Recombined chicken Marek's disease virus vaccine strain for expressing infectious bursaldisease virus VP2 gene and construction method and application of recombined chicken Marek's disease virus vaccine strain

A chicken Marek's disease and Marek's disease technology, applied in the direction of viruses, viral peptides, viruses/bacteriophages, etc., can solve the problems of difficult removal of BAC sequences, complicated construction process, and potential safety hazards

Active Publication Date: 2016-06-22
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

The BAC method is based on homologous recombination, which also has the disadvantages of complex construction process and long cycle; moreover, the sequence of BAC itself is difficult to remove, so that the recombinant virus obtained later carries unnecessary foreign gene sequences, which poses a safety hazard

Method used

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  • Recombined chicken Marek's disease virus vaccine strain for expressing infectious bursaldisease virus VP2 gene and construction method and application of recombined chicken Marek's disease virus vaccine strain
  • Recombined chicken Marek's disease virus vaccine strain for expressing infectious bursaldisease virus VP2 gene and construction method and application of recombined chicken Marek's disease virus vaccine strain
  • Recombined chicken Marek's disease virus vaccine strain for expressing infectious bursaldisease virus VP2 gene and construction method and application of recombined chicken Marek's disease virus vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] The construction of embodiment 1 MDV vaccine 814 strain genome Fosmid libraries

[0091] Marek's disease virus vaccine strain 814 (Zhang, F., Liu, C.J., Zhang, Y.P., et al. Comparative full-length sequence analysis of Marek's disease virus vaccine 814. Arch Virol. 2012, 157 (1): 177-183. Institute preserved, provided. The GenBank accession number of the whole genome sequence of the 814-strain attenuated Marek's disease virus vaccine is JF742597.

[0092] The MDV genome Fosmid library was constructed according to the instructions of the Epicentre CopyControlTMFosmidLibraryProductionKit kit. Methods as below:

[0093] Genomic DNA of 814 strains of MDV vaccine was aspirated repeatedly 50-100 times with a 200 μ L pipette tip by physical means, and passed through pulsed field electrophoresis (CHEF of BioRad Company). XAPulsedField ElectrophoresisSystem system, the conditions are: electrophoresis buffer 0.5 × TBE, gel concentration 1%, program 5-220kb) analysis, until its...

Embodiment 2

[0095] Embodiment 2 virus rescue

[0096] According to the sequencing analysis of recombinant cosmid ends, 6 groups of 5 cosmid combinations were selected. The 5 cosmids in each combination were cloned with 814 genomic DNA fragments of MDV vaccine, which contained overlapping regions and could be spliced ​​to cover the complete MDV genome. The selected cosmid DNA was extracted with the extraction kit from QIAGEN Company. The extracted cosmids were linearized with NotI (NEB Company): NotI endonuclease 100U, cosmids 10 μg, 37°C for 2h. The digested product was extracted with phenol-chloroform-isoamyl alcohol, and precipitated with ethanol to prepare MDV genomic DNA for transfection.

[0097]The five MDV genomic DNA fragments were co-transfected into the secondary CEF cells by calcium phosphate transfection method. The preparation method of CEF cells is as follows: take 9-10 day-old SPF chicken embryos, aseptically take out the chicken embryos, place them in a plate filled wit...

Embodiment 3

[0101] Example 3 Construction of a recombinant mutant cosmid inserting the VP2 gene expression framework (SEQ ID No.1) inside the US2 gene of the MDV genome

[0102] Based on the above established MDV multi-fragment cosmid rescue system, within the US2 gene of the MDV genome in the selected 5-cosmid group member p814-5, specifically, the US2 gene has a total of 813bp, and in this study, the US2 gene was deleted. The 15th to 630th nucleotides are replaced by inserting the CAG-VP2 expression framework (the nucleotide sequence of the CAG-VP2 expression framework is SEQ ID NO.1, and its structure is CMV enhancer-chicken β-actin promoter-VP2 gene -sv40PolyA) to construct a recombinant mutant cosmid p814-5US2VP2 (p814VP2 for short).

[0103] The construction process of the recombinant mutant cosmid p814-5US2VP2 is briefly described as follows:

[0104] 3.1 Construction of pKSKanccdB plasmid

[0105] Three pairs of primers shown in Table 1 were used for multiplex PCR amplification ...

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Abstract

The invention discloses a recombined chicken Marek's disease virus vaccine strain for expressing an infectious bursaldisease virus VP2 gene and a construction method and application of the recombined chicken Marek's disease virus vaccine strain, and belongs to the technical field of medicine or veterinary medicine. By means of the recombination and clone technology, a gene segment CAG-VP2 containing the infectious bursaldisease virus VP2 gene and a CAG promoter sequence is inserted in a US2 gene of the strain 814 of the chicken Marek's disease virus, a recombined cosmid with a CAG-VP2 expression frame inserted in a US2 gene is constructed, and the recombined chicken Marek's disease virus vaccine strain for expressing the infectious bursaldisease virus VP2 gene is obtained through salvation of the recombined cosmid. Research shows that the obtained vaccine strain has the same in-vitro replication ability as a parent virulent vaccine strain 814 and good hereditary stability, and can resist attacks of a very virulent MDV strain and a very virulent IBDV strain at the same time. It can be seen that the obtained recombined MDV vaccine strain can be used for preparing medicine for preventing or treating infectious bursaldisease and the chicken Marek's disease.

Description

technical field [0001] The present invention relates to a recombinant chicken Marek's disease virus vaccine strain and its construction method and application, in particular to a recombinant chicken Marek's disease virus vaccine strain expressing infectious bursal virus VP2 gene and its construction method and application. The invention belongs to the field of medical or veterinary technology. Background technique [0002] my country is a big poultry country. Chicken infectious bursal disease and Marek's disease are the two most important immunosuppressive infectious diseases that endanger the healthy development of my country's poultry industry. Their widespread prevalence has brought huge economic losses to my country's poultry industry. [0003] Infectious bursal disease (IBD) is an acute and highly contagious disease caused by infectious bursal disease virus (IBDV) that mainly harms 3-6 week-old chicks. The disease first appeared in the Gumboro area of ​​the United Sta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01
CPCC07K14/005C12N7/00C12N2710/16034C12N2710/16043C12N2710/16044C12N2720/12022C12N2720/12034
Inventor 王笑梅李凯刘长军张艳萍崔红玉祁小乐高立高玉龙王永强高宏雷
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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