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A ca16 infectious clone with green fluorescent protein gene and its construction method and application

A green fluorescent protein and infectious cloning technology, applied in the application field of eGFP-CA16 reporter virus, can solve the problems of lack of epidemiological research data, lagging research of vaccines and therapeutic drugs, etc., to avoid operational difficulties and ensure fidelity Effect

Inactive Publication Date: 2015-09-30
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is a lot of investment in EV71-related research, and EV71 vaccines and drugs have been developed rapidly, but there is little understanding of the infection and pathogenic mechanism of CA16, lack of epidemiological research data, and research on its vaccines and therapeutic drugs. Relatively lagging behind, so in-depth research on CA16 is of great significance

Method used

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  • A ca16 infectious clone with green fluorescent protein gene and its construction method and application
  • A ca16 infectious clone with green fluorescent protein gene and its construction method and application
  • A ca16 infectious clone with green fluorescent protein gene and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0055] A kind of preparation that has the CA16 infectious clone of green fluorescent protein gene, its steps are:

[0056] 1. Extraction of CA16 / GD09 / 24 viral RNA:

[0057] Take 140ul parental CA16: CA16 / GD09 / 24 virus solution (from Jian-Feng Han, Nan Yu, Yu-Xian Pan, Si-Jie He, Li-Juan Xu, Rui-Yuan Cao, Yue-Xiang Li, Shun- Ya Zhu, Yu Zhang, E-De Qin, Xiao-Yan Che, Cheng-Feng Qin. Phenotypic and genomic characterization of humancoxsackievirus A16 strains with distinct virulence in mice. Virus Re s .2014Jan22;179:212-9. According to the requirements of the QIAamp Viral RNA Mini Kit (purchased from Qiagen) kit instructions, CA16 RNA was extracted and stored at -80°C for future use;

[0058] 2. Segmented amplification of the genome sequence of the CA16 / GD09 / 24 virus strain:

[0059] Construct a cDNA infectious clone with the full-length genome of CA16: According to the sequence of the CA16 / GD09 / 24 virus strain (GenBank accession no.KC117317), use the viral RNA in step 1 as a ...

Embodiment 2

[0072] The CA16 infectious clone with the green fluorescent protein gene successfully rescued the virus with the same growth tendency as the recombinant CA16 virus:

[0073] 1. Linearization and phenol-chloroform extraction of plasmids: 10 μg of plasmid pACYC177-CA16-FL and plasmid pACYC177-eGFP-CA16-FL were digested with MluI (described above), digested at 37°C for two hours, and washed with 0.8% agar After sugar gel electrophoresis to identify the complete digestion, add 100 μl saturated phenol (purchased from Sinopharm Chemical Reagent Company) to the digested product, shake and mix, centrifuge at 17000g for 5min, draw the supernatant into a new centrifuge tube, and centrifuge to the original Add 100 μl sterile water to the tube, shake and mix well, and centrifuge at 17,000 g for 5 minutes; absorb the supernatant and combine it with the supernatant obtained in the previous step (total volume is about 200 μl), add 200 μl chloroform (purchased from Sinopharm Chemical Reagent C...

Embodiment 3

[0083] A kind of application of CA16 infectious clone with green fluorescent protein gene in antiviral drug screening, its steps are:

[0084] 1. Inoculate 1×10 cells in each well of a 12-well cell culture plate 5 vero cells at 37°C, 5% CO 2 Under culture conditions, when the confluence reaches 60%, according to the multiplicity of infection (MOI) of 0.1, add 200 μl of diluted parental CA16, recombinant CA16 and eGFP-CA16 virus solution to each well, 37 ° C, 5% CO 2 After adsorption in the incubator for 1 hour, the virus solution was discarded, and 1ml of DMEM medium containing 2% fetal bovine serum was added to each well;

[0085] 2. Dilute the stored NITD008 at a concentration of 20mM to 0, 1, 2, 3, 4mM with DMSO, and add 1μl of diluted NITD008 to the wells respectively, that is, the final concentrations are 0, 1, 2, 3, 4μM respectively . 37°C, 5% CO 2 cultured in an incubator for 48 hours, and observed the expression of eGFP under the action of different concentrations ...

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Abstract

The invention discloses a CA16 infectious clone with a green fluorescent protein gene as well as a construction method and an application of the CA16 infectious clone. The method comprises the steps of firstly, inserting full-length cDNA (complementary deoxyribonucleic acid) of a CA16 / GD09 / 24 virus strain by taking a low copy number plasmid pACYC177 as a carrier; then, inserting an eGFP (enhanced green fluorescent protein) reporter gene and adding a 2A protease cleavage site (AITTL) between 5'UTR and VP4 by taking the full-length infectious clone as a skeleton so as to obtain a full-length infectious clone of the CA16 with the eGFP reporter gene. The CA16 infectious clone with the green fluorescent protein gene, constructed by the method, disclosed by the invention, can be used for saving an eGFP-CA16 reporter virus of which the growth tendency is similar to that of a recombinant CA16 virus and a parent CA16, and can also be used for screening antiviral drugs, thereby providing a convenient platform for deepening virus replication and pathogenesis researches and laying a solid foundation for screening and developing novel vaccines in future.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CA16 infectious clone with a green fluorescent protein gene, a method for constructing the clone, and an eGFP-CA16 reporter rescued from the clone by reverse genetics technology Virus application. Background technique [0002] Coxsackievirus A16 type 16 (Coxsackievirus A16, CA16) belongs to the genus Enterovirus in the family Picornaviridae. and capsid composition. Its nucleic acid is a single-stranded positive-strand RNA with a total length of about 7410 nucleotides, and the genome consists of an open reading frame (ORF) and 5', 3' non-coding regions (UTR). ORF encodes a polymeric precursor protein, which is hydrolyzed into three precursor proteins P1, P2 and P3 by proteolysis. P1 encodes the structural proteins VP1, VP2, VP3 and VP4 of the virus, and the three polypeptides of VP1, VP2 and VP3 are exposed in On the surface of the virus shell, VP4 is embedded in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C12N7/01C12Q1/70C12Q1/02C12R1/93
Inventor 张波邓成林徐琳琳秦成峰李晓丹袁志明叶寒青
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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