Chicken Marek's disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof

A chicken Marek's disease, gene deletion vaccine technology, applied in the field of biomedicine, can solve the problem that MD vaccine cannot provide effective protection

Inactive Publication Date: 2012-04-04
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

[0003] In recent years, our research group has continuously isolated more than 20 virulent MDV strains from chicken farms where MD immunization failed. The immune challenge tests on some of the strains have shown that some strains can break through HVT vaccine, HVT+SB1 The immune protection of the bivalent vaccine and the serotype 1 CVI988 vaccine proves that the virulence of the wild MDV virus prevalent in my country is gradually increasing, and all existing MD vaccines cannot provide effective protection

Method used

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  • Chicken Marek's disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof
  • Chicken Marek's disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof
  • Chicken Marek's disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Construction of recombinant virus rMSΔmiR9-12 strain:

[0040] 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)), MDV Md5 (see literature : Marek virus super strain (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), 814 strains of chicken Marek's disease virus (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 Pathogen (SPF) chicken embryos and SPF chickens were provided by the Animal Cente...

Embodiment 2

[0066] Example 2 Recombinant Virus Biological Characteristics and Immunoprotective Efficacy Analysis

[0067] 1) Detection of the growth characteristics of the recombinant virus: draw the growth curve of the purified virus, and the specific steps are as follows: Dilute the MS strain and the recombinant virus rMSΔmiR9-12 to 100PFU / ml with 3% cell culture medium, and use a dose of 1ml per well Inoculate a 6-well plate of CEF primary cells that has been spread into a single layer. After the virus grows for 1, 2, 3, 4, 5, 6, and 7 days, the virus is digested with trypsin, and each virus is collected in 3 replicate wells per day. Extract the total DNA of the recombinant virus with the genomic DNA extraction kit of Tiangen Company, and measure the copy number (Copies / 106cells) per million cells of the virus with the method of double real-time fluorescent quantitative PCR, such as Figure 11 shown.

[0068] 2) Detection of genetic stability of recombinant virus: MS strain and recomb...

Embodiment 3

[0072] Embodiment 3 Recombinant Virus Immunoprotective Efficacy Analysis

[0073]160 1-day-old SPF white Laihang chickens were randomly divided into 6 groups, of which the first 5 groups had 30 chickens in each group, and the sixth group had 10 chickens. The first group was the recombinant virus immune group, and the second group was the recombinant immune super MDV Md5 challenged virus. The third group is the 814 virus immune group, the fourth group is the 814 virus immune super MDV Md5 challenge group, the fifth group is the super MDV Md5 direct challenge group, and the sixth group is the blank control group. The chicks were reared in a negative pressure isolator, and the chicks had free access to food and water. The immunization dose of recombinant virus rMSΔmiR9-12 and 814 virus was 2000PFU per mouse. On the 7th day after immunization, the challenge dose of supervirulent Md5 was 1000 PFU per mouse, intraperitoneally injected. From the date of inoculation, observe and rec...

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Abstract

The invention discloses a chicken Marek's disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof. The invention is characterized in that: a recombinant strain rMSdeltamiR9-12 strain is constructed by taking a recombinant MDV circulating strain rMS-LacZdeltaMeq which carries a beta-galactosidase reporter gene and deletes a Meq gene as a parent strain, and deleting mdv-miR-M9, mdv-miR-M5 and mdv-miR-M12. The invention further discloses the application of the gene deletion vaccine strain to the prevention and treatment of the chicken MDVs and the application effect. The microbial collection number of the gene deletion vaccine strain is CGMCC No.4613. The miRNA deletion vaccine strain has stable heredity and high safety, has a good immune protection effect on the chicken Marek's disease, and can be prepared into a single vaccine or a combined vaccine and the like for preventing and treating the chicken MDVs.

Description

technical field [0001] The invention relates to a chicken Marek's disease virus gene deletion vaccine strain and its application, in particular to a chicken Marek's disease virus miRNA deletion vaccine strain and its application, and belongs to the field of biomedicine. Background technique [0002] Chicken Marek's disease (MD) is a highly contagious and neoplastic disease that seriously endangers the development of 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 occurs, the loss is often particularly huge. Vaccine is the main means to control the disease. MD vaccine must be used in breeder flocks and laying hens. The use of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
Inventor 刘长军张艳萍李志杰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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