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Whole genome infectious clone of mink enteritis parvovirus and its construction method and application

A technology of enteritis parvovirus and infectious cloning, applied in the fields of viruses, applications, genetic engineering, etc., can solve the problems that few people study, it is difficult to clone the full-length genome, and affect the establishment of reverse genetics operation platform, etc., to achieve high accuracy Sex, simple effect

Active Publication Date: 2022-02-18
SHANDONG AGRICULTURAL UNIVERSITY
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AI Technical Summary

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 structure of autonomously replicating parvovirus, it is difficult to clone its full-length genome, which seriously affects the establishment of its reverse genetics operation platform

Method used

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  • Whole genome infectious clone of mink enteritis parvovirus and its construction method and application
  • Whole genome infectious clone of mink enteritis parvovirus and its construction method and application
  • Whole genome infectious clone of mink enteritis parvovirus and its construction method and application

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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 whole genome infectious clone of mink enteritis parvovirus and its construction method and application. The method is to digest the complete genome of the MEV replication form, and sequentially and directional clone it into the vector pUC18‑M in the form of segments to obtain the recombinant plasmid. After mixing the recombinant plasmid and the transfection reagent, the CRFK cells can be transfected to obtain the rescued virus. The mink enteritis parvovirus infectious clone constructed by using the reverse genetic technology in the present invention transfects CRFK cells in vitro, and can induce the cells to produce the same cytopathic and growth tendency as the parental virus. This method is simple, fast, time-saving and labor-saving, and has higher accuracy than the traditional PCR method. The application of this cloning system can quickly and conveniently carry out base mutations at any position in the MEV genome, thereby providing a basis for the subsequent development of MEV molecular biology. Research and the development of vaccines provide effective ways and means.

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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Patent Type & Authority Patents(China)
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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