Study On The Expression And Genetic Immunization Of Chimeric Competent Fragments Of Hantaan Virus M And S Segments

Hemorrhagic Fever with Renal Syndrome (HFRS), which is caused by hantavirus, is an acute infectious disease characterized by fever, vascular hemorrhage and kidney dysfunction. I~ China about 50,000-100,000 cases of HFRS are reported yearly, with the severe symptoms and high mortality. There are still no effective therapeutical drugs and prophylactic vaccines directed to HFRS until now. Hantaviruses are tripartite negative-stranded RNA viruses in the Bunyaviridae family. The viral RNA segments, L, M and 5, encode an RNA dependent RNA polymerase (IWRP), two envelope glycoproteins (OP. G1 and G2), and nucleoprotein (NP). respectively, it is indicated that the OP could 7 stimulate neutralization antibodies and could protect infected animal and human body from Hantavirus lethal infection. Moreover, the neutralization sites of GP mainly exist in G2. But the immunogenicity of OP is weak. The antibodies stimulated by GP are produced later and the titers are low. The prophase work in our laboratory showed that the main antigenic sites of NP existed in the amino proximal of NP (0.7Kb fragment of S segment). Besides, the related study demonstrated that among the structural proteins, NP had the strongest immunogenicity and could stimulate high titers and long-lasting antibodies. In addition, it 憊as confirmed that NP could induce cellular immunity activities against HTNV infection. Therefore GP and NP may play equal important role in stimulating the humoral and cellular immunity .How to bring them into full play in stimulating immune responses is a problem waiting to solve in the research of Hantavirus genetic engineering vaccine. In this research, we fused expressed G2 fragment of M segment and 0.7Kb fragment of S segment. We expected it could make up for each other抯 deficiencies. First, the prokaryotic expression vectors pGEX-4T- I -62S0. 7 and pGEX-4T-l-S0.7G2 were constructed. After inducing expressed the fusion proteins, SDS-PAGE, Western-blot and ELISA methods were used to identify the activities of the fusion proteins. SDS-PAGE results showed that the fusion proteins expressed by the chimeric genes could not be observed. Western-blot analysis showed that fusion proteins (3ST-G2NO.7and GST-N0.7G2 were induced and their molecular weights were about 1 OOKD. ELISA results 8 showed that fusion proteins had high combination activities with HTNV NP specific mAb and low combination activities with HTNV GP specific niAb. It is suggested that the fusion proteins kept the activities of their parental proteins. Then the recombinant eukaryotic expression vectors pcDNA3. I -02S0.7, pcDNA3.I-SO.702, pcDNA3.I-02 and pcDNA3.l-SO.7 were constructed. After transfecting COS-7 cells by lipofectin, RT-PCR, immunoflourescence and immunoprecipation were used to identify the expressions. The results showed that the recombinant eukaryotic expression vectors transient expressed in COS-7 cells and the products of chimeric genes were fused proteins. On the basis of the prophase work, BALB/c mice were vaccinated by recombinant eukaryotic expression vectors. ELISA and cell microculture neutralization test in vitro were used to detect the humoral immunity i...