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Active vaccine, and preparation method and application thereof

A technology of live vaccines and genes, which is applied in the direction of pharmaceutical formulas, medical preparations containing active ingredients, antibody medical ingredients, etc., can solve problems such as serious epidemics, mixed infections, and economic losses in the poultry industry, and achieve the effect of easy operation

Active Publication Date: 2016-10-19
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Marek's disease is a neoplastic disease that can be controlled by vaccines. The disease occurs in many areas of my country from time to time. Even immunized chickens will also have the disease. MDV infection will induce immunosuppression and secondary or mixed infection , making the outbreak of MD more serious, causing higher morbidity and mortality, and causing huge economic losses to the poultry industry

Method used

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  • Active vaccine, and preparation method and application thereof
  • Active vaccine, and preparation method and application thereof
  • Active vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The construction of embodiment 1 transfer vector

[0050] 1. Extraction of H9 subtype avian influenza virus genome RNA: Inoculate H9 subtype AIVA155-1-2014 strain into 10-day-old SPF chicken embryos, collect the allantoic fluid of dead chicken embryos within 24 to 72 hours, and use Trizol reagent for urine extraction. Extraction of total RNA from cyst fluid. The specific operation was carried out according to the instructions of the reagents.

[0051] 2. RT-PCR: Design a pair of primers, the sequence of which is: upstream primer: 5'-GAAGCGGCCGCAAAATGGAGACAATATCACTAATAAC-3', downstream primer: 5'-GGTGGGCCCTTATACAAATGTTGCATCTGC-3'. Not I and Apa I were introduced at the 5' end of the upstream primer and downstream primer respectively, and the length of the amplified fragment was expected to be 1700bp, and then cloned into the T vector, and the positive recombinant plasmid containing the HA gene was identified by restriction analysis and PCR.

[0052] 3. Construction of ...

Embodiment 2

[0056] Construction, screening and identification of embodiment 2 recombinant Marek's virus

[0057] Chicken embryo fibroblasts (CEF) were prepared according to conventional methods, and after they grew into 80% monolayer, they were inoculated with HVT, the dose was 50 PFU per well in a 24-well plate, and after acting at 37°C for 4 hours, they were treated with Lipofectamine TM 2000 instructions, transfect the cells with the transfer vector plasmid, after 2 hours of action, pour off the transfection solution, add MEM culture solution containing 5% fetal bovine serum, and incubate at 37°C CO 2 After culturing in the incubator for 4 to 5 days, when typical cell lesions appear, add a nutrient solution containing X-Gal (200ug / ml), observe blue plaques, digest the infected CEF cells in 24 wells with 0.25% trypsin, Seed onto 96-well culture plates with fresh CEF cell monolayers. Observe the blue spots, digest the cells with blue holes and connect them to new cells, repeat this cycl...

Embodiment 3

[0061] Embodiment 3 Identification of recombinant virus expressing HA protein

[0062] 1. Indirect immune enzyme reaction Infect the CEF cells in 96 wells with the recombinant virus (rHVT-HA), 4-5 days later. Pour off the culture medium and fix the cells with pre-cooled 95% ethanol and acetone (4:6) mixture; perform immunoenzyme reaction with SPF chicken anti-H9N2 serum and HRP-labeled rabbit anti-chicken IgY.

[0063] Results: After staining with X-Gal, the plaques of CEF cells containing the recombinant virus were blue. The results of immunoenzyme reaction showed that the coeruleus formed by the recombinant virus could specifically react with H9N2 serum.

[0064] 2. Western-blot detection Infect CEF cells with the recombinant virus (rHVT-HA). After 4-5 days, the collected cells were lysed, and the supernatant was taken for 12% SDS-PAGE electrophoresis, and then transferred to the membrane. Anti-H9N2 serum and HRP-labeled rabbit anti-chicken IgY were analyzed by Western-blo...

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Abstract

The present invention provides an active vaccine rHVT-HA for prevention and treatment of avian influenza and Marek's disease. The HVT is recombined with a HA gene of avian influenza virus, and the HA gene can express HA protein. The live vaccine can rapidly generate HI antibody in the body, has more effective prevention effect on avian influenza effect than the traditional inactivated vaccine, and has good protective effect on the infection of Marek's virus.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a live vaccine for treating bird flu, a preparation method and application. Background technique [0002] H9 subtype avian influenza virus (Avian Influenza Virus, AIV) can cause respiratory diseases and egg production decline in chickens, and has higher morbidity and mortality in the case of mixed infection or secondary infection of other pathogens. cause serious economic losses. Its genome consists of 8 single-stranded antisense RNAs, encoding 10 proteins, 8 of which are structural proteins, including HA and NA, and the other two are non-structural proteins. It is generally believed that HA is its main antigenic gene, which can elicit protective antibodies in birds. However, the mutation rate of H9 subtype AIV is fast, and the existing vaccine strains cannot effectively protect the circulating strains, and most of the existing vaccines are inactivated ...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61P31/16A61P31/22
Inventor 张许科孙进忠郭丽霞田克恭
Owner PU LIKE BIO ENG
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