Marek's disease virus vaccine CV1988 strain VP22 gene

A virus vaccine, Marek's technology, applied in genetic engineering, plant genetic improvement, viruses/bacteriophages, etc., can solve problems such as the difficulty of introducing them into specific types of cells, introducing cells, and toxic and side effects

Inactive Publication Date: 2004-07-21
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In the past, an obvious defect of both nucleic acid vaccines and protein immunity was that they could not amplify and spread in vivo like viral vector vaccines. It was difficult for people to introduce genes into cells, and it was even more difficult to direct them into specific types of cells
Even if they are introduced, they are difficult to stay and express in a controlled manner for a long time, and they will produce toxic side effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0027] Embodiment of the invention

[0028] 1. Cloning of MDV VP22 gene

[0029] 1.1 Genomic DNA extraction

[0030] Use DEF prepared from clean duck embryos to amplify MDV GA strains, Md5, Md11, RB1B, CVI988, FC126, Z4, and 648A. When more than 90% of the cells have CPE, digest with 0.25% trypsin, 0.01MPBS (pH7.2) Wash once, suspend it in 500 μl of digestion solution (100 mM NaCl, 25 mM EDTA, 10 mM Tris~Cl pH8.0, 0.5% SDS, 0.1 mg / ml proteinase K), and digest at 37° C. for 4 hr or overnight. After digestion, mix with an equal amount of TE-saturated phenol / chloroform-isoamyl alcohol, shake well, extract by centrifugation twice, and extract once more with chloroform. After extraction, precipitate with 2 times the volume of absolute ethanol at -20°C for 30 min or overnight, centrifuge at 12,000 rpm for 20 min, wash the precipitate with 70% ethanol, dry it naturally in the air, and suspend it in 50 μl double-distilled H2O, -20 ℃ frozen.

[0031] 1.2 VP22 (amplification of ULA9...

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Abstract

The UL49h gene fragment in the fibroblast genome of the duck embryo or chicken embryo infected by weak-toxic vaccine strain CV1988 / Rispens is amplified by DNA PCR method to obtain the PCR product, chich is then cloned and coded. After compared with the classical MDV strong-toxic GA strain, it is determined that said UL49h of weak-toxic CV1988 has 3 site-specific mutations and a deficient mutation. Said CV1988 VP22 gene or part of its gene sequence can be used to develop the protective antigen and medicine, and increase the immune protection effect of protein and the curative effect of medicine.

Description

field of invention [0001] The invention belongs to the technical field of gene engineering production medicine in biotechnology pharmaceutical industry. Background technique [0002] In the past, an obvious defect of both nucleic acid vaccines and protein immunization was that they could not amplify and spread in vivo like viral vector vaccines. It was difficult for people to introduce genes into cells, and it was even more difficult to direct them into specific types of cells. Even if they are introduced, it is difficult for them to stay for a long time and express them in a controlled manner, and they will produce toxic side effects. Green M et al. (1988) and Frankel A et al. (1988) when studying the TAT protein of human type I acquired immunodeficiency (HIV-1) respectively, found that after adding TAT in the medium of in vitro cultured cells, the The protein can quickly enter the cell and localize in the nucleus without any toxic side effects. Subsequently, some natural...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/255C12N7/01C12N15/38C12N15/62C12Q1/68
Inventor 秦爱建陈鸿军金文杰刘岳龙
Owner YANGZHOU UNIV
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