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GPV (gosling plague virus) subunit vaccine as well as preparation method and application thereof

A technology of gosling plague virus and subunit vaccine, which is applied in the field of veterinary biological products, can solve the problems that no subunit vaccine of gosling plague has been successfully developed and applied, and is beneficial to the control and elimination of the disease, and the immunogenicity Good, low production cost effect

Inactive Publication Date: 2018-01-09
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, there is no report on the successful development and application of gosling plague subunit vaccine, nor is there any report on the expression of VP3 using the BAC TOBAC baculovirus expression system

Method used

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  • GPV (gosling plague virus) subunit vaccine as well as preparation method and application thereof
  • GPV (gosling plague virus) subunit vaccine as well as preparation method and application thereof
  • GPV (gosling plague virus) subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of recombinant baculovirus

[0035] 1. Genome extraction of goose plague virus: extract the total DNA of goose plague virus TZ10 strain with TRIzol Reagent kit;

[0036] 2. Primer design and synthesis: Design a pair of specific primers based on the VP3 gene sequence of gosling plague virus published in GenBank, and add Sal I and Not I restriction sites at both ends of the primers to amplify the 1605bp target fragment. The primer sequences are:

[0037] P1: ACGCGTCGACATGGCAGAGGGAGGAG

[0038] P2: ATAAGAATGCGGCCGCTTACAGATTTTGAGTTAG

[0039] 3. Amplification and recovery of VP3 gene fragments

[0040] (1) PCR amplification: use the extracted DNA as a template, and perform PCR with the primers in step 2. The PCR reaction system is (total volume 25 μl): 0.5 μl of DNA template, 0.5 μl of P1 and P2, 12.5 μl of DNA polymerase and 11 μl of sterile water. The PCR reaction conditions are: 95°C, 5min; 30 cycles of 95°C for 30s, 65°C for 30s, 72°C fo...

Embodiment 2

[0048] 7. Recombinant bacmid transfection of Sf9 cells: transfect the purified recombinant bacmid into Sf9 cells by using liposome transfection method, the specific operation method refers to the Cellfectin of Thermo Fisher Scientific (China) Co., Ltd. According to the instructions of the transfection reagent, the F1 generation recombinant baculovirus GPVP3 / Bac strain was obtained. Embodiment 2: Preparation of recombinant VP3 protein

[0049] 1. Recombinant baculovirus amplification: Inoculate the recombinant baculovirus GPVP3 / Bac strain into insect cells Sf9, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the F2 generation recombinant baculovirus;

[0050] 2. Identification of expressed protein:

[0051] (1) Insert the above-mentioned F2 generation recombinant baculovirus into insect cell Sf9 at an inoculum amount of MOI=5-10, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the rec...

Embodiment 3

[0056] Embodiment 3: vaccine preparation

[0057] 1. Inactivation: Add the recombinant VP3 protein prepared in large quantities in Example 2 into an inactivation tank, add an inactivator BEI at a final concentration of 0.2% to 0.5%, and inactivate at 37° C. for 24 hours.

[0058] 2. Inspection of semi-finished products

[0059] (1) Sterility test: carry out sterility test according to the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0060] (2) Determination of protein content: Serially dilute the BSA standard with known concentration, and perform SDS-PAGE together with the recombinant VP3 protein. After electrophoresis, use QuantityOne software to perform grayscale analysis, and compare the grayscale of the target protein and the standard protein To quantify the target protein. VP3 protein content is not less than 60μg / ml.

[0061] (3) Inactivation test: Insect cell Sf9 was taken from the inactivated protein liquid, and cultured at 27° C. for 72 hours. No ...

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Abstract

The invention provides a preparation method of a GPV (gosling plague virus) subunit vaccine. The preparation method comprises steps as follows: a GPV VP3 protein complete gene is cloned; a recombinantbaculovirus of the antigen protein is constructed by utilizing an insect cell-baculovirus expression system (BAC TO BAC) and the recombinant virus efficiently expresses the GPV VP3 antigen protein ininsect cells Sf9; after extraction, purification and BEI inactivation, an adjuvant is added for emulsification, and the vaccine is prepared. The preparation method is simple, a large amount of the GPV VP3 protein can be prepared, short time is consumed, expression quantity is high, production cost is greatly reduced, and large-scale production is facilitated. The GPV subunit vaccine containing the VP3 protein prepared with the method is good in immunization effect, low in immunizing dose and high in safety and can effectively prevent GPV infection.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a preparation method of a gosling plague virus subunit vaccine, the prepared subunit vaccine and the application of the subunit vaccine. Background technique [0002] Gosling plague is an acute or subacute septic infectious disease of goslings caused by Gosling plague virus (GPV), also known as goose parvovirus. The disease mainly affects susceptible goslings aged 4 to 20 days, and has the characteristics of rapid transmission, high morbidity and mortality. As goslings age, their morbidity and mortality rates decrease. [0003] The anti-infective drugs currently used in goose plague mainly include antibiotics, chemical synthetic drugs, traditional Chinese medicine preparations, attenuated live vaccines, and egg yolk antibodies and other anti-viral biological agents. It has a certain curative effect on goose plague, but it also has certain disadvantages....

Claims

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Application Information

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IPC IPC(8): A61K39/23A61K39/39A61P31/20C12N15/866C12N15/35
CPCY02A50/30
Inventor 丁国伟宋庆庆魏荣荣李玉安李甜甜杨豫蒙
Owner 扬州优邦生物药品有限公司
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