CN101210241A - Method for assembling bacteriophage gene engineering antibody library gene - Google Patents
Method for assembling bacteriophage gene engineering antibody library gene Download PDFInfo
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- CN101210241A CN101210241A CNA2006101052935A CN200610105293A CN101210241A CN 101210241 A CN101210241 A CN 101210241A CN A2006101052935 A CNA2006101052935 A CN A2006101052935A CN 200610105293 A CN200610105293 A CN 200610105293A CN 101210241 A CN101210241 A CN 101210241A
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- variable region
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Abstract
The invention relates to a gene assembly method of phage genetically engineered antibody library, belonging to the field of biomedical study and clinical applications. The method aims to overcome the problems of the prior art, including poor stability, low efficiency, high mutation probability and low fidelity, and indirect application to humanized single chain antibody study. The technical proposal adopted by the invention comprises the following steps of: obtaining heavy chain variable region gene and light chain variable region gene of a humanized antibody by RT-PCR, and ligating the antibody heavy chain variable region, a linker and the antibody light chain variable region with the pre-designed restriction enzyme cutting sites on two ends of primers using PCR and restriction enzyme method at the same time.
Description
Affiliated technical field:
The present invention relates to biomedical research and clinical application field, be specifically related to a kind of method of phage gene engineered antibody Ku Jiyin assembling.
Background technology:
Genetic engineering technique is developed fast in recent years, and series of genes engineering medicine has been widely used in clinical, and its range of application mainly concentrates on the genetically engineered drug and the monoclonal antibody of tumour, acquired immune deficiency syndrome (AIDS).At present, the biotech drug in the development of the whole world surpasses 2200 kinds, and wherein kind has entered clinical experimental stage surplus in the of 1700.The U.S. has more than 300 kind of bio-pharmaceutical to be in the clinical experiment stage in later stage.Calendar year 2001 FDA has ratified 8 kinds of biotech drugs and vaccine and 3 kinds of indications that medicine is new; FDA in 2002 have ratified the new indication of 20 true tumor technical agents and 15 biotech drugs altogether.FDA had ratified just to comprise in 35 new drugs 14 true tumor preparations in 2003.But the research and development at the single-chain antibody of fungi yet there are no report.Because that people source monoclonal antibody remains is unstable in preparation difficulty, hybridoma, yield poorly and avidity a little less than etc. problem, the monoclonal antibody overwhelming majority commonly used at present is a mouse source antibody, mouse source monoclonal antibody is reused at human body can produce human antimouse antibody, the existence of Fc section can make again its with body in have shortcomings such as the non-specific tissue of Fc acceptor and cell combine, therefore limited its application clinically.
Along with the genetic engineering antibody technology is increasingly mature, the genetically engineered small molecular antibody that utilizes molecule clone technology preparation provides new thinking for the immunodiagnosis and the immunotherapy of disease.Wherein, the small molecules single-chain antibody (single-chain Fv Fragment, scFv) because of its molecule is little, the solid tissue penetration power is strong, and plasma half-life is short, the affinity and the specificity of the antibody that can be kept perfectly again, and extremely pay attention to.The scFv structure is very simple, is formed by connecting by an elasticity linker by antibody heavy chain variable region and variable region of light chain, and be minimum antibody fragment with complete antigen binding site.Can in bacterium, express, be easy to genetic manipulation and genetically engineered mass production, and available gene engineering method makes up the antineoplastic amalgamation protein that is connected with other effector molecules.Therefore, scFv becomes the of great value molecule with medicine, toxin, radiotoxin guiding fungi, and very big clinical application potentiality are arranged in fungus therapy.
Usually phage antibody library technique is to be based upon phage surface to present on technology and the antibody dna recombinant technology, a complete set of variable region gene with pcr amplification antibody, particularly in the light chain of antibody variable region and being connected of heavy chain, adopt the PCR method of attachment of Amersham Bioscieences company design, realized the commercialization of test kit, but the test kit of Amersham Bioscieences company mainly designs at the mouse antibodies variable region, although having removed, scFv has higher antigenic antibody constant region position, but, therefore unavoidably still there is certain immunogenicity owing to there is the difference of kind.This has just limited its application in the research of humanized's single-chain antibody greatly.
The principle of classical phage gene engineered antibody Ku Jiyin assembling is: based on the mouse antibodies gene order, design corresponding primer, by round pcr is the platform amplification of finishing antibody variable gene and be connected, and finally obtains complete single-chain antibody gene library.Its process is divided into three phases: both by RT-PCR increase respectively light chain, heavy chain variable region gene; The PCR of light chain and variable region of heavy chain is connected; Utilize PCR to dose restriction enzyme site.
The method of classical phage gene engineered antibody Ku Jiyin assembling is: at first utilize obtain antibody variable gene with the mouse antibodies heavy chain that designs and variable region of light chain degenerate primer by RT-PCR after, obtain the scFv fragment of total length with SOE-PCR, adding restriction enzyme site by PCR at last, the PCR product is directly connected in the expression vector after enzyme is cut.
The shortcoming of classical phage gene engineered antibody Ku Jiyin assembling is: because the randomness of SOE-PCR method is bigger, in connection procedure except the fragment that 5 '-VH-Linker-VL (VK)-3 ' about 800bp occurs, the junction fragment that 5 '-VH-VL (VK)-3 ' also may occur therefore can disturb the segmental acquisition of purpose.Again because PCR itself also can be subjected to Taq enzymatic amplification efficient, the restriction of multiple conditions such as Mg2+ concentration, so the problem that exists of prior art be less stable, the efficient of experiment low, have bigger sudden change probability, influence the fidelity of gene.
Summary of the invention:
The present invention will provide a kind of method of phage gene engineered antibody Ku Jiyin assembling, to overcome the less stable that prior art exists, efficient is low, has bigger sudden change probability and Lo-Fi, and the problem that can not be directly used in the research of humanized's single-chain antibody.
For overcoming the problem that prior art exists, the technical solution adopted in the present invention is: a kind of method of phage gene engineered antibody Ku Jiyin assembling: be to utilize PCR and enzyme to cut simultaneously to be connected two kinds of methods, after utilizing RT-PCR to obtain humanized's heavy chain of antibody and chain variable region gene, the restriction enzyme site that designs in advance by the primer two ends is realized the splicing of antibody heavy chain variable region, Linker and antibody chain variable region.
In the such scheme:
Characteristics according to the human antibodies variable region have designed 65 ' end variable region of heavy chain primers and 73 ' end variable region of light chain primers, article 6,5 ' hold variable region of light chain primer and 13 3 ' end variable region of light chain primers, amount to 32 primers, design and synthesize the Linker (just having introduced XhoI and two restriction enzyme sites of ApaI respectively) of a pair of XhoI of including and two restriction enzyme sites of ApaI simultaneously at the light chain of antibody variable region and the connecting portion of heavy chain and Linker; The method that adopts enzyme to cut connection realizes the splicing of antibody heavy chain variable region, Linker and antibody chain variable region, and described each primer is as follows:
Variable region of heavy chain upstream primer: protect sequence-SfiI at random---antibody heavy chain variable region sequence
Variable region of heavy chain downstream primer: protect sequence-XhoI at random---antibody heavy chain variable region sequence
Variable region of light chain upstream primer: protect sequence-ApaI at random---antibody chain variable region sequence
Variable region of light chain downstream primer: protect sequence-NotI at random---antibody chain variable region sequence
Linker：XhoI——(G
4S)
3——ApaI
In experiment, can also utilize pGEM-11ZF (+) carrier that has SfiI, XhoI, ApaI and NotI restriction enzyme site simultaneously to preserve the scFv fragment that has been connected as cloning vector.The repetition that helps testing, the stability of assurance product.
