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Chicken Marek's disease Meq gene deleted vaccine strain, construction method thereof, and application thereof

A gene-deletion vaccine, chicken Marek's disease technology, applied in the field of biomedicine, to achieve the effect of stable genetics and great application value

Active Publication Date: 2012-02-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method also has some disadvantages. One is that a foreign gene (BAC component) is inserted into the viral genome, and there are potential unfavorable factors.

Method used

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  • Chicken Marek's disease Meq gene deleted vaccine strain, construction method thereof, and application thereof
  • Chicken Marek's disease Meq gene deleted vaccine strain, construction method thereof, and application thereof
  • Chicken Marek's disease Meq gene deleted vaccine strain, construction method thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Recombinant virus transfer plasmid construction:

[0066] 1. Test materials: MDV MS strain (see literature: Construction of Marek's virus miRNA deletion strain and its growth in vitro, Yang Wenchuang, Liu Changjun, etc., Chinese Veterinary Science. 2011, 41 (03)), MDVMd5 (see literature: Marek virus supervirulent strains (Md5, RB1B) virulence test, Gan Junji Liu Xiufan Wu Changxin, Proceedings of the Eleventh Academic Symposium of the Poultry Disease Branch of the Chinese Society of Animal Husbandry and Veterinary Medicine. 2002), chicken Marek disease virus 814 strains (see literature : Cloning and sequence analysis of gE, gI, gp82 genes of chicken Marek's disease virus 814 strains. Zhang Yanping, Liu Changjun, etc. Advances in Animal Science, 2007 (3)) Preserved and provided by the Laboratory of Avian Infectious Diseases, Harbin Veterinary Research Institute; no specific pathogens (SPF) chicken embryos and chickens were provided by the Animal Center of Harbi...

Embodiment 2

[0075] Example 2 The acquisition and identification of recombinant virus-rMSΔMeq

[0076] The first recombination: 1) Co-transfection: The total genomic DNA of MDV MS strain and the transfer vector plasmid pALacZB were co-transfected into CEF by calcium phosphate precipitation method. In the case of CEF, passage to a cell culture dish with a diameter of 60 mm after digestion, and the inoculum amount of CEF is 9×10 per dish. 6 indivual. First, add 388 μL of nuclease-free deionized water into a clean and sterile 1.5 mL centrifuge tube; then add 10–18 μL of the prepared MS strain-infected CEF genomic total DNA (0.8 μg μL-1) and plasmid pALacZB in sequence (1μg·μL-1)2μL; add 30~36μL TE (pH7.4); slowly add 62μL 2mol·L-1CaCl 2 , mix slowly; use a 1mL pipette to slowly add 500μL of 2×HEPES from the bottom, use a micropipette with a 200μL range to blow about 20 bubbles from the bottom to mix well, and incubate in a 37°C incubator for 30min. In the meantime, pack it into fine pellet...

Embodiment 3

[0082] Embodiment 3 biological characteristics of recombinant virus

[0083] 1) Detection of growth characteristics of recombinant virus

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Abstract

The invention discloses a chicken Marek's disease (MD) Meq gene deleted vaccine strain, a construction method thereof, and an application thereof. The chicken Marek's disease rMS delta Meq gene deleted vaccine strain has a microbe reservation number of CGMCC No. 4612. According to the invention, on a basis of a parent strain which is MDV MS strain, a 468bp base sequence on a front part of a main oncogene is deleted thorough two times of homologous recombination, such that the chicken Marek's disease Meq gene deleted vaccine strain is obtained. The invention also relates to the application of the gene deleted vaccine strain in preparation of medicine used for controlling chicken Marek's disease, and in detection and disgnosis methods used for distinguishing a vaccine strain and a chicken Marek's disease wild strain, wherein the methods are designed aiming at the deleted gene sequence of the gene deleted strain. The gene deleted vaccine strain provided by the invention has good safety and good immuno-protection effect upon chicken Marek's diseases. The vaccine strain can be used for preparing monovalent vaccines or combined vaccines used for controlling chicken Marek's disease.

Description

technical field [0001] The invention relates to an attenuated vaccine strain obtained by gene deletion, in particular to a chicken Marek's disease virus Meq gene deletion vaccine strain, and also relates to its application in preparing medicines for preventing and treating chicken Marek's disease, which belongs to the field of biomedicine. Background technique [0002] Chicken Marek's disease (MD, Marek's Disease) is caused by infection with Marek virus (MDV, Marek's Disease virus), and is a highly contagious and neoplastic disease that seriously endangers the development of the poultry industry. The disease can cause multiple lymphomas in chickens, leading to failure and death of chickens. At the same time, the immune organs of the chicken body are damaged, resulting in severe immunosuppression, and other diseases are easily complicated. The course of the disease is long, and after the disease occurs, it often leads to the elimination of the whole flock. Once the disease o...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/01C12N15/09C12N15/63C12Q1/70C12Q1/68A61K39/245A61P31/22C12R1/93
Inventor 刘长军张艳萍李志杰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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