Duck virus hepatitis suicide DNA vaccine, as well as constructing method and application thereof

A technology for duck viral hepatitis and DNA vaccine, applied in the field of animal virology and animal infectious disease, can solve the problems of immune failure, strong virulence, unstable effect, etc., and achieve the effect of small immune dose and convenient operation.

Inactive Publication Date: 2012-07-18
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The first purpose of the present invention is to provide a kind of suicidal DNA vaccine of duck viral hepatitis, to solve the problem that existing DVH vaccines in the prior art are general

Method used

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  • Duck virus hepatitis suicide DNA vaccine, as well as constructing method and application thereof
  • Duck virus hepatitis suicide DNA vaccine, as well as constructing method and application thereof
  • Duck virus hepatitis suicide DNA vaccine, as well as constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the construction method of duck viral hepatitis suicide DNV vaccine-pSCA1-VP1 suicide DHV vaccine of the present invention, comprises the following steps:

[0063] 1) Extraction and purification of total RNA of DHV virus: DHV ZJ08 virus strain was injected intramuscularly into the legs of two-day-old ducklings. After the ducklings died, the liver was dissected, and the liver was cut into pieces with scissors and ground, and added 1% double-antibody, after freezing and thawing the grinding solution 3 times, use TakaRa Catrimox-14 TM The kit extracts RNA, and the specific steps refer to the kit instructions.

[0064] 2) Design specific primers: design a pair of specific primers according to the sequence analysis of the DHV virus VP1 protein gene, and introduce the Kozak sequence in the upstream primers:

[0065] VP1-F: 5'-CG GGATCC BamH I GCCACCATG kozak序列 GGTGATTCCAACCAGTT-3',

[0066] VP1-R: 5'-TCC CCCGGG Sma I TTCAATTTCCAGATTGAGTTCAGA-3'.

[00...

Embodiment 2

[0081] Example 2: Identification of transient expression of exogenous VP1 gene in vitro of pSCA1-VP1DHV vaccine

[0082] 1) In vitro mRNA expression identification of VP1 gene: RT-PCR detection

[0083]Transfection was performed when BHK-21 cells grew to 80% in the six-well plate. The transfection process is as follows: (1) Add 5 μg pSCA1-VP1 plasmid and 10 μg liposomes to 250ul Opti-MEM culture medium, mix gently, and let stand for 5 minutes; (2) Add the Opti-MEM culture medium containing the plasmid Add it into the Opti-MEM culture solution containing liposomes, mix gently, and let it stand for 20 minutes; (3) Aspirate and discard the culture solution in the six-well plate, wash the cells with PBS for 3 times, and then add pSCA1- VP1 plasmid and liposome Opti-MEM mixture 500ul, add 500ul Opti-MEM in a six-well plate, shake gently; (4) put in 37 ℃, 5% CO 2 In the incubator, after 5 hours, the full culture medium containing FBS was changed to continue the cultivation. After...

Embodiment 3

[0086] Example 3: Detection of pSCA1-VP1 DHV vaccine T lymphocyte proliferation in ducklings

[0087] Twenty-four one-day-old ducklings were randomly divided into 4 groups, 6 in each group, respectively DHV / attenuated vaccine, pSCA1 / VP1, pSCA1 empty vector, and PBS groups. In the second week, lymphocytes were collected from the jugular vein and diluted to 1×10 with RPMI 1640 containing 10% FBS. 7 / mL. In a 96-well cell culture plate, 100 μL of cell suspension was added to each well, and RPMI 1640 self-control was set at the same time, and 8 replicate wells were set for each sample. Place the culture plate at 37°C, 5% CO 2 After culturing under saturated humidity conditions for 68 hours, add 10 μL of MTT (5 mg / mL) to each well, incubate for 4 hours, add 50 ul of DMSO solution to terminate the reaction, measure the OD value at λ570 nm after the blue precipitate dissolves, and perform statistical analysis, as shown in Table 1 with Image 6 , the results showed that there was ...

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Abstract

The invention relates to the technical field of animal virology and animal infectious disease, and particularly relates to duck virus hepatitis suicide DNA vaccine, and the building method and the application thereof. The duck virus hepatitis suicide DNA vaccine provided by the utility model comprises DHV-VPI gene and alphavirus copying sub carrier pSCA 1, the constructing of the DNA vaccine comprises the following steps: the total RNA of DHV virus is extracted and purified, specific primers are designed, through the augmentation of RT-PCR, the total length cDNA gene order of DHV-VPI can be cloned, and finally, a purified VP 1 total length Cdna segment is restructured into the eukaryotic expression plasmid of alphavirus copying sub carrier pSCA 1. Compared with the prior art, the required immunizing dose of the vaccine provided by the invention is small, and the vaccine is safe and efficient; the vaccine can be eliminated by an organism with the Apoptosis of cells. Therefore, DNA plasmid are prevented from being possibly integrated into host cell chromosomes or causing hidden troubles such as immune tolerance and the like, and the duck virus hepatitis suicide DNA vaccine is efficient and stable, and the operation is convenient and quick.

Description

technical field [0001] The invention relates to the technical field of animal virology and animal epidemiology, in particular to a duck viral hepatitis suicide DNA vaccine and its construction method and application. Background technique [0002] Duck viral hepatitis (DVH) is a highly lethal and rapidly spreading viral infectious disease of ducklings caused by duck viral hepatitis virus (DHV). The disease is characterized by acute onset, short course and high mortality. The clinical symptoms are mainly characterized by neurological symptoms such as convulsions, convulsions, and opisthotonus, and the pathological changes are mainly characterized by hepatomegaly and spot hemorrhage. DHV is divided into three serotypes, namely type I, type II and type III, and there is no cross-protection among the three serotypes. Type I DHV is distributed worldwide and is often mixed with other viruses and bacteria. It is very harmful to the duck industry and has caused huge economic losses...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/29A61P31/14C12N15/51C12N15/86
Inventor 刘光清
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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