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Expression of Nucleocapsid and Small E Proteins of SARS Coronavirus and Preparation for Their Specific Monoclonal Antibodies

Author: WangPing
Tutor: XuZhiKai;HuangQingSheng;DingTianBing
School: Fourth Military Medical University
Course: Pathogen Biology
Keywords: Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Envolope Monoclonal antibody
CLC: R392
Type: Master's thesis
Year: 2005
Downloads: 59
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Abstract

Severe acute respiratory syndrome (SARS) is a potentially fatal atypical pneumonia that arose in Guangdong Province of the People’s Republic of China in November 2002. Within 6 months SARS spread to over 30 countries and killed over 800 people. This outbreak has had a profound impact on public health and economies worldwide and reinforced the danger of emerging infectious diseases in densely populated societies. There were over 8000 SARS patients in the world, and this contagious disease’s death rate is high( the average rate is about 10% in the world,and is about 15% in China). After then, scientists confirms that SARS is a highly infectious disease caused by a novel coronavirus ,SARS-CoV, SARS-associated coronavirus has been identified as the etioiogic agent of SARS.There is only one thing to do the clinical treatment for the SARS patients before the special medicine anti-SARS-CoV bedevelopped out. The research on the vaccine of SARS-CoV still needs a longer process.The availability of monoclonal antibodies (MAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection.As the SARS breaked out, the global scientisties did the detailed research to the coronal virus. By study the nucleocapsid (N) and the small envelope ( E) gene stabilities of the SARS coronal viruses, scientisties discover them mutation rate is low. Nucleocapsid package and protect the virus genome, and it’s monoclonal antibody probably have the neutralization activity; The small E protein is placed in the virus envelope, the gene structure is simple, easy operation, but the function is still not clear at present.We adopt the method of RT- PCR, amplified the coding gene of N protein by extracting out from virus specimen. Amplified out one 1,269bps DNA sequence from the specimen. Result of DNA sequencing confirmed that the amplified fragment was N protein gene. After being confirmed by DNA sequencing, the gene was sub-cloned into prokaryotic expression vector. The recombinant N protein was expressed in E. coli by induction with isopropyl-β -D-thiogalactopyranoside (IPTG) and recovered from SDS-PAGE gel and served as immunogen to immunized BALB/c mouse for the preparation of the mAb. SDS-PAGE analysis showed that the expressed protein in E.coli was located at approximately Mr being 43×103 on the gel. It is confirmed that this expressed protein canreact to special immunity respond with the convalescent period SARS patients’ serum by the Western blot. The recombinant N protein was recovered from SDS-PAGE gel and immunized in BALB/c mice. Western blot analysis proved that three MAbs obtained could reacted specifically to the recombinant N protein. The recombinant N protein and it’s mAbs lay the foundation for further in development of early diagnosis of SARS coronavirus infection and of research on neutralized antibody activities.The gene sequence coding E protein is selected from the Genebank ,and the complete E gene was used with link-PCR method to synthesize. After confirmed with DNA sequencing it linked with the human immunio-globulin IgG sequence Fc segment, and then the fused gene was recombined into prokaryotic expression vector, pPRO EX Hta plasmid. EFc protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen to immunized BALB/c mouse in the preparation of the mAb. Result of DNA sequencing confirmed that the amplified fragment was E protein gene. SDS-PAGE analysis showed that the expressed protein in E.coli was located at approximately Mr being 37×103 on the gel. This protein could be further confirmed by Western-blot with serum of a SARS patient. The recombinant EFc protein was recovered from SDS-PAGE gel and immunized in BALB/c mice. There is 2 mAbs for anti-E protein. Western blot analysis proved that 2 mAbs obtained could reacted specifically to the recombinant EFc protein. The recombinant EFc protein and mAbs lay the foundation for

Expression of Nucleocapsid and Small E Proteins of SARS Coronavirus and Preparation for Their Specific Monoclonal Antibodies

Abstract

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