Experimental Study On Recombinant DNA Vaccine Against SARS-Associated Coronavirus M Gene

SARS(Severe Acute Respiratory Syndrome)is a contagious disease that has high infectivity and mortality as well as heavy damage on public health.The World Health Organization (WHO) announced in April 2003 that A novel coronavirus has been identified as the main causative agent of SARS. This virus is named SARS-Associated coronavirus (SARS-CoV). SARS-CoV is a new coronavirus which entirely differs from other coronavirus. It has a high homogeneity with coronavirus coming from animals and strong virulence which results in respiration disfunction, even death. At present, there isn t specifically effective therapy to cure viral infection. The best method to control this kind of disease is prevention. Among the methods, developing vaccine is a key step. DNA vaccine with incomparable advantage is the third generation vaccine which follows pathogeny vaccine and subunit vaccine. Thus, this study choose the subject of SARS-CoV DNA vaccine and membrane-protein gene of SARS-CoV as candidate vaccine gene. we constructed recombinant DNA vaccine containing SARS-CoV M geneand observed the production of SARS-CoV specific immune response in immunized mice, so as to investigate the feasibility of SARS vaccine .This study includes two parts.In the first part, we acquired M gene sequence of SARS-CoV SIN2774 from GenBank. According to this sequence, the primers of M gene are designed by software primer Primier 5.0. Among the primers, we chose the best one and appended enzyme sites of EcoR I and Xho I at the 5 end of the primer, which are included in clone sites of pcDNA3.1(+). Using cDNA of SIN2774 from Singapore as template, PCR amplified SARS-CoV M gene at appropriate conditions. The amplified DNA was digested by restriction endonuclease EcoR I and Xho I at the same time, and ligated into pcDNA3.1(+) vector. The recombinant plasmid including SARS-CoV M gene was named pcDNA3.1(+)/M. The recombinant plasmid was identified by restriction endonuclease digestion and sequence analysis.In the second part, the recombinant plasmid pcDNA3.1(+)/M was largely extracted and was used to immunize the BALB/c mice intramuscularly. Mice were divided into three groups randomly: pcDNA3.1(+) control plasmid group, low dosage (50ug/mouse) and high dosage(100ug/mouse) of pcDNA3.1(+)/M groups. After 2> 4^ 6 weeks, the SARS-CoV antibodies in the serum of mice were detected with ELISA kit. And the level of cytokine IFN-yin spleen cell culture supernatants and capability of lymphocyte proliferative response is determined.This results showed that the sequence of inserted M gene which came from recombinant plasmid was entirely consistent with thesequence in the GenBank and their ORF was not changed. The results suggested that M gene had been inserted correctly into pcDNA3.1(+) plasmid. Animal test revealed that specific anti SARS-CoV antibodies can be detected in serum of all animals inoculated with recombinant plasmid and the level of IFN-y in spleen cell culture supernatants ascended timely and capability of lymphocyte proliferative response increased. Contrasted with control group inoculated empty pcDNA3.1(+)/M plasmid, it has significant difference. Conclusion: This study successfully constructed recombinant plasmid pcDNA3.1(+)/M which can induce specific antibodies and enhance the functions of cell-immunity in mice.This might be benefit for further research on the safe and efficient SARS DNA vaccine.