CN104673822A - Recombinant vector and its preparation method and use - Google Patents
Recombinant vector and its preparation method and use Download PDFInfo
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- CN104673822A CN104673822A CN201310612966.6A CN201310612966A CN104673822A CN 104673822 A CN104673822 A CN 104673822A CN 201310612966 A CN201310612966 A CN 201310612966A CN 104673822 A CN104673822 A CN 104673822A
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Abstract
The invention provides a recombinant vector and its preparation method and use. The recombinant vector is constructed from a yeast secretory expression vector pPICZaA, contains a human-derived CTLA4 extracellular region gene for expression of a human-derived CTLA4 fusion protein, also contains a Kex2 protease cleavage site, His label and c-myc label, can release the expressed CTLA4 fusion protein in a secretory protein form out of cells, realizes purification of the protein, and provides a simple method for CTLA4 protein industrialization.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of recombinant vectors and its preparation method and application.
Background technology
Cytotoxic t lymphocyte-associated antigen 4 (CTLA4) is the acceptor of T lymphocytic cell surface, it is the costimulatory molecules of suppressor T cell activation, after the B7 molecule on T cell surface receptor CTLA4 and APC combines, the activation of meeting suppressor T cell, thus the overactivity of suppressor T cell.The disease that a lot of excessive immune reaction causes all has extremely close relation with the low expression of CTLA4 albumen, as the disease such as immunological rejection, chronic bronchial asthma, rheumatic arthritis (RA), acquired immune deficiency syndrome (AIDS), systemic lupus erythematous, psoriatic, endotoxin shock disease that allotransplantation causes, therefore CTLA4 albumen or fusion rotein have very wide application prospect in the specific treatment of these diseases.In addition, the overexpression of CTLA4 albumen in body and a lot of disease particularly tumour have close relationship, as melanoma, leukemia, part cancer of the stomach and esophagus cancer, leishmaniasis etc.The monoclonal antibody CTLA4mAb of CTLA4 can be widely used in antineoplastic immunotherapy, is study hotspot and the direction of current therapy of tumor.Acquisition has biological function, is the key of CTLA4 specific treatment disease close to the recombinant protein of the natural CTLA4 albumen of the mankind.
Therefore, be necessary to provide a kind of expression vector at eukaryotic expression CTLA4 recombinant protein.
Summary of the invention
For solving the problem, first aspect present invention provides a kind of recombinant vectors, and described recombinant vectors is the recombinant vectors of yeast secreted expression carrier pPICZaA, and this recombinant vectors contains people source CTLA4 Extracellular domain, can be used for expressing people source CTLA4 fusion rotein.This recombinant vectors is also containing Kex2 protease cleavage site, His label and c-myc label, the CTLA4 fusion rotein of expressing can be made to be discharged into outside born of the same parents with the form of secretory protein, and purifying is carried out to albumen, the suitability for industrialized production for CTLA4 albumen provides a kind of easy method.Second aspect present invention provides a kind of recombinant bacterium, and this recombinant bacterium comprises the recombinant vectors described in first aspect.Third aspect present invention provides a kind of preparation method of recombinant vectors.Fourth aspect present invention provides a kind of gene, and described gene is the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1.Fifth aspect present invention provides a kind of application of recombinant vectors.
First aspect, the invention provides a kind of recombinant vectors, comprises the gene as shown in SEQ ID NO:1, and described recombinant vectors is restructuring pPICZaA plasmid, and described gene inserts the multiple clone site of described pPICZaA plasmid.
The pPICZaA plasmid that the present invention adopts contains His label (histidine-tagged) and c-myc label, these two kinds of labels on the protein band after expression can be made, His label is conducive to the separation and purification expressing rear fusion protein, c-myc label is conducive to fusion rotein analysis in an experiment and tracking, such as analysis during immunoblot experiment.
Preferably, gene as described in relation to the first aspect inserts Xho I and the Xba I site of pPICZaA plasmid.
Second aspect, the invention provides a kind of recombinant bacterium, comprises recombinant vectors as described in relation to the first aspect, and described recombinant bacterium is restructuring Pichia pastoris GS115 bacterial strain.
Nucleotide sequence as shown in SEQ ID NO:1 provided by the invention comprises the encoding sequence of Kex2 protease cleavage site and encoding sequence (the Gene Bank clone number: NM-005214.3) of CTLA4 extracellular region.
Kex2 proteolytic enzyme is arranged in yeast cell film, it is the nickase of α-factor signal peptide, it can effectively identify restriction enzyme site Lys-Arg, obtained the CTLA4 albumen of natural N end (upstream due to Kex2 restriction enzyme site provided by the invention is Xho I site of plasmid by the cutting to the signal peptide before restriction enzyme site, next-door neighbour CTLA4 protein extracellular, downstream sequence, therefore, natural N can be obtained after cutting and hold CTLA4 protein extracellular; This natural N end refers to that the N end in CTLA4 protein extracellular does not contain other any unnecessary series).
