Expression Of The S1 Gene Of SARS And Establishment Of The ELISA Detection Method

The main proteins of SARS coronavirus are RNA polymerase, S protein, E protein, N protein, in which the S protein is mainly in charge of the infection. The difference of S protein will not only cause the change of the host but will also lead to the cold, peritonitis, gastroenteritis and many diseases of the host. Meanwhile, S protein is also the main part which influences the level of the infection. We amplified the S1 genes of AY304492 by PCR, cloned and successfully expressed the SI protein in E.coli. Then, we purified the expression protein and established the ELISA method of detecting the SARS antibodies by the recombinant protein. Also, we cloned the SI genes of AY274119 and then established the eukaryotic expression vector, which is the basis of the further study and produce of nucleic acid bacterin.1 .Referring to the sequence of AY304492 published by GeneBank, we designed a set of primer. The PCR amplification product of AY304492 SI protein was analyzed by agarose gel electrophoresis and demonstrated to be a specific strain about 86bp. The recycle was cloned into the pGEM T-Easyl vector, and digested by BamH I and Xho I. Then we recycled the target fragments, ligated them respectