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HuNoV GII.4 clinical isolate sequence and construction and application of infectious clone thereof

An infectious cloning and sequencing technology, applied in genetic engineering and biological fields, can solve the problem of no HuNoV infectious cloning, etc., and achieve the effect of simple construction method and high reliability

Active Publication Date: 2019-08-20
CHENGDU VIROGEN BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early studies abroad have tried to construct a reverse genetic system of HuNoV, and achieved some success [7, 8], but so far there has been no report of an infectious clone of the Chinese strain of HuNoV

Method used

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  • HuNoV GII.4 clinical isolate sequence and construction and application of infectious clone thereof
  • HuNoV GII.4 clinical isolate sequence and construction and application of infectious clone thereof
  • HuNoV GII.4 clinical isolate sequence and construction and application of infectious clone thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Replication and toxin-producing ability of HuNoV infectious clones

[0055] Spread eukaryotic cells A, B, and C on 10 cm cell culture dishes, transfect eukaryotic cells A, B, and C with 10 μg of HuNoV plasmid after 16 hours, and collect cells after 3 days; or spread eukaryotic cells C on 6-well plates, 16 hours Afterwards, 3 μg of HuNoV plasmid was transfected into eukaryotic cell C, and the cells were collected 4, 8, 12, 24, and 36 hours after transfection; after centrifugation at 1100 rpm for 5 minutes:

[0056] Cell pellet:

[0057] 1. Take 10 7 cells, and the total RNA in the cells was extracted with the RNA extraction kit from MN Company.

[0058] 2. Using the RNA as a template, use the HuNoV GII nucleic acid detection kit of Sun Yat-Sen University Daan Gene Co., Ltd. to detect the replication of HuNoV RNA in various eukaryotic cells by real-time quantitative PCR (such as figure 2 );

[0059] Cell supernatant:

[0060] 1. Use a 100KD ultrafiltratio...

Embodiment 2

[0070] Example 2: Infectivity of Progeny Virions Produced by HuNoV Infectious Clones

[0071] 1. After transfecting C cells with 10 μg pCMV-HuNoV plasmid for 3 days, collect the cell supernatant, centrifuge at 1100 rpm for 5 minutes, pipette the cell supernatant into clean collection tubes, aliquot 1 mL in each tube and store in a -80°C refrigerator for later use.

[0072] 2. Spread 6-well plates with C cells, add 1 mL of virus supernatant to each well after 16 hours and infect for 4 hours, wash with 2 mL of 1×PBS for 3 times, then replace with fresh medium and culture for 24 hours.

[0073] 3. Remove the cell supernatant, add 2mL 1×PBS to each well to wash 3 times, scrape off the cells with a cell scraper, transfer to a 1.5mLEP tube, centrifuge at 1100rpm for 5min, remove the supernatant, and keep the cell pellet.

[0074] 4. Use the RNA extraction kit of MN company to extract the total RNA in the cells.

[0075] 5. Using the RNA as a template, use the HuNoV GII nucleic acid...

Embodiment 3

[0076] Example 3: Infectious replication ability of progeny virions produced by HuNoV infectious clones

[0077] 1. After transfecting C cells with 10 μg pCMV-HuNoV plasmid for 3 days, collect the cell supernatant, centrifuge at 1100 rpm for 5 minutes, pipette the cell supernatant into clean collection tubes, aliquot 1 mL in each tube and store in a -80°C refrigerator for later use.

[0078] 2. Spread 35mm glass-bottomed dishes with C cells, add 1mL virus supernatant to each well after 16 hours and infect for 4 hours, wash with 2mL 1×PBS for 3 times, then replace with fresh medium and culture for 24 hours.

[0079] 3. Remove the cell supernatant, add 2mL 1×PBS to each small dish and wash 3 times.

[0080] 4. Add 1 mL of 4% paraformaldehyde to each well to fix at room temperature for 10 min.

[0081] 5. Remove paraformaldehyde, add 1mL 1×PBS to each small dish and wash once.

[0082] 6. Remove PBS, add 1mL 2% TritonX-100 to each small dish, and permeabilize at room temperatur...

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Abstract

The present invention discloses a human norovirus (HuNoV) GII.4 Chinese clinical isolate sequence and an infectious clone thereof, as well as a construction method and an application of the infectiousclone. Certain differences exist between the clinical isolate sequence and currently disclosed domestic and foreign HuNoV GII.4 sequences, besides, the clone can effectively proliferate in a varietyof eukaryotic cell lines and can also produce progeny infectious virions; and the inactivated virions can be used as a novel vaccine of HuNoV for development study.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology. Specifically, the invention relates to the construction and application of a human norovirus (HuNoV) GII.4 Chinese clinical isolate sequence and its infectious clone. The clone is capable of efficiently proliferating in eukaryotic cells and capable of producing infectious progeny virions. Background technique [0002] Human Norovirus (HuNoV) is the main cause of acute non-bacterial epidemic gastroenteritis. It often breaks out in semi-closed communities such as nursing homes, schools, hospitals, cruise ships and disaster relief agencies, and is susceptible to all age groups. Many people, especially infants, the elderly, and immunocompromised individuals [1], still lack NoV vaccines. Once an outbreak of HUNoV is difficult to control, the virus can be transmitted from person to person through the aerosol-like dispersed virus particles formed by infected person's vomit or feces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/63C12N15/66A61K39/12A61P31/14
CPCA61K39/12A61K2039/5252A61P31/14C12N7/00C12N2770/16021C12N2770/16034
Inventor 胡勤学章牡丹
Owner CHENGDU VIROGEN BIOPHARMACEUTICAL CO LTD
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