Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction and application of recombinant Marek's disease virus MDLV21 strain

A technology of MDLV21 and Marek virus, applied in recombinant DNA technology, applications, viruses, etc., can solve problems such as differences in virus virulence, and achieve the effect of reducing impact, good biological safety, and promoting purification

Active Publication Date: 2021-11-19
SHANDONG AGRICULTURAL UNIVERSITY
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

"Quasispecies" are ubiquitous in viruses, and MDV is no exception. British scholars have confirmed that although the genomes of two clones of the same MDV strain are highly similar, the virus virulence is significantly different

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of recombinant Marek's disease virus MDLV21 strain
  • Construction and application of recombinant Marek's disease virus MDLV21 strain
  • Construction and application of recombinant Marek's disease virus MDLV21 strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1——construction of recombinant Marek virus MDLV21 strain:

[0068] 1. Construction of recombinant shuttle plasmids for replacing RLORF6 and Meq genes

[0069] Referring to the published full gene sequence of MDV standard strain Md5 (GeneBank accession number is No.NC002229), primers RLORF6-F / R and Meq-F / R were designed respectively for the flanking sequences of the RLORF6 and Meq genes as the homology arms. The EGFP-F / R amplified EGFP protein expression cassette was designed:

[0070] RLORF6-F: 5'-acgacggcca gtgccaagct tagttctctt cccctactta ccatttc-3'47

[0071] RLORF6-R: 5'-gccaaaaccg catcactagt tttcatagtt tcgggaagat ca-3'42

[0072] Meq-F: 5'-ctctttacac ctgtaccgtg ccc-3'23

[0073] Meq-R: 5'-tatgaccatg attacgaatt cccgcccccg aacactttc-3'39

[0074] EGFP-F:5'-actagtgatg cggttttggc agtac-3'25

[0075] EGFP-R: 5'-cacggtacag gtgtaaagag taagatacat tgatgagttt ggacaaac-3'48

[0076] Using the whole genome DNA of MDV SCA13 strain (the strain has been deposited...

Embodiment 2

[0123] Example 2——Differential diagnosis of recombinant virus MDLV21, vaccine strain CVI988 / Rispens, and wild-type strain Md5 For the RLORF6 and Meq genes deleted in recombinant virus MDLV21, primers MDV-F / R were designed on both sides:

[0124] MDV-F: 5'-ccgagtctaa gctacacggt aagg-3'24

[0125] MDV-R: 5'-tgattcctag gcaggcgtct c'-3'21

[0126] The PCR amplification system is as follows: 1 μL of each primer (10 μM), 12.5 μL of 2xMax Master Mix buffer, 1 μL of template DNA, ddH 2 O to make up 25 μL. 95°C for 30s, 95°C for 15s, 57°C for 15s, 72°C for 1min, 72°C for 5min. 25 loops.

[0127] A 194bp band can be amplified with MDLV21 virus DNA as a template, a 1461bp band can be amplified with CVI988 / Rispens virus DNA as a template, and a 1283bp band can be amplified with Md5 virus DNA as a template ( figure 2 ).

[0128] For the CamR gene deleted by the recombinant virus MDLV21, primers Cam-F / R were designed on both sides, and the sequence is as follows:

[0129] Cam-F: 5'-tc...

Embodiment 3

[0135]Example 3—Proliferation and stability of recombinant virus MDLV21 in chicken embryo fibroblasts

[0136] The MDLV21 and SCA13 of Example 1 were respectively inoculated in a 6-well plate covered with a single layer of CEF cells at a dose of 100 PFU / well, and placed in a 6-well plate containing 5% CO 2 , Cultured in a cell culture incubator at 37°C. The number of MDV plaques was calculated at 0, 24, 48, 72, 96, 120, and 144 hours after inoculation, respectively. The proliferation curve of MDV was drawn, and the proliferation of MDLV21 was analyzed. After culturing for 3-5 days, after MDV-specific plaques appeared, they were digested with trypsin and transferred to a 6-well cell culture plate covered with monolayer chicken embryo fibroblasts, subcultured for 15 generations in turn, and the morphology of plaques was observed. The virus DNA was extracted every 5 generations, and the stability of MDLV21 was identified by PCR amplification with primer MDV-F / R. The PCR amplif...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to construction and application of a recombinant Marek's disease virus MDLV21 strain. The constructed recombinant virus simultaneously deletes two major genes RLORF6, Meq and CmR related to MDV pathopoiesis and tumorigenesis, compared with deletion of a single gene, the vaccine safety is greatly improved, and the capacity of obtaining deleted genes in the field is reduced; the genome of the recombinant virus strain does not have resistance genes and meets the strict requirements of domestic and overseas biological product industries; and the recombinant virus has good biological safety, has a more effective anti-MDV immune protection effect, and is especially suitable for MDV immunization of chicken flocks in China. The recombinant virus can be effectively distinguished from a wild virus, the horizontal transmission capacity is weak, the recombinant virus not only has great application value for immunization of chicken flocks MDV, but also is beneficial to promotion of MDV purification, the genome of the recombinant virus strain does not have an FRT sequence, the influence of exogenous genes on the biological activity of the virus is reduced, any shearing of virus gene segments among FRT sequences is avoided, and research and development of recombinant live virus vector vaccines are facilitated.

Description

【Technical field】 [0001] The invention relates to the construction and application of a recombinant Marek virus MDLV21 strain, belonging to the field of molecular biology of veterinary biological products. 【Background technique】 [0002] Chicken Marek's disease (MD) is an important immunosuppressive and tumor disease caused by chicken Marek's disease virus (MDV) infection. Immunosuppression caused by MDV infecting the host is an important factor leading to the failure of vaccine immunity for other diseases. The use of vaccines can effectively prevent the occurrence of MDV tumors in chicken flocks. However, with the selection of immune pressure and the evolution of MDV itself, the commercial MDV vaccine CVI988 / Rispens, which is widely used in clinical practice, presents a trend of incomplete protection. In recent years, MD outbreaks often broke out even in chicken farms immunized with MDV vaccine, indicating that the virulence of wild MDV strains in chicken flocks in my coun...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/65C12N15/38A61K39/245A61K47/46A61P31/22C12Q1/70C12N15/11C12R1/93
CPCC12N7/00C12N15/85C12N15/65C07K14/005A61K39/12A61K47/46A61P31/22C12Q1/705C12N2710/16321C12N2710/16352C12N2710/16322C12N2710/16334C12N2710/16362C12N2800/106A61K2039/552A61K2039/5254Y02A50/30
Inventor 苏帅崔宁成子强王淑雯李久庆
Owner SHANDONG AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com