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Method for constructing coronavirus infectious clone and application thereof

A technology for infectious cloning and coronavirus, which is applied in the direction of viruses, antiviral agents, viruses/phages, etc., can solve the problems of unstable preservation of coronavirus cDNA, inability to obtain a complete genome, and low efficiency of enzymatic cleavage and connection. Integrity, high rescue efficiency, avoiding unstable effects

Active Publication Date: 2019-08-02
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the above problems, and provides a method for constructing coronavirus infectious clones using medium and low copy plasmid vectors, which overcomes the problem that coronavirus cDNA cannot be stored stably in bacteria in the prior art, and overcomes the traditional method of enzymatic digestion. The problems of low connection efficiency and low rescue rate overcome the problem that the traditional method cannot obtain a complete genome, and the method includes the following steps:

Method used

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  • Method for constructing coronavirus infectious clone and application thereof
  • Method for constructing coronavirus infectious clone and application thereof
  • Method for constructing coronavirus infectious clone and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Construction of infectious clone of avian infectious bronchitis virus IBV-H120

[0064] 1) Primers and gene synthesis

[0065] According to the H120 infectious clone design sequence and rescue strategy, design linear vector amplification primers, point mutation primers, auxiliary plasmid construction primers and identification and detection primers. The primers and genes were synthesized by BGI (Table 1).

[0066] Table 1 Primers used in this study

[0067] Table 3.1 Nucleotide sequences of oligos used in this work

[0068]

[0069]

[0070]

[0071]

[0072] Note: The bolded sequence in italics is the T7 promoter, and the underlined sequence is the restriction endonuclease recognition site

[0073] 2) RNA extraction of avian infectious bronchitis virus vaccine strain H120

[0074] Avian infectious bronchitis virus vaccine strain H120 virulent allantoic cavity was inoculated with 9-day-old SPF chicken embryos, and the allantoic fluid of the remaining chicken embryos was harvested ...

experiment example 1

[0102] Rescue virus detection

[0103] 1) RT-PCR detection

[0104] Take 200μL of rH120 F5 strain virus liquid and extract viral RNA according to the instructions of Axyprep Body Fluid Virus DNA / RNA Small Extraction Kit. The extracted RNA is dissolved in 40μL RNase-free TE buffer, and 10μL RNA is taken according to Recombinant DNase I (RNase-free) (Takara) Instructions to remove DNA. Using the above RNA as a template, the primers IBV-S1-F / R, IBV-MF / R and H120-3ab-F / R in Table 3.1 are expanded according to the instructions of PrimeScript OneStep RT-PCR Kit Ver.2 Increase S1, M and 3ab genes. RT-PCR products were observed by 1% agarose gel electrophoresis Figure 5 , And sent to Huada Gene Company for sequencing. See the sequencing results Image 6 .

[0105] 2) Western blotting detection of M protein

[0106] Inoculate the rH120 F5 and female H120 virus liquids into the cell culture six-well plate monolayer CK cells, adsorb at 37°C for 2h, then change to 2% serum DMEM medium, and pl...

experiment example 2

[0112] Rescue the biological characteristics of the virus

[0113] 1) Determination of rH120 virus growth

[0114] Dilute the rH120 F5 virus liquid and the mother virus H120 virus with normal saline, and inoculate 30 10-day-old SPF chicken embryos through the allantoic cavity. 2 EID50 / embryo. The virus allantoic fluid of 5 chicken embryos was harvested at 6h, 12h, 24h, 36h and 48h respectively after inoculation, mixed and aliquoted and stored at -80℃.

[0115] ( Picture 9 ) The results showed that the proliferation trend of rH120 F5 virus was basically the same as that of the female parent strain H120. The overall titer of rH120 F5 was slightly lower than that of the female parent strain H120, and the difference in virus titers was not significant (p>0.05). The strain H120 is basically the same. It shows that rH120 F5, like the female parent strain H120, is highly adapted to chicken embryos.

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Abstract

The invention discloses a method for constructing a coronavirus infectious clone and an application thereof. The method comprises the following steps: firstly, acquiring a cDNA segment covering a selected full-length genome of coronavirus; performing segmental cloning; adopting a RED / ET recombination technology for assembling the cDNA segment containing the full-length genome of coronavirus onto aplasmid vector; screening, thereby acquiring the infectious clone containing the full-length cDNA of coronavirus. The invention has the beneficial effects that a medium / low copy plasmid vector is firstly utilized to successfully construct the infectious bronchitis coronavirus infectious clone; a new method is established for the coronavirus infectious clone; the method is capable of solving the problems of difficulty in selecting coronavirus vector and instability of viral macro-genome in bacteria; the positive cloning efficiency is high; the acquired reverse genetic vaccine strain cloning has integrity, passage stability, infectivity and high rescuing efficiency; an effective tool is supplied for researching nosogenesis of coronavirus, developing a novel vaccine, and the like.

Description

Technical field [0001] The invention belongs to the technical field of animal genetic engineering, and more specifically, relates to a method for constructing an infectious clone of a coronavirus and its application. Background technique [0002] Reverse genetics technology is a method to study the structure and function of viruses by constructing infectious molecular clones of viruses and performing molecular operations at the DNA level. Due to the large genome of coronaviruses, it is difficult to have suitable vectors that can accommodate such a large Genome to get the virus. Therefore, the research of coronavirus genome has been limited to temperature-sensitive mutant strains, defective viruses, and recombinant viruses constructed using targeted RNA homologous recombination technology. Among them, targeted RNA homologous recombination technology was the first to be used in the research of coronavirus reverse genetics. Technology, the reverse genetic manipulation technology of...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/86A61K39/215A61P31/14
CPCC12N15/66C12N15/86A61K39/12A61P31/14C12N2770/20043A61K2039/5254Y02A50/30
Inventor 谢青梅封柯宇邵冠明张新珩邵洋洋
Owner SOUTH CHINA AGRI UNIV
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