Coronal virus genetic engineering protein and use thereof

A genetic engineering and protein technology, applied in the field of coronavirus genetic engineering protein and its application, can solve the problems of long time, complicated operation, increased difficulty of specimens and the risk of laboratory infection

Inactive Publication Date: 2007-07-04
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus isolation method takes a long time to obtain the result, and the operation is complicated, and the isolation rate is low
Although the RT-PCR detection method saves time and only takes a few hours, it has high sensitivity and specificity, but it is prone to false positive results.
Both of the above detection methods require clinical specimens containing the virus, which greatly increases the difficulty of obtaining specimens and the risk of laboratory infection

Method used

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  • Coronal virus genetic engineering protein and use thereof
  • Coronal virus genetic engineering protein and use thereof
  • Coronal virus genetic engineering protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The preparation of embodiment 1 full-length SARS-CoV Spike gene

[0077] The structure of the full-length SARS-CoV Spike gene is shown in Figure 1.

[0078] 1. Construction of recombinant plasmid pUCm-T-S(n) containing SARS-CoV Spike gene N fragment

[0079] Take out the E.coli strain containing the plasmid pSn (containing the gene sequence 1-1469bp encoding the Spike protein) preserved with glycerol from the refrigerator, streak it on the LB plate containing kanamycin (100 μg / mL), and culture it overnight at 37°C . Pick a single colony with a sterile toothpick, inoculate into 3 mL of LB liquid medium containing kanamycin (100 μg / mL), cultivate overnight at 37° C., 220 rpm, and extract plasmid pSn by alkaline lysis. Digest pSn with Xba I and Kpn I, recover a 1521bp fragment, and then ligate it with the same double-digested plasmid pUCm-T (Shanghai Sangong), and transform the ligation product into Escherichia coli JM109 to obtain a recombinant plasmid pUCm-T-S (n) Pos...

Embodiment 2

[0085] Embodiment 2 Contains the construction of the recombinant baculovirus transfer vector of full-length SARS-CoV Spike gene

[0086] Amplification Primer Synthesis: Upstream Primer:

[0087] TTCTCgAgATgTTTATTTTCTTATTATTTTCTTACTCTCACTAg (the underline is the Xho I restriction site), and the downstream primer gCgAATTCTTATgTgTAATgTAATTTgACACCCTTg (the underline is the EcoR I restriction site). pUCm-T-S is used as a template, and the target gene SARS-CoV S is amplified by conventional PCR method, and the size of the PCR product is checked by gel electrophoresis to see if it is in line with the expectation. The PCR product was recovered and cloned into the pUCm-T vector, and transformed into Escherichia coli E.coli JM109, and the colony was picked to quickly extract the plasmid for enzyme digestion identification, and the obtained positive clone was named pUCm-TFS.

[0088] The recombinant plasmid pUCm-TFS DNA was extracted and the nucleotide sequence was determined by the dideo...

Embodiment 3

[0092] Example 3 Construction of recombinant insect baculovirus containing full-length SARS-CoV Spike gene

[0093] 1. Preparation of Insect Cell Culture

[0094] Add 4 mL of complete insect cell culture medium containing 10% fetal bovine serum to a 25 cm2 culture flask. Take out an ampoule of cryopreserved sf9 cells, and quickly place it in a 37°C water bath. Break the neck of the ampoule and transfer the contents to a 25cm2 culture bottle. Shake the flask gently by hand to disperse the cells evenly, and incubate at 27°C for 2-3h until the cells adhere to the wall. Remove the old culture medium and replace with 5 mL of fresh complete culture medium containing 10% fetal bovine serum. Continue to incubate and change the medium every 3 days (remove the old medium and replace it with fresh one) until the cells grow confluent. Prepare a new 25cm2 culture flask and add 4mL of complete culture solution containing 10% fetal bovine serum. When the sf9 cells have grown to confluen...

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Abstract

The invention discloses a genetic engineering protein of coronavirus and its application. That is genetic engineering protein FSPA relevant to S protein of coronavirus SARS- CoV and nucleotide sequences amnio sequence and code said FSPA. The invention also discloses a recombinant insect virus strain containing SARS- CoV Spike- recombinant autographa californica nucleopolyhe-drovirus AcNPV- FSPA, CCTCC No.V200513, inserts into the expression box of SARS- CoV Spike. The inventiton also discloses the application of FSPA in checking the coronavirus SARS- CoV, which is the pathogen for acute respiratory syndrome.

Description

technical field [0001] The present invention relates to the recombinant genetically engineered protein FSPA related to coronavirus SARS-CoV, and also relates to the performance of the genetically engineered protein FSPA in detecting acute respiratory syndrome (Severe acute respiratory syndrome, SARS, also known as infectious atypical pneumonia, AP). Pathogen—Application in SARS-CoV. Background technique [0002] Severe acute respiratory syndrome (Severe acute respiratory syndrome, SARS), also known as infectious atypical pneumonia (Atypical pneumonia, AP). It can cause symptoms such as fever, dry cough, and dyspnea in the infected person, and severe infection can lead to death [Marra M A, Jones S J, Astell C Ret al. The genome sequence of the SARS-associated coronavirus [J]. Science, 2003; 300: 1399.][Lew T W, KwekT K, Tai D et al.Acute respiratory distress syndrome incritically ill patients with severe acute respiratory syndrome[J].JAMA,2003;290:374...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/86C12N7/01
Inventor 徐进平孟小林王健鲁伟
Owner WUHAN UNIV
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