Fusion protein of SVV and FMDV, encoding gene of fusion protein, expression vector, cell line, engineering bacteria, vaccine and application

A technology of fusion protein and coding gene, which is applied in the field of biomedicine, can solve problems such as indistinguishability, difficulty in diagnosis and treatment, and achieve the effect of improving safety and increasing antibody titer

Active Publication Date: 2019-10-11
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The clinical symptoms of swine vesicular disease caused by SVV are similar to those of vesicular diseases such as foot-and-mouth disease (FMD), and it is difficult to distinguish them
The diagnosis and treatment of the above two diseases have certain difficulties, and there is no good technical means to avoid infection of the two diseases at the same time. Therefore, combining the characteristics of the pathogens of the two diseases, the recombinant subunit vaccine of SVV and FMD is designed to deal with the disease Prevention and control are crucial

Method used

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  • Fusion protein of SVV and FMDV, encoding gene of fusion protein, expression vector, cell line, engineering bacteria, vaccine and application
  • Fusion protein of SVV and FMDV, encoding gene of fusion protein, expression vector, cell line, engineering bacteria, vaccine and application
  • Fusion protein of SVV and FMDV, encoding gene of fusion protein, expression vector, cell line, engineering bacteria, vaccine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1 expresses fusion protein by CHO cell expression system

[0092] 1 Materials and methods

[0093] 1.1 Cell culture

[0094] The CHO cell line and Geneticin (50 mg / ml) were purchased from Invitrogen Company of the United States, and the culture temperature was 37° C., and the culture medium (Hycell CHO medium) was purchased from Hyclone Company of the United States. CHO expression system and related reagents were purchased from Invitrogen, USA.

[0095] Cell density and viability were counted and observed by trypan blue staining. Cell viability was calculated as the percentage of viable cells in total cells at different times after infection. Cell suspension culture, cell density 5×10 6 / ml, and the cell viability was greater than 95%.

[0096] 1.2 VP1 gene synthesis and construction of recombinant positive plasmid

[0097] According to the whole genome of SVV and FMDV, the VP1 gene sequence (GenBank accession number MF615510.1 and NC_001623) and C3d se...

Embodiment 2

[0120] Example 2 Transformation of Mammalian CHO Cells Improves the Production of Recombinant Antigens

[0121] 1 method

[0122] The construction of the recombinant plasmid with the highest expression level is shown in Example 1.

[0123] 1.1.1 Plasmid preparation: extract the recombinantly constructed plasmid using an extraction kit (according to the kit instructions), add 75% ethanol to the obtained plasmid, and perform aseptic treatment to ensure that the plasmid transfected into the cell is sterile. The detector measures the plasmid concentration.

[0124] 1.1.2 Lipofectamine transfection: 5×10 6 CHO cells / ml, 1μg / μl final concentration of plasmid; transfection conditions and culture: prepare solution A, gently add plasmid DNA to 900μl OptiPRO SFM for dilution, and let it stand for 5-10min; prepare solution B, add 80μl liposome reagent to 920μl Dilute with OptiPRO SFM, place for 5-10min, add solution B to solution A, gently invert to mix, leave for 3-5min, add cells to...

example 3

[0137] The preparation of example 3 SVV-FMDV dual vaccine

[0138] Dilute the harvested SVV-FMDV fusion protein with PBS solution to different concentrations, mix the diluted fusion protein solution with SEPPIC ISA206 adjuvant according to the ratio of antigen component and adjuvant 1:1, and finally prepare 0.5 μg Different antigen gradient vaccines per head, 5μg / head, 10μg / head, 30μg / head. Stir for 8-10min at a rotating speed of 8000r / min, add 0.01% (volume ratio) thimerosal solution before terminating the stirring, after fully oscillating and mixing, perform sterility testing, viscosity determination, After the stability test is qualified, it is placed at 4°C for later use.

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Abstract

The invention relates to the technical field of biomedicine, and particularly provides a fusion protein of an SVV and an FMDV, an encoding gene of the fusion protein, an expression vector, a cell line, engineering bacteria, a vaccine and application. The fusion protein is obtained by replacing an epitope capable of inducing the body to produce a neutralizing antibody with a decoy epitope and comprises SVV VP1 protein fragments, FMDV VP1 protein fragments and complement C3d protein fragments. The fusion protein combines antigens for inducing the body to produce the neutralizing antibody, of twopathogens of SVV and FMDV, and the safety of the antigens is improved while the antigenic epitopes of the SVV and the FMDV are reserved. The complement C3d molecules can excite nonspecific body fluidand cellular immune reactions in the body, and play an important role in increasing the antibody titer of the vaccine, and activating the cellular immune response of the body. The vaccine prepared bymeans of the fusion protein is safe and effective, and hydroa and foot-and-mouth diseases can be effectively prevented.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fusion protein of SVV and FMDV and its coding gene, expression vector, cell line, engineering bacteria, vaccine and application. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV). Mainly against artiodactyls, occasionally in humans and other animals. It is clinically characterized by blistering of the oral mucosa, hooves, and breast skin. The disease has many transmission routes and fast speed, and has repeatedly broken out in the world, causing huge political and economic losses. In view of this, the World Organization for Animal Health (OIE) ranks it as the first class A infectious disease. At present, FMD is prevalent in two-thirds of OIE member countries, threatening the safety of livestock and the trade of livestock products in n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N15/70C12N5/10C12N1/21A61K39/295A61K39/125A61K39/135A61P31/14C12R1/19
CPCA61K39/12A61K2039/552A61K2039/70A61P31/14C07K14/005C12N2770/32022C12N2770/32034C12N2770/32122C12N2770/32134
Inventor 贺笋程兰玲张伟李延涛李俊辉
Owner 天康生物制药有限公司
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