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Bovine akabane disease virus vaccine

A technology for Akabane disease virus and vaccines, applied in the direction of viruses, virus peptides, antiviral agents, etc., can solve the problems of potential risks in vaccine safety, complex production processes, and many manpower and material resources, and achieve low production costs, high purity, The effect of strong antigen immunity

Pending Publication Date: 2022-05-17
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

The above two methods are conventional methods, the production process is complicated, it requires a lot of manpower and material resources, and the introduction of animal-derived protein poses potential risks to the safety of the vaccine
[0003] At present, the disease is widespread in China and has caused significant economic losses.

Method used

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  • Bovine akabane disease virus vaccine
  • Bovine akabane disease virus vaccine
  • Bovine akabane disease virus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Vector construction and immunogenicity verification of G1 protein and G2 protein

[0026] 1. Construction of cell lines expressing G1 protein and G2 protein

[0027] 1.1 Gene synthesis

[0028] 1.1.1 G1 gene synthesis

[0029] The bovine Akabane disease gene sequence selected from Geenbank was compared and analyzed. According to the partial tropism of CHO cell codons, the codon optimization and modification of the G1 gene sequence was carried out, and the restriction site was designed, and the C-terminal of the G1 sequence was added. Different signal peptide sequences, and with a His tag, finally got SEQ ID NO.1.

[0030] The nucleotide sequence encoding the G1 protein was inserted into the eukaryotic transfer vector pcDNA3.1 through the BamHI and NotI sites, ligated with T4 DNA ligase overnight at 16°C, transformed with Escherichia coli competent DH5a and coated on On the LB plate containing ampicillin, pick positive colonies after culturing overnight at 3...

Embodiment 2

[0046] Determination of Example 2 Antigen Ratio

[0047] After decellularization, the expressed proteins were quantified separately, and mixed with appropriate adjuvants to prepare vaccines. The final finished vaccines contained different doses of G1+G2 proteins (10μg+10μg, 20μg+20μg, 30μg+30μg, 40μg+40μg, 50μg+50μg, 60μg+60μg, 70μg+70μg, 80μg+80μg) / mL, immunize 3-4 month old calves (healthy susceptible, AKAV antibody negative), 2ml / head, 21 days after immunization, blood test for neutralizing antibody To determine the optimal immunization dose, the results showed that the fifth group (50μg+50μg) could produce good neutralizing antibodies, the sixth group, the seventh group, and the eighth group (60μg+60μg, 70μg+70μg, 80μg+80μg) And the antibody highest value, lowest value, average value to analyze, three neutralizing antibody titer differences are not significant, in order to ensure the effectiveness of this invention product, the antigen dose in this invention is determined ...

Embodiment 3

[0050] The immunoprotective effect of embodiment 3 vaccine

[0051] After decellularized fragments, the expressed proteins were quantified separately, and then mixed with appropriate adjuvants to prepare vaccines. The final protein content of G1 and G2 in the finished vaccine was 60 μg+60 μg / mL. Ten healthy 3-4 month-old calves (healthy and susceptible, AKAV antibody negative) were used, among which 5 calves were immunized with 2ml / head of the vaccine, and the other 5 calves were immunized with the same dose of normal saline as a control group. On the 7th, 14th, and 21st day after immunization, and on the 7th and 14th day after the challenge, the blood of the immunized group and the control group was collected, and the neutralizing antibody of bovine Akabane disease virus was measured respectively. The test results are shown in Tables 4 and 5.

[0052] After the immunization, the virus was challenged at the same time, and the oral and anal swabs were collected after the challe...

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Abstract

The invention relates to the technical field of biology, and particularly provides a cattle akabane disease virus vaccine. The protein for preparing the bovine akabane disease virus vaccine comprises G1 protein of bovine akabane disease virus and G2 protein of bovine akabane disease virus, a nucleotide sequence for coding the G1 protein is shown as SEQ ID NO.1, and a nucleotide sequence for coding the G2 protein is shown as SEQ ID NO.2. The invention further provides a preparation method of the bovine akabane disease virus vaccine. The protein is suitable for a eukaryotic cell expression system, the process for preparing active ingredients of bovine akabane disease virus vaccines is changed, and meanwhile the cost is reduced. In addition, the protein has broad-spectrum antigenicity, and a good cross protection effect can be achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bovine Akabane disease virus vaccine. Background technique [0002] Akabane disease, also known as Akabane disease, is a polymorphic infectious disease of cattle, sheep and goats caused by Akabane virus (AKAV). Characterized by stillbirth and congenital arthrogryposis and hydrocephalus (Arthrogryposis–Hydraencephaly, AH syndrome), it is one of the most important infectious viruses that harm ruminants. The current bovine Akabane disease virus vaccine mainly has the following forms: 1. The live vaccine is an attenuated vaccine that is attenuated on Hmlu-1 cells at 30°C; 2. The inactivated vaccine is to inoculate Hmlu-1 cells with OBE-1 strain AKAV, Collect the cell culture medium when cell lesions appear, centrifuge at 3000rpm for 10min, take the supernatant for use or store it at 8°C for later use, and add 0.1% formalin when preparing the vaccine. The above two methods are convent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/40C12N15/85A61K39/12A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2760/12022C12N2760/12034C12N2800/107
Inventor 贺笋李俊辉程兰玲张伟张淑琴
Owner 天康制药股份有限公司
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