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Feline calicivirus reverse genetic platform and construction method thereof

A feline calicivirus and reverse genetic technology, applied in the field of molecular biology, can solve the problems of time-consuming, labor-intensive and complicated process of PCR cloning

Pending Publication Date: 2021-10-12
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the research of FCV reverse genetics platform, the whole genome is cloned according to the single restriction nuclease recognition site of FCV. However, the traditional PCR cloning is time-consuming and labor-intensive, and the process is relatively complicated.

Method used

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  • Feline calicivirus reverse genetic platform and construction method thereof
  • Feline calicivirus reverse genetic platform and construction method thereof
  • Feline calicivirus reverse genetic platform and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 sequence analysis and the design of primer

[0026] According to the FCV (DL19) strain sequence, DNA MAN and DNA Star were used to design primers, and the online biological software NovoPro (https: / / www.novopro.cn / tools / rest_summary.html) was used to limit the FCV (DL19 strain) sequence 4505 XholⅠ restriction sites were selected for homology arm primer design. The primers were synthesized by Beijing Huada Biotechnology Co., Ltd., and the primer sequences are shown in Table 1.

[0027] Table 1 FCV genome primer sequences

[0028]

[0029]

Embodiment 2

[0030] The extraction of embodiment 2 viral total RNA and the acquisition of cDNA

[0031] The extraction of total virus RNA was carried out according to the instructions of Trizol Reagent, and the specific steps were as follows:

[0032] 1) Take out the sample stored in the -80°C refrigerator, and wait for the sample to completely melt and return to room temperature;

[0033] 2) Add chloroform according to 200 μL chloroform / mL Trizol, vortex and oscillate for 15 seconds to fully mix the sample, and let stand at room temperature for 3 minutes;

[0034] 3) After centrifugation at 12,000rpm for 15min at 4°C, the sample was divided into three layers, and the uppermost aqueous phase containing RNA (about 450μL) was slowly drawn into a new 1.5mL EP tube, and pre-cooled isopropanol was added at a ratio of 1:1. , mix the liquid gently and place it on ice for 10 minutes;

[0035] 5) Take out the mixed liquid and centrifuge at 12,000 rpm at 4°C for 10 minutes. After discarding the su...

Embodiment 3

[0044] Example 3 Expression of eGFP recombinant vector construction

[0045] In order to further verify whether the pBluescript II SK (+) modified vector system is available, the whole eGFP genome was inserted after the pBluescript II SK (+) modified vector FCV 5'UTR to construct a recombinant vector expressing eGFP, and to verify the rescue system minigenome system.

[0046] Expression of eGFP recombinant vector transfection:

[0047] Inoculate BHK-T7 into a 6-well cell plate, and when the cells cover about 80% of the bottom of the well, use Lipofectamine 2000 to transfect the constructed pBluescript II SK(+)-eGFP, and observe the eGFP gene expression under a fluorescent microscope 48 hours after transfection Condition.

[0048]The constructed pBluescript II SK(+)-eGFP was transfected with Lipofectamine 2000, and 48 hours after transfection, the expression of the green fluorescent protein reporter gene was observed under a fluorescent microscope. As shown in Figure 2, the ex...

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Abstract

The invention provides a feline calicivirus reverse genetic platform and a construction method thereof. A feline calicivirus DL19 strain genome is divided into two segments to be connected to a modified pBluescript II SK (+) vector, a feline calicivirus DL19 strain full-length infectious clone plasmid capable of serving as the reverse genetic platform is constructed, and a foundation is laid for subsequent virus rescue and genome structure research.

Description

technical field [0001] The application belongs to the fields of molecular biology and veterinary medicine. Specifically, the application provides a feline calicivirus reverse genetics platform and a construction method thereof. Background technique [0002] Feline calicivirus (Feline calicivirus, FCV) used to belong to the calicivirus genus of the Picornaviridae family. In 1981, the International Committee on Taxonomy of Viruses (ICTV) listed it as a separate Caliciviridae. At present, the family Caliciviridae consists of five genera, namely Sapovirus, Lagovirus, Vesivirus, Neubervirus, and Norovirus. FCV was isolated for the first time in 1957. It mainly infects domestic cats, and can also infect cats such as lions, tigers, and leopards, posing a certain threat to the health of cats. FCV existed for many years in a non-pathogenic form before 1998 with a low mortality rate. Since 1998, malignant systemic diseases with extremely high mortality caused by FCV have been report...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/40C12N7/00
CPCC12N15/85C12N7/00C07K14/005C12N2800/107C12N2770/16022C12N2770/16021
Inventor 梁瑞英崔尚金梁琳赵静杰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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