Compared with prior art, advantage of the present invention is:
One, good stability, the sudden change probability is little, help improving the fidelity of gene: in the process that makes up the genome project antibody library, owing to should guarantee the fidelity of antibody variable gene, improve the joint efficiency of scFv again, so the stability requirement for experiment is very high, the present invention has improved experiment feasibility taking into account on the two the basis just, its raising to stability comprise following some:
1, two restriction enzyme sites of XhoI and ApaI seldom exist on the mankind's antibody variable gene, and these two restriction enzyme sites of design on the primer of the light chain of antibody variable region and heavy chain can not destroy the integrity of antibody variable gene.
2, at (the G of classics
4S)
3On the basis of Linker, after introducing XhoI and two restriction enzyme sites of ApaI, can add leucine, L-glutamic acid and glycine, proline(Pro) respectively in the both sides of Linker, although four amino acid whose addings increase the length of Linker to some extent, but because all there is not bigger side chain in this four seed amino acid, therefore there is not tangible influence in the snappiness for Linker, can not influence the stability of correct folding, the interaction of light, heavy chain and weight chain heterozygote and discern antigenic ability.
3, be to have used sfiI, SalI, XhoI, NotI restriction enzyme site ready-made on pGEM-11zf (+) cloning vector, avoided the segmental appearance of junction fragment of 5 '-VH-VL (VK)-3 ' in the connection procedure, and then guaranteed the reliability of experimental result to prevent the generation of invalid fragments.
4, in the method owing to only need a PCR reaction and 3 enzymes to cut ligation, therefore purpose antibody variable gene fragment has not only effectively increased, also strong avoidance repeatedly PCR react caused transgenation, experimental implementation that the application of business-like pGEM-11zf (+) cloning vector simultaneously is also easy has greatly guaranteed the repeatability of testing.
Two, applied widely: as to utilize current up-to-date genetic engineering technique, get bone-marrow-derived lymphocyte immune antibody variable region gene structure humanized single-chain antibody library in the blood samples of patients by angling, not only can be used for the single-chain antibody research of various external source infectious diseases, also can be used for the research and the treatment of diseases such as tumour.
Three, experimental cost is cheap, and economy is strong.Owing to utilized genetic engineering technique amplification people antibody variable gene, testing required primer, restriction endonuclease, carrier etc. can realize recycling, simultaneously needing in the Monoclonal Antibody also to avoid the situation of a large amount of laboratory animal, can reduce R﹠D costs greatly.
Embodiment:
To do detailed explanation to the present invention by embodiment below.
Method principle of the present invention: be to utilize PCR and enzyme to cut simultaneously to be connected two kinds of methods, after utilizing RT-PCR to obtain humanized's heavy chain of antibody and chain variable region gene, the restriction enzyme site that designs in advance by the primer two ends is realized the splicing of antibody heavy chain variable region, Linker and antibody chain variable region.
The present invention specifically is that the characteristics according to the human antibodies variable region have designed 65 ' end variable region of heavy chain primers and 73 ' end variable region of light chain primers, article 6,5 ' hold variable region of light chain primer and 13 3 ' end variable region of light chain primers, amount to 32 primers, design and synthesize the Linker (just having introduced XhoI and two restriction enzyme sites of ApaI respectively) of a pair of XhoI of including and two restriction enzyme sites of ApaI simultaneously at the light chain of antibody variable region and the connecting portion of heavy chain and Linker; The method that adopts enzyme to cut connection realizes the splicing of antibody heavy chain variable region, Linker and antibody chain variable region, and described each primer is as follows:
Variable region of heavy chain upstream primer: protect sequence-SfiI at random---antibody heavy chain variable region sequence
Variable region of heavy chain downstream primer: protect sequence-XhoI at random---antibody heavy chain variable region sequence
Variable region of light chain upstream primer: protect sequence-ApaI at random---antibody chain variable region sequence
Variable region of light chain downstream primer: protect sequence-NotI at random---antibody chain variable region sequence
Linker：XhoI——(G
4S)
3——ApaI
In experiment of the present invention, can also utilize pGEM-11ZF (+) carrier that has SfiI, XhoI, ApaI and NotI restriction enzyme site simultaneously to preserve the scFv fragment that has been connected as cloning vector.The repetition that helps testing, the stability of assurance product.
