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Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS)

A technology of homology and biological samples, applied in the fields of immunology and molecular biology, can solve the problems that no anti-SARSCoV vaccine has been developed

Inactive Publication Date: 2005-04-27
THE CHINESE UNIVERSITY OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, no protein-based vaccine against SARS CoV has been developed

Method used

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  • Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS)
  • Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS)
  • Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS)

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Experimental program
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Embodiment

[0236] The present invention confirms that the nucleocapsid protein of SARS CoV is the main antigen recognized by the immune response of individuals infected with SARS CoV. The present invention also determined that the N-terminal half (SEQ ID NO: 2) and the C-terminal half (SEQ ID NO: 6) of the nucleocapsid protein are part of this immune recognition. As described herein, these two protein parts function as diagnostic tools and prophylactic drugs against SARS infection

[0237] general method

[0238] 1. Cell Culture and Virus Production

[0239] Vero (monkey kidney fibroblast) cells were grown in Dulbecco modified Eagle medium (DMEM, Gibco BRL) containing 5% fetal bovine serum, 100 U / ml penicillin G, and 100 g / ml streptomycin at 37°C, 5% CO 2 Cultured in a tissue culture incubator with saturated humidity. The coronavirus strain CUHK-W1 was prepared by infecting Vero cells and collecting the supernatant when significant cytopathic changes (CPE) we...

example 1

[0264] Example 1 Identification of Nucleocapsids as Important Diagnostic Antigens by Western Blot (WB) Analysis

[0265] By detecting the antibody response of SARS patients to SARS CoV infection, it is possible to identify which SARS virus antigen the patient's immune response is directed against. A natural mixture of viral antigens is extracted from cultured cells containing viral growth and separated on a 10% polyacrylamide gel. figure 1 A shows the gel protein profile of the extracted protein after gel electrophoresis. The proteins on this gel were transferred to polyvinylidene difluoromembrane and incubated with the patient's sera, then the patient's antibody bound to the transferred protein was detected by an enzyme-linked antibody directed against the patient's antibody, and detected with an enzyme-linked fluorophore reagents for its analysis. The method is as described in paragraph (4a) above.

[0266] Such as figure 1 As shown in B, the antigens with the strongest ...

example 2

[0267] Example 2 ELISA studies using recombinant viral antigens to examine the importance of nucleocapsid proteins

[0268] In order to check that the nucleocapsid is the main SARS virus antigen recognized by the SARS sensitized immune system, SARS nucleocapsid recombinant antigen (N-terminal half and C-terminal half), spike protein are produced in Escherichia coli bacteria (3 subunits) and nonstructural proteins (NSP12 [2 subunits], NSP9 [1 intrinsic segment], NSP13 [all]). image 3 A shows the relationship between these virion molecules. image 3 B shows the regions produced recombinantly for each protein. image 3 C is the molecular weight of each recombinantly expressed protein that can be roughly observed by Coomassie brilliant blue stained gel.

[0269] These recombinant antigens were analyzed by ELISA with patient sera as described above. In this analysis, recombinant N-terminal nucleocapsid antigen (rNa) reacted with the same patient sera as natural virus extracts, ...

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Abstract

The present invention relates to the fields of immunology and molecular biology and describes compositions and methods for using proteins, peptides and nucleic acids related to the SARS CoV nucleocapsid protein and the spike glycoprotein. In particular, the present invention provides immunostimulatory preparations, prophylactic pharmaceutical preparations, diagnostic assays and kits for identifying and preventing SARS infections.

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of Patent Application No. 60 / 507,207 filed September 29, 2003, claiming the benefit of that prior application. field of invention [0003] The present invention relates to the fields of immunology and molecular biology, and describes compositions and methods of using proteins, peptides and nucleic acids related to SARS CoV nucleocapsid protein and spike glycoprotein. Background technique [0004] Severe acute respiratory syndrome (SARS) is caused by a disease known as SARS-CoV (Poutanen SM et al., 2003. N Engl J Med. Published at www.nejm.org March 31, 2003; Peiris JSM et al., 2003. Lancet 361, 1319-1325 ; Ksiazek TG et al, 2003.N Engl J Med.Published at www.nejm.org April 10, 2003; and DrostenC et al, 2003.N Engl J Med.Published at www.nejm.org April 10, 2003) novel coronavirus A new infectious disease caused by a virus. According to its genome structure of 29,751bp (Marra ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215C07K4/00C07K14/165C07K16/10C12Q1/70G01N33/53G01N33/532G01N33/547G01N33/552G01N33/569
CPCC12N2770/20034C07K2319/23G01N2469/20G01N2333/165C07K16/10C07K14/005G01N33/56983A61K39/215C12N2770/20022A61K2039/55566A61K2039/6031A61K39/12
Inventor 梁子明谭志恒马振雄林栢良陈基湘
Owner THE CHINESE UNIVERSITY OF HONG KONG
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