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Bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and identification method thereof

A technology for bovine viral diarrhea and rhinotracheitis virus, which can be applied to vaccines, viruses, and viral peptides, and can solve problems such as difficult differential diagnosis, high content of foreign proteins, and large side effects

Active Publication Date: 2020-12-25
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are dual inactivated vaccines for bovine viral diarrhea and bovine infectious rhinotracheitis in China, which may control the epidemic to a certain extent, but because they are inactivated vaccines, they have high protein content and large side effects. High difficulty

Method used

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  • Bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and identification method thereof
  • Bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and identification method thereof
  • Bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The target genes of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 were synthesized by the biological company, and the primer sequences of SEQ ID NO.5-SEQ ID NO.12 were amplified respectively. Add the above target gene. The result is as figure 1 Shown (where, M: marker, 1: BVDV-E2, 2: BVDV-NS3, 3: IBRV-VP8, 4: control, 5: IBRV-gB).

Embodiment 2

[0083] Connect and Transform

[0084] 1. Plasmid pET-30a (+), BVDV-NS3 gene (SEQ ID NO.1), BVDV-E2 gene (SEQ ID NO.3), IBRV-VP8 gene (SEQ ID NO.2), IBRV-gB gene (SEQ ID NO.4) were subjected to KpnI and BamHI double enzyme digestion respectively;

[0085] 2. Perform 1% agarose gel electrophoresis on the digested product, recover the target band from the gel and purify it;

[0086] 3. T4-DNA ligase connects the plasmid and each gene respectively;

[0087] 4. Kanamycin (100ml / L) solid medium to screen BVDV-NS3-pET-30a (+), BVDV-E2-pET-30a (+), IBRV-VP8-pET-30a (+) and IBRV- gB-pET-30a(+) was transformed into Escherichia coli BL21 positive strain;

[0088] 5. Identification of recombinant prokaryotic expression vector by double enzyme digestion.

[0089] Prokaryotic expression and purification

[0090] 1. Pick a single colony of Escherichia coli BL21 containing the expression vector in LB solid medium (containing 100mg / L kanamycin) and culture it overnight in 20mL of LB liqui...

Embodiment 3

[0098] immune experiment

[0099] 1. Take 300 μg of purified prokaryotically expressed BVDV-NS3 and 500 μg of IBRV-VP8 protein, mix and emulsify with adjuvant ISA206VG, ​​and prepare 10 vaccines for cattle.

[0100] 2. Randomly select 20 cows over 3 months old from cattle farms negative for bovine viral diarrhea and bovine infectious rhinotracheitis, and randomly divide them into an experimental group and a control group, with 10 cattle in each group. The experimental group was inoculated intramuscularly, with 1ml of each head containing 30 μg of BVDV-NS3 and 50 μg of IBRV-VP8 protein; the control group was inoculated with normal saline, and each head was inoculated with 1ml.

[0101] 3. Continue to observe for 14 days after immunization, and monitor body temperature changes, appetite, and mental changes. The results found that compared with the control group, the body temperature of the cattle in the experimental group fluctuated 24 hours after vaccination, with a fluctuatio...

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Abstract

The invention relates to the technical field of biology, and particularly provides a bovine viral diarrhea-infectious bovine rhinotracheitis bivalent subunit fusion vaccine and an identification method thereof. The inventor optimizes genes of BVDV-NS3 and IBRV-VP8, and performs fusion expression on antigen epitope proteins of the BVDV-NS3 and the IBRV-VP8 to obtain polypeptide fragment compositions coded by SEQ ID NO.1 and SEQ ID NO.2. The polypeptide fragment compositions can be used for preparing bivalent subunit fusion vaccines, good protective efficacy can be generated after animals are immunized, the purpose of preventing bovine viral diarrhea and infectious bovine rhinotracheitis at the same time is achieved, and the effect of preventing two diseases by one injection is achieved. Meanwhile, a detection kit provided by the invention can be used for rapidly identifying and diagnosing whether cattle are immunized with vaccine strains or infected wild strains, so that the purification of bovine viral diarrhea virus and infectious bovine rhinotracheitis virus is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bovine viral diarrhea-bovine infectious rhinotracheitis dual subunit fusion vaccine and an identification method thereof. Background technique [0002] Bovine viral diarrhea is an infectious disease caused by bovine viral diarrhea virus (BVDV) with main symptoms of cattle immunosuppression, diarrhea, and fever. Causing great losses to the cattle industry. [0003] Infectious bovine rhinotracheitis (IBR) is a bovine respiratory contact disease caused by bovine herpesvirus type I (BHV-1, also known as bovine infectious rhinotracheitis virus IBRV). At the same time, it is accompanied by clinical symptoms such as conjunctivitis and mastitis, which have a great impact on milk production and bull fertility. [0004] Bovine viral diarrhea and bovine infectious rhinotracheitis occur in most cattle farms in my country, and the infection in some important pastoral areas reaches about 80%, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/06C07K14/18G01N33/569A61K39/295A61K39/12A61K39/245A61P31/14A61P31/22
CPCC07K14/005G01N33/56983G01N33/56994A61K39/12A61K39/39A61P31/14A61P31/22A61K2039/70A61K2039/552C12N2770/24334C12N2770/24322C12N2710/16034C12N2710/16022G01N2469/20G01N2333/183G01N2333/06
Inventor 贺笋任立松杜久斌李延涛任郭子君
Owner 天康制药股份有限公司
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