Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof

An enteritis parvovirus, infectious cloning technology, applied in applications, viruses, genetic engineering, etc., can solve the problems of few people's research, difficulty in cloning full-length genome, affecting the establishment of reverse genetics operation platform, etc., to overcome the capacity Small, simple method, high accuracy effect

Active Publication Date: 2020-06-26
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although GenBank has reported the full genome sequence of MEV, few examples of full-length clones have been studied, especially full-length infectious clones
Due to the special genome

Method used

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  • Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof
  • Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof
  • Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof

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Embodiment Construction

[0030] In order to better understand the present invention, the present invention will be further described below in conjunction with specific biological materials and parameters, but the present invention is not further limited. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all the reagents involved can be obtained from commercial sources.

[0031] 1. Transformation of vector pUC18

[0032]First linearize pUC18 with Hind Ⅲ, perform 1% agarose gel electrophoresis at 37°C for 1 hour, cut out the DNA of the target band, recover and purify it; then use Blunting blunting enzyme to treat the purified vector, and act at 37°C for 10 minutes 1% agarose gel electrophoresis at 70°C for 5 minutes, and the DNA of the target band was excised, recovered and purified. This step changed the two ends of the vector fragment from sticky ends to blunt ends, and named it pUC18-M; The vector purified in the previ...

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Abstract

The invention provides a mink enteritis parvovirus whole genome infectious clone and a construction method and application thereof. The method comprises the following steps of: carrying out enzyme digestion on a complete genome in an MEV replication form, and sequentially and directionally cloning to a vector pUC18 M in a segmented form to obtain a recombinant plasmid; and mixing the recombinantplasmid with a transfection reagent, and transfecting CRFK cells to obtain a rescue virus. The mink enteritis parvovirus infectious clone constructed by utilizing a reverse genetic technology transfects the CRFK cells in vitro, and can induce the cells to generate cytopathic effects and growth trends the same as those of a parent virus. The method is simple, rapid, time-saving and labor-saving, and has higher accuracy compared with a traditional PCR method, and base mutation can be rapidly and conveniently carried out at any position of the MEV genome by applying the cloning system, so that aneffective way and means are provided for subsequent research and development of molecular biology of MEV and research and development of vaccines.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a whole genome infectious clone of mink enteritis parvovirus using reverse genetic manipulation technology, a construction method and application thereof. Background technique [0002] Mink enteritis parvovirus (MEV) can cause mink viral enteritis, a highly contagious infectious disease characterized by gastrointestinal mucosal bleeding and severe diarrhea, with high morbidity and mortality, among which young minks are more susceptible . The disease began to break out in my country in the 1980s, causing huge economic losses to my country's mink breeding industry, and it is also one of the three major diseases recognized worldwide as endangering the mink breeding industry. Although there are vaccines for the prevention of mink viral enteritis at present, the disease is still relatively frequent in production practice and seriously endangers the health of mink. [0003] Reverse...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/35C12N7/00
CPCC12N15/85C12N7/00C12N2800/107C12N2750/14352C12N2750/14321
Inventor 谢之景朱倩
Owner SHANDONG AGRICULTURAL UNIVERSITY
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