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TK gene deletion type goat pox virus as well as preparation method and application of TK gene deletion type goat pox virus

A goat pox virus and gene technology, applied in the biological field, can solve the problems of difficult site-specific knockout, non-infectious DNA, etc., and achieve the effects of good genetic stability, safety and good safety.

Active Publication Date: 2017-11-24
吉林省凯博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the goat pox virus genome is very large and its DNA is not infectious, it is not easy to specifically knock out the corresponding gene by direct gene shearing method

Method used

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  • TK gene deletion type goat pox virus as well as preparation method and application of TK gene deletion type goat pox virus
  • TK gene deletion type goat pox virus as well as preparation method and application of TK gene deletion type goat pox virus
  • TK gene deletion type goat pox virus as well as preparation method and application of TK gene deletion type goat pox virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Goat Poxvirus Shuttle Vectors pGVTK and pGVTKα

[0035] According to the complete gene sequence of goat pox virus published in GenBank, after analyzing and comparing the homology of the thymidine kinase gene TK of goat pox virus GTPV-AV41, two segments were selected according to the thymidine kinase gene TK of goat pox virus and its flanking sequences Nucleotide sequence as homologous recombination left arm TKL (422bp, SEQ ID No: 1) and homologous recombination right arm TKR (402bp, SEQ ID No: 5), and between homologous left arm and homologous right arm Add poxvirus universal early and late promoter PE / L (SEQ ID No: 2), enhanced green fluorescent protein EGFP (SEQ ID No: 3), transcription termination signal T5NT (SEQ ID No: 4), to obtain goat pox virus shuttle vector pGVTK. Another kind of sheeppox virus shuttle vector pGVTKα also contains the above-mentioned homologous recombination left arm TKL (422bp) and homologous recombination right arm ...

Embodiment 2

[0037] Example 2 Screening and Identification of Goat Pox Virus with TK Gene Deletion Type

[0038] 1. Preparation of goat pox shuttle plasmid

[0039] Use the "Endotoxin-Free Plasmid Extraction Kit (Spin Column Type)" (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to prepare the shuttle plasmid, and use spectrophotometry to measure the concentration of nucleic acid, and the OD260 / 280 is between 1.8-2.0 Store at -40°C for later use.

[0040] 2. Cell culture and digestion passage

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Abstract

The invention provides a TK gene deletion type goat pox virus and a preparation method thereof. The preparation method comprises the following steps: firstly, constructing two kinds of goat pox virus shuttle vectors pGVTK and pGVTKalpha, wherein core elements contained in a plasmid pGVTK are a TK gene homologous left arm, a pox virus general early and late promoter PE / L, a fluorescent marker gene, a transcription terminator and a TK gene homologous right arm sequentially, and core elements contained in a plasmid pGVTKalpha are a TK gene homologous left arm, an insignificant sequence and a TK gene homologous right arm sequentially; and then specifically knocking down a TK gene by utilizing a secondary homologous recombination technology, wherein a selection marker can be knocked down together. The toxicity of the constructed TK gene deletion type goat pox virus is reduced to some extent, but virus proliferation is not influenced, the growth characteristics of a parent strain are still kept, and the TK gene deletion type goat pox virus also has good genetic stability, is applicable to preparation of a goat pox vaccine and has good safety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a TK gene-deleted goat pox virus and its preparation method and application. Background technique [0002] Goat pox (goat pox) is a highly contagious disease of goats, characterized by fever, generalized pox, nodules and death. Because the disease can cause serious economic losses and hinder international trade, it is listed as a reportable infectious disease by the World Organization for Animal Health (OIE), while my country's Ministry of Agriculture lists it as a first-class animal disease. [0003] Although the existing attenuated vaccine can effectively prevent the occurrence of the disease, there are still relatively large safety hazards in use, and it is difficult to distinguish vaccination from virulent infection. my country is a country where goat pox frequently occurs, and the only way to control the disease fundamentally is to start the eradication plan, so it ...

Claims

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Application Information

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IPC IPC(8): C12N15/863C12N7/01A61K39/275A61P31/20
CPCA61K39/12A61K2039/552C12N7/00C12N15/86C12N2710/24021C12N2710/24034C12N2710/24243
Inventor 不公告发明人
Owner 吉林省凯博生物技术有限公司
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