Coronal virus genetic engineering protein and use thereof

A technology of genetic engineering and coronavirus, applied in the direction of genetic engineering, application, virus/bacteriophage, etc., can solve the problems of increasing the difficulty of specimens and the risk of laboratory infection, low separation rate, long time, etc.

Inactive Publication Date: 2009-09-16
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

[0073] The preparation of embodiment 1 full-length SARS-CoV Spike gene

[0074] For the structure of the full-length SARS-CoV Spike gene, see figure 1 .

[0075] 1. Construction of recombinant plasmid pUCm-T-S(n) containing SARS-CoV Spike gene N fragment

[0076] 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) Posi...

Embodiment 2

[0082] Embodiment 2 Contains the construction of the recombinant baculovirus transfer vector of full-length SARS-CoV Spike gene

[0083]Synthesis of amplification primers: upstream primer: TTCTCgAgATgTTTATTTTCTTATTATTCTTACTCTCACTAg (the underline is the Xho I restriction site), downstream primer gCgAATTCTTATgTgTAATgTAATTTgACACCCTTg (the underline is the EcoRI restriction site). Using pUCm-T-S as a template, the target gene SARS-CoV S was amplified by conventional PCR method, and the size of the PCR product was checked by gel electrophoresis to see if it was 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, the colony was picked and the plasmid was quickly extracted for enzyme digestion identification, and the positive clone obtained was named pUCm-TFS.

[0084] The recombinant plasmid pUCm-TFS DNA was extracted and the nucleotide sequence was determined by the dideoxy method. The s...

Embodiment 3

[0088] Example 3 Construction of recombinant insect baculovirus containing full-length SARS-CoV Spike gene

[0089] 1. Preparation of Insect Cell Culture

[0090] 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 coronavirus genetically engineered protein and its application. The genetically engineered protein FSPA related to the S protein of coronavirus SARS-CoV and the nucleotide sequence and amino acid sequence encoding the FSPA. A recombinant insect virus strain containing SARS-CoV Spike gene - recombinant Autographa californica nuclear polyhedrosis virus strain AcNPV-FSPA, CCTCC NO.V200513, the recombinant virus strain is inserted into the SARS-CoV Spike expression cassette. Application of genetically engineered protein FSPA in detecting the pathogen of severe acute respiratory syndrome—coronavirus SARS-CoV.

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--SARS-CoV application. 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 incriticallyill patients with severe acute respiratory syndrome[J].JAMA,2003;290:374.][Holmes KV.SARS2associate...

Claims

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Application Information

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Patent Type & Authority Patents(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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