Concrete operation steps is:
1, design of primers
Design of primers is with reference to the Daniele method
[3](slightly changing) design heavy chain V
HThe section primer, light chain V
LAnd V
KThe section primer.Heavy chain section primer 5 ' end is introduced the SfiI restriction enzyme site, and 3 ' end is introduced the XhoI restriction enzyme site.Light chain section primer 5 ' end is introduced the ApaI restriction enzyme site, and 3 ' end is introduced the NotI restriction enzyme site.The protection base of light chain and heavy chain is 12, and sequence is identical.Link chain 5 ' end is introduced the XhoI restriction enzyme site, and 3 ' end is introduced the ApaI restriction enzyme site.
2, the total RNA of whole blood extracts
Get moniliosis reconvalescent peripheral blood 45 examples, the lymphocyte separation medium isolated lymphocytes is extracted the total RNA of lymphocyte with test kit.RNA sex change electrophoresis is identified the integrity of RNA, nucleic acid-protein analysis-e/or determining content.Liquid nitrogen is preserved standby.
3, RT-PCR amplification V
H, V
LAnd V
KChain
Pressing the explanation of reverse transcription test kit, is template with total RNA, and cDNA first chain is synthesized in reverse transcription, is template with first chain, pcr amplification V
H, V
LAnd V
KChain.
PCR reaction: with synthetic cDNA first chain of reverse transcription is the template V that increases respectively
H, V
LAnd V
KGene is with V
H, V
LAnd V
K3 ' end primer of gene mixes the back respectively and increases respectively with 5 ' section each primer.Reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, and 35 circulations are extended 8min for back 72 ℃.Reaction system: in the reaction system of 20 μ l, contain cDNA template 1 μ l, 10 * PCR buffer, 0.2 μ l, dNTP 0.2mmol/L, Mg
2+2.5mmol/L, each 10pmol/L of upstream and downstream primer, Taq enzyme 1.0U.
2% agarose gel electrophoresis detects the PCR product, and DL2000 Marker compares.Observing the 370bp place has band to occur, and cuts glue and reclaim.Reclaim product cloning in the T carrier, and order-checking is identified.
4, heavy chain, Linker chain are connected with light chain
Heavy chain gene and light chain gene are got off with SfiI, XhoI and ApaI, NotI double digestion from the T carrier respectively, after electrophoresis reclaims, be connected step by step with carrier respectively, transform with the Linker chain.
4.1 heavy chain gene is connected into pGEM-11zf (+) carrier: from T carrier SfiI, the XhoI double digestion gets off with heavy chain gene, is connected in the pegm-11zf carrier through cutting with same enzyme, and transformed into escherichia coli extracts plasmid and identifies correct through double digestion.
4.2 light chain gene is connected into: with light chain gene from the T carrier with behind ApaI and the NotI double digestion, be connected in pGEM-11ZF (+) carrier through cutting with same enzyme, transformed into escherichia coli extracts plasmid and identifies correctly through double digestion.
4.3Linker the access of chain: the Linker chain forms ApaI and the XhoI site of its two ends for having cut by two oligonucleotide annealing of synthetic.Synthetic Linker chain connects in the plasmid of the second step gained, obtains complete antibody gene.
Preserve the clone that all connect by cloning vector, can lay the foundation for the research in later stage.
For being illustrated more clearly in the difference that the present invention and classical phage gene engineered antibody storehouse make up, please referring to following table:
| Classical way | Present method |
| Have 3 groups of primers to constitute, comprise antibody variable region amplification degenerate primer, scFv connects primer and adds the restriction enzyme site primer. | Be made of one group of primer, primer can be finished the variable region amplification simultaneously and add the effect of restriction enzyme site. |
| Hold on the primer at 5 ' of 3 ' end of amplification antibody heavy chain variable region and antibody chain variable region and not introduce restriction enzyme site. | Hold on the primer at 5 ' of 3 ' end of amplification antibody heavy chain variable region and antibody chain variable region and to introduce ApaI and XhoI restriction enzyme site respectively. |
| Utilize the method acquisition scFv connection product whole process of PCR to need 3 PCR. | The method of utilizing enzyme to cut connection obtains scFv and connects product, and whole process needs 3 enzymes to cut ligation. |
| In whole experiment, only need a kind of carrier | In experiment, except that the needs expression vector, also need to utilize pGEM-11ZF (+) carrier. |
| Design at the mouse antibodies variable region gene | Design at people's antibody variable gene |
Claims (3)
1. the method for phage gene engineered antibody Ku Jiyin assembling: be to utilize PCR and enzyme to cut simultaneously to be connected two kinds of methods, after utilizing RT-PCR to obtain humanized's heavy chain of antibody and chain variable region gene, the restriction enzyme site that designs in advance by the primer two ends is realized the splicing of antibody heavy chain variable region, Linker and antibody chain variable region.
