Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein

A fusion protein and cell line technology, applied in chemical instruments and methods, biochemical equipment and methods, fusion polypeptides, etc., can solve the problems of short antibody production time, immune death, poor immune effect, etc.

Active Publication Date: 2017-03-22
TANGSHAN YIAN BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is how to improve the poor immune effect of classical swine fever E2 protein in the Escherichia coli expression system; or the short production time of the antibody produced by the expression product of other expression systems and the defects of immune death caused by the residual prokaryotic endotoxin

Method used

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  • Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein
  • Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein
  • Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the acquisition of PigFC-pigSCFVE2 mutant and its coding gene

[0067] In order to improve the long-term effect of wild-type E2, the protein sequence of classical swine fever E2 was fused with porcine FC antibody fragments. The specific protein sites of specific fusion proteins are shown in Table 1.

[0068] Table 2 Fusion protein design

[0069] name PigFC-pigSCFVE2 porcine FC fragment 22-242 link peptide 243-258 E2 259-631 E2 epitope optional

[0070] Synthesis of the PigFC-pigSCFVE2 gene shown in SEQ ID No.1 is the fusion of the N-terminus of the E2 protein with the porcine FC fragment. The indicated protein PigFC-pigSCFVE2. E2 mutants can also be obtained by fusing the E2 immune epitope to the C-terminus of the E2 protein and keeping the amino acid residues of E2 unchanged.

Embodiment 2

[0071] Embodiment 2, the preparation of E2 expression antigen

[0072] 1. Construction of recombinant PigFC-pigSCFVE2 recombinant expression vector

[0073] Use NheI and XhoI to double digest the PMD18-PigFC-pigSCFVE2 carrier to obtain the DNA synthesis DNA nucleic acid molecule shown in SEQ ID No.1. The fragment of about 2000bp is the target fragment, which contains the DNA fragment of the coding gene of PigFC-pigSCFVE2 or the mutation of the coding gene DNA fragments were recovered and purified. The result is as figure 1 .

[0074] The purified DNA fragment containing the coding gene of PigFC-pigSCFVE2 was digested with NheI and XhoI double-digestion eukaryotic expression vector pcCND3.1(-) respectively, the effect is as follows figure 2 , the synthetic gene was ligated to the vector segment (the ligation system was 0.5 μl pcCND3.1(-); 4.5 μl PigFC-pigSCFVE2 digested fragment, 2xT4 quick ligase, room temperature for 3 hours). The ligation product was transformed into D...

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Abstract

The invention relates to a vaccine production technology in the technical field of biology, in particular to a CHO cell strain which is established by utilizing a gene engineering means and is used for expressing recombinant protein PigFC-pigSCFVE2, and a preparation method and application of the recombinant protein. The recombinant fusion protein PigFC-pigSCFVE2 provided by the invention is A1) or A2) shown as follows, wherein A1) is protein of which the amino acid sequence is as shown in SEQ ID No.2, and A2) is protein which is obtained by substituting, losing and/or adding one or several amino acid residues in the amino acid sequence of the protein of the A1) and has PigFC-pigSCFVE2 activity. A monoclonal cell strain which is obtained through the method and capable of carrying out secretory expression on PigFC-pigSCFVE2 is higher in fusion protein expression quantity, fusion protein obtained through affinity separation and purification of an antibody can be combined with a monoclonal antibody, animals can be immunized, the immunity of a generated neutralizing antibody is higher than that of a present market product, the fusion protein can be used for swine classical fever preventive vaccine, and the production cost and the immunity failure loss can be reduced.

Description

technical field [0001] The invention relates to vaccine production technology in the field of biotechnology, in particular to a CHO cell strain expressing recombinant protein PigFC-pigSCFVE2 constructed by means of genetic engineering, and a preparation method and application thereof. Background technique [0002] Classical swine fever, commonly known as "rotten intestinal fever", is an acute, febrile, contagious infectious disease caused by the swine fever virus of the family Flaviviridae, which is highly contagious and fatal. Pigs are the only natural host of the virus. The infectious disease was first discovered in Ohio, USA in 1833, and its prevalence has spread all over the world for more than a hundred years. The International Office of Epizootics defines it as a Class A infectious disease, and China's "Animal Epidemic Prevention Law" lists it as a Class I infectious disease. It is currently one of the main diseases that endanger the development of China's pig industr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85G01N33/569A61K39/187A61K47/68A61P31/14
CPCA61K39/12A61K2039/552C07K14/005C07K2319/30C12N15/85C12N2770/24322C12N2770/24334C12N2800/107G01N33/56983G01N2333/183
Inventor 李润马广鹏孙爱娟李琳史明冯丽芳杨丽聪陈蕾程水生
Owner TANGSHAN YIAN BIOLOGICAL ENG CO LTD
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