After nucleotides sequence as shown in SEQ ID NO:1 provided by the invention is listed in and expresses in yeast, the CTLA4 extracellular region fusion rotein containing Kex2 protease cleavage site can be obtained, this Kex2 protease cleavage site cuts through the Kex2 proteolytic enzyme of yeast cell film, impels CTLA4 extracellular region fusion rotein to be secreted into outside born of the same parents.
The third aspect, the invention provides a kind of preparation method of the recombinant vectors as described in second aspect, comprises the steps:
(1), the upstream primer of design CTLA4 gene extracellular domain fragment and downstream primer, the base sequence of described upstream primer and downstream primer is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3;
(2), provide or prepare CTLA4 gene template, and with step (1) gained upstream primer and downstream primer for PCR primer, amplification CTLA4 gene extracellular domain fragment;
(3), get pPICZaA plasmid, step (2) increased the CTLA4 gene extracellular domain fragment obtained and described pPICZaA plasmid utilize identical restriction endonuclease to carry out double digestion reaction respectively, and purifying connects after reclaiming, and obtains described recombinant vectors.
Preferably, in described step (2), described CTLA4 gene template is the recombinant vectors pGEM-T-CTLA4 containing CTLA4 gene.
It is NM-005214.3 that the pGEM-T-CTLA4 plasmid that the present invention adopts contains human CTLA 4 full length gene ORF(Gene Bank clone number)
Preferably, in described step (3), it is Xho I restriction endonuclease and Xba I restriction endonuclease that described double digestion reacts the restriction endonuclease adopted.
Described upstream primer as shown in SEQ ID NO:2 is specially:
5’-CCGCTCGAGAAAAGAAAAGCAATGCACGTGGCC-3’
Described downstream primer as shown in SEQ ID NO:3 is specially:
5’-GCTCTAGAGCGTCAGAATCTGGGCACGGTT-3’
This upstream primer (CTLA4up) and downstream primer (CTLA4down) composed as follows:
Fourth aspect, the invention provides a kind of gene, and described gene is the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1.
5th aspect, the invention provides the application of preparation method in preparation immunity or tumor disease medicine of the recombinant vectors as described in second aspect or the recombinant vectors as described in fourth aspect.
The recombinant vectors that the present invention builds can be used for expressing CTLA4 protein extracellular, people source, coded by this CTLA4 protein extracellular, albumen behaviour source full-length gene order, the pPICZaA-CTLA4 recombinant vectors constructed by the present invention and yeast strain GS115/pPICZaA-CTLA4 can be widely used in the fields such as bio-pharmaceuticals, antibody drug, protein production.
Recombinant vectors that the invention provides and its preparation method and application has following beneficial effect:
(1) recombinant vectors provided by the invention adopts pPICZaA plasmid, by the encoding gene of Kex2 protease cleavage site and gene constructed in this expression vector containing human CTLA 4 extracellular region, can be used for the human CTLA 4 fusion rotein of expression-secretion type.
(2) secondly, described recombinant vectors is His label and c-myc label also, contributes to carrying out purifying and analysis to albumen;
(3) in addition, the preparation method of recombinant vectors provided by the invention is easy, is easy to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is pPICZaA-CTLA4 construction of recombinant vector schema provided by the invention;
Fig. 2 is the agarose gel electrophoresis figure of the Kex2-CTLA4 gene that the embodiment of the present invention obtains;
Fig. 3 is the plasmid map of the recombinant vectors that the embodiment of the present invention obtains.
Embodiment
The following stated is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Plasmid vector pGEM-T-CTLA4, pPICZaA plasmid that the embodiment of the present invention uses and the yeast strain GS115 used are commercial goods, and the reagent used is commercial goods.
PPICZaA-CTLA4 construction of recombinant vector schema shown in composition graphs 1, embodiments provides a kind of preparation method of recombinant vectors, comprises the following steps:
(1) clone of Kex2-CTLA4 fusion gene
A) provide upstream primer and downstream primer, wherein, the base sequence of described upstream primer is as shown in SEQ ID NO:2, and the base sequence of described downstream primer is as shown in SEQ ID NO:3;
B) provide pGEM-T-CTLA4 plasmid as DNA profiling, adopt pcr amplification CTLA4 Extracellular domain sequence, configuration amplification system is as follows, the upstream primer described in primer employing step (1) in system and downstream primer:
PCR reaction system
Pcr amplification program is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 40s, and after 30 circulations, 72 DEG C extend 10min.Get 5 μ L reaction product 1.5% agarose gel electrophoresis to identify, after qualification is correct, glue reclaims PCR primer.