2. the method for a kind of phage gene engineered antibody Ku Jiyin assembling as claimed in claim 1, it is characterized in that: be that characteristics according to the human antibodies variable region have designed 65 ' end variable region of heavy chain primers and 73 ' end variable region of light chain primers, article 6,5 ' hold variable region of light chain primer and 13 3 ' end variable region of light chain primers, amount to 32 primers, design and synthesize the Linker (just having introduced XhoI and two restriction enzyme sites of ApaI respectively) of a pair of XhoI of including and two restriction enzyme sites of ApaI simultaneously at the light chain of antibody variable region and the connecting portion of heavy chain and Linker; The method that adopts enzyme to cut connection realizes the splicing of antibody heavy chain variable region, Linker and antibody chain variable region, and described each primer is as follows:
Variable region of heavy chain upstream primer: protect sequence-SfiI at random---antibody heavy chain variable region sequence
Variable region of heavy chain downstream primer: protect sequence-XhoI at random---antibody heavy chain variable region sequence
Variable region of light chain upstream primer: protect sequence-ApaI at random---antibody chain variable region sequence
Variable region of light chain downstream primer: protect sequence-NotI at random---antibody chain variable region sequence
Linker： XhoI——(G
4S)
3——ApaI
3. the method for a kind of phage gene engineered antibody Ku Jiyin assembling as claimed in claim 1 or 2 is characterized in that: utilize pGEM-11ZF (+) carrier that has SfiI, XhoI, ApaI and NotI restriction enzyme site simultaneously to preserve the scFv fragment that has been connected as cloning vector.
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| CNA2006101052935A CN101210241A (en) | 2006-12-27 | 2006-12-27 | Method for assembling bacteriophage gene engineering antibody library gene |
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| CNA2006101052935A CN101210241A (en) | 2006-12-27 | 2006-12-27 | Method for assembling bacteriophage gene engineering antibody library gene |
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| CN103361742A (en) * | 2013-07-08 | 2013-10-23 | 中国人民解放军第三军医大学第一附属医院 | Human agkistrodon acutus venom immune ScFv antibody library and construction method thereof |
| CN103740684A (en) * | 2014-02-10 | 2014-04-23 | 赛业（广州）生物科技有限公司 | Site-specific mutagenesis Lambda phage Int recombinant protein and preparation method thereof |
| CN103740684B (en) * | 2014-02-10 | 2015-11-25 | 赛业（广州）生物科技有限公司 | Lambda phage Int recombinant protein of a kind of rite-directed mutagenesis and preparation method thereof |
| CN104017080A (en) * | 2014-06-13 | 2014-09-03 | 华南农业大学 | Canine single-chain antibody, and construction method and application thereof |
| CN105219780A (en) * | 2015-10-30 | 2016-01-06 | 中国人民解放军第三军医大学第一附属医院 | Anti-many subgenotype HCV antibody gene R3-19 and application thereof |
| CN105177017A (en) * | 2015-10-30 | 2015-12-23 | 中国人民解放军第三军医大学第一附属医院 | Antibody gene R4-85 resisting kinds of sub-gene type HCV and application thereof |
| CN105177017B (en) * | 2015-10-30 | 2020-06-02 | 中国人民解放军第三军医大学第一附属医院 | Anti-multi-subgenomic HCV antibody gene R4-85 and application thereof |
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