing in the 2YTAG flat board of 100ug/mL penbritin; The DNA that collects all cloned plasmids is heavy chain antibody storehouse;
(6) adopt ApaLI and NheI to carry out double digestion, heavy chain variable region gene is cut out from the DNA of heavy chain antibody storehouse, be connected with light chain lambda storehouse DNA and be connected with light chain kappa storehouse DNA respectively; After dividing the conversion TG1 competent cells that shock by electricity for four times, transformed bacteria is evenly coated with and contains in the 2YTAG flat board of 100ug/mL penbritin; Collect all clones' plasmid DNA, be antibody library DNA;
(7) carry out the diversity analysis of natural human source Fab phage antibody library.
2. the monoclonal antibody being screened by method described in claim 1.
3. the monoclonal antibody screening according to claim 2, it is anti-CD 6 monoclonal antibody, and the aminoacid sequence of the VH-CH1 of its Fab antibody is as shown in SEQ ID NO:1, and the aminoacid sequence of Vkappa-Ckappa is as shown in SEQ ID NO:3.
4. monoclonal antibody according to claim 3, it is characterized in that, the process of the biological screening of anti-CD 6 monoclonal antibody is: enrichment screening kappa chain phage antibody library → phage library ELISA → mono-clonal Phage-ELISA evaluation → PCR finger printing authentication → mono-clonal bacterium colony order-checking → Phage-ELISA result verification.
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