Method for preparing infective hog cholera virus cDNA carrier and use thereof

A kind of swine fever virus and infectious technology, applied in the field of genetic engineering, can solve the problems of inactivated virus vaccine application, non-attenuated vaccine application, pathological changes, etc.

Inactive Publication Date: 2005-04-06
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to build a new generation of swine fever vaccine, Zhang Chuyu and others have completed the full genome sequence of three strains with my country's independent intellectual property rights, namely the standard virulent Shimen strain of swine fever virus, the rabbit attenuated vaccine strain (C strain) and the low-virulence strain 39. Later, the whole genome infectious cDNA clone (Wu Haixiang, Zhang Chuyu, etc., Science Bulletin No. 8, April, 2003) of the standard virulent Shimen strain of classical swine fever virus was successfully constructed, but the expression of this whole genome infectious cDNA clone produced The most important thing is to interfere with defective particles, which cannot cause pathological changes in experimental animals alone, and cause immune responses in animals. It must have a helper virus to cause cytopathic diseases
More importantly, it cannot be directly used as an inactivated virus vaccine, nor can it be used as an attenuated vaccine

Method used

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  • Method for preparing infective hog cholera virus cDNA carrier and use thereof
  • Method for preparing infective hog cholera virus cDNA carrier and use thereof
  • Method for preparing infective hog cholera virus cDNA carrier and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 The cloning of the whole gene of classical swine fever virus and the construction of the expression vector

[0042] ①Dilute the freeze-dried virus of CSFV Shimen strain 10 times with normal saline (provided by China Veterinary Drug Control Institute, Beijing), and inoculate 2-month-old non-immune pigs (provided by Hubei Institute of Animal Husbandry and Veterinary Medicine) into the ear vein. 24 hours after inoculation, the body temperature of pigs reached 40°C, and clinical symptoms of classical swine fever appeared. When the body temperature turned from 42°C to drop on the 7th day, blood was collected from the anterior vena cava immediately, and EDTA anticoagulant was added as the experimental material.

[0043] ② Total RNA was isolated from anticoagulated whole blood with the RNA extraction kit produced by Promega. On the basis of the whole genome sequence of Shimen strain virus, 7 pairs of primers were designed, and 7 overlapping fragments were used to amp...

Embodiment 2

[0045] Embodiment 2 Confirmation of the whole genome expression vector of classical swine fever virus

[0046] The identification of the whole genome cloned plasmid pT7SM was carried out simultaneously by PCR amplification and enzyme digestion identification. Seven pairs of primers randomly distributed on the genome were selected for PCR identification, and three restriction enzymes, BamHI, KpnI and HindIII, were used for restriction endonuclease identification of the whole genome.

Embodiment 3

[0047] Example 3 Preparation of expression vector-deficient classical swine fever virus vector

[0048] Construction of Defective CSF Virus Deleting NS2 Gene Based on Obtaining Complete Gene Cloning of CSFV

[0049] ① Amplification of NS3 gene. In the genome of swine fever virus, the NS2 gene is closely connected with the NS3 gene. For the deletion of the NS2 gene, the PCR method was first used to amplify the downstream NS3 gene, and then the NS2 gene was excised by enzyme cutting behind the upstream P7 gene of the NS2 gene. , and fuse the NS3 gene in-frame to the downstream of P7. When amplifying the NS3 gene, pair the upstream primer NS3(+) with the downstream primer P3D of cDNA3 (as shown in the figure), use the plasmid pGEMT3-1 as a template, and use Pfu DNA polymerase to perform PCR amplification. The target product is in 3 The 'end contains the single restriction site BamHI in the middle of the genome. The Pfu PCR reaction system was prepared in an ice bath, 1ul of th...

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Abstract

The invention discloses a pig acute communicable disease virus infectious DNA carrier preparation method and the application, this carrier includes T7 to start sub- and pig acute communicable disease virus standard full strength Shimen gene group's DNA, starts sub- and in downriver DNA in T7, in the flaw NS2 gene 1,260 alkali base to, retains the 5' end 3752nd alkali base to to the PshAI position spot 3881st alkali base to the between 130bp NS2 gene fragment, other DNA sequence and a Shimen infectiousness clone DNA to be same. The invention method is simple, the ease of operation, may construct the double price or the multi- prices genetic engineering vaccine, suits the pig acute communicable disease virus the distinction diagnosis and in the vaccine research application.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. Specifically, the invention relates to a method for preparing an infectious cDNA vector of classical swine fever virus, a virus produced by vector expression, and its application in differential diagnosis of classical swine fever virus and vaccine research. Background technique [0002] Swine fever is one of the most important acute and contagious diseases of pigs, with a mortality rate of over 90%. In the "International Animal Health Code" formulated by the International Office of Epizootics, swine fever is listed as one of the 16 legal infectious diseases. my country is a country with a high incidence of swine fever. Frequent outbreaks of swine fever have caused huge economic losses to the pig industry and seriously hindered the development of animal husbandry and export trade in our country. [0003] Swine fever is widespread worldwide, especially in so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12C12N7/01C12N15/86C12Q1/68C12Q1/70
Inventor 张楚瑜吴海祥郑从义屈三甫
Owner WUHAN UNIV
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