(2) containing the structure of the recombinant vectors pPICZaA-CTLA4 of Kex2-CTLA4 fusion gene
1. contain the double digestion of Kex2-CTLA4 fusion gene and pPICZaA plasmid;
Get pPICZaA plasmid, by step (1) the gene fragment obtained that increases utilize identical enzyme to carry out endonuclease reaction respectively with pPICZaA plasmid, it is respectively as follows that enzyme cuts system:
The connection of 2.Kex2-CTLA4 fusion gene and pPICZaA;
After step 1 gained double digestion product is carried out 1% agarose gel electrophoresis, reclaim object fragment, connected by Kex2-CTLA4 fusion gene with pPICZaA, 22 DEG C connect 5h, and ligation system is as follows:
3. enzyme is cut and checks order qualification
To connect product conversion DH5 α competent cell, and coat the LB solid plate substratum containing Amp, 37 DEG C of overnight incubation, picking list bacterium colony extracts plasmid enzyme cuts qualification after cultivating, and it is as follows that enzyme cuts system:
37 DEG C of enzymes cut through night, 1% agarose gel electrophoresis is identified, enzyme cut after agarose gel electrophoresis figure as shown in Figure 1, in fig. 2, swimming lane M is DNA molecular amount marker(1Kb Plus DNA Ladder, invitrogen), swimming lane 1 is that the recombinant vectors pPICZaA-CTLA4 built carries out double digestion (Xho I and Xba I) result, wherein, swimming lane 1 has obvious band at about 400bp, this stripe size is consistent with the size (about 400bp) of Kex2-CTLA4 fusion gene, shows that Kex2-CTLA4 fusion gene is successfully building up on carrier;
The positive colony of qualification is checked order, after nucleotide fragments and the CTLA4 fragment comparison of NCBI people source will be inserted, on all four for base sequence carrier is preserved, recombinant vectors pPICZaA-CTLA4(3898bp provided by the invention) plasmid map as shown in Figure 3.
Claims (8)
1. a recombinant vectors, is characterized in that, comprises the gene as shown in SEQ ID NO:1, and described recombinant vectors is restructuring pPICZaA plasmid, and described gene inserts the multiple clone site of described pPICZaA plasmid.
2. a recombinant bacterium, is characterized in that, comprises recombinant vectors described in claim 1.
3. recombinant bacterium according to claim 2, is characterized in that, described recombinant bacterium is restructuring Pichia pastoris GS115 bacterial strain.
4. a preparation method for recombinant vectors as claimed in claim 1, is characterized in that, comprises the steps:
(1), the upstream primer of design CTLA4 gene extracellular domain fragment and downstream primer, the base sequence of described upstream primer and downstream primer is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3;
(2), provide or prepare CTLA4 gene template, and with step (1) gained upstream primer and downstream primer for PCR primer, amplification CTLA4 gene extracellular domain fragment;
(3), get pPICZaA plasmid, step (2) increased the CTLA4 gene extracellular domain fragment obtained and described pPICZaA plasmid utilize identical restriction endonuclease to carry out double digestion reaction respectively, and purifying connects after reclaiming, and obtains described recombinant vectors.
5. the preparation method of a kind of recombinant vectors as claimed in claim 4, is characterized in that, in described step (2), described CTLA4 gene template is the recombinant vectors pGEM-T-CTLA4 containing CTLA4 gene.
6. the preparation method of a kind of recombinant vectors as claimed in claim 4, is characterized in that, in described step (3), it is Xho I restriction endonuclease and Xba I restriction endonuclease that described double digestion reacts the restriction endonuclease adopted.
7. a gene, is characterized in that, described gene is the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1.
8. the application of recombinant vectors as claimed in claim 1 in preparation immunity or tumor disease medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108893487A (en) * | 2018-07-19 | 2018-11-27 | 中国农业科学院北京畜牧兽医研究所 | A kind of construction method of plant expression plasmid carrier containing C-Myc protein fusion label and its carrier |
| CN113234760A (en) * | 2021-06-04 | 2021-08-10 | 长沙爱科博生物科技有限公司 | Recombinant adenovirus 5 vector containing porcine reproductive and respiratory syndrome ORF5 gene and preparation method and application thereof |
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| CN113234760A (en) * | 2021-06-04 | 2021-08-10 | 长沙爱科博生物科技有限公司 | Recombinant adenovirus 5 vector containing porcine reproductive and respiratory syndrome ORF5 gene and preparation method and application thereof |
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Application publication date: 20150603 |