Monoclonal antibody of SARS coronavirus N protein and its application

A SARS virus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problems of long time required, high technical difficulty, and high equipment requirements

Inactive Publication Date: 2005-09-28
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the technical difficulty is high, the operation is complicated, the time required is long, and the equipment requirements are high, so it is not suitable for mass detection.

Method used

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  • Monoclonal antibody of SARS coronavirus N protein and its application
  • Monoclonal antibody of SARS coronavirus N protein and its application
  • Monoclonal antibody of SARS coronavirus N protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: the expression of SARS N protein

[0077] After inserting the full-length gene of SARS N protein (SEQ ID NO: 1) into the expression vector pETs, transfect Escherichia coli BL21 (DE3). Add IPTG to a final concentration of 0.5mmol / L to induce bacteria to express N protein, then purify by Ni-NTA affinity chromatography according to the operation manual, and then detect by SDS-PAGE.

Embodiment 2

[0078] Embodiment 2: Preparation of polyclonal antibody

[0079] Animal immunization: Rabbits were immunized three times with the purified N protein expressed in Example 1. For the first immunization, use Freund’s complete adjuvant and N protein in equal amounts and then emulsify it, and inject it at multiple points under the skin, 1 mg / cause; for the second and third immunizations, use Freund’s incomplete adjuvant and N protein in equal amounts, and then emulsify , The immunization method and dosage are the same as the first immunization. The interval between each immunization is about one month, and the blood is collected one week after the third immunization.

[0080] Indirect enzyme-linked immunosorbent assay (indirect ELISA) to detect the titer of antiserum:

[0081] Coat the ELISA detection plate with N protein, block with PBS blocking solution containing 10% bovine serum and 0.1% Tween, then add different dilutions of antiserum, incubate at 37°C for 2 hours, and set t...

Embodiment 3

[0085] Example 3: Establishment of anti-N protein hybridoma cell lines and preparation of monoclonal antibodies thereof

[0086] 1. Animal immunization: BALB / C mice were immunized with recombinant N protein three times. The immunization method was the same as that of rabbits, and one month after the third immunization, the mice were recalled. The titer of antiserum detected by indirect ELISA was 64000.

[0087] 2. Preparation and screening of hybridomas: Take mouse spleen cells 3 days after memory stimulation and NS-1, SP2 / 0 myeloma cells were fused with 50% PEG according to conventional methods, and after screening and cloning by indirect ELISA method, we obtained Hybridoma cell strains, in which the S-39-2 clone was used in this work (the deposit number is CCTCC NO: -C200407). The antibody subtype secreted by this cell line is IgG1.

[0088] 3. Preparation of ascites and purification of antibodies: each mouse was intraperitoneally injected with 0.5ml pristane, 7-10 days l...

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Abstract

The present invention relates to one kind monoclonal antibody with specificity on the serial proteins shown in SEQ ID No. 2, hybrid tumor cell train generating the monoclonal antibody and its preparation process. The monoclonal antibody of the present invention may be used in diagnosing and treating diseases caused by SARS virus infection, especially SARS. As the application, the present invention also proposes at least one kind of diagnosis kit with the monoclonal antibody of the present invention.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal antibody to the N protein of SARS coronavirus and an application thereof. Background technique [0002] From November 2002 to February 2003, atypical pneumonia of unknown etiology broke out in Guangdong, my country, and then similar cases appeared in Hong Kong, Vietnam, Canada, Singapore, the United States, China and other parts of the world, causing panic among people all over the world And the high attention of governments and researchers of various countries. On March 5, 2003, the World Health Organization (WHO) issued a global health alert on atypical pneumonia and named the disease severe acute respiratory syndrome (SARS). Scientists have shown that severe acute respiratory syndrome (SARS) is caused by a new coronavirus (sars). This new coronavirus is an enveloped positive-strand RNA virus with a full-length genome of 29,727 nucleotides and 11 open reading frames e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P11/00A61P31/14C07K16/10C07K19/00C12N5/16G01N33/577
Inventor 孙兵尚勃季永镛田林李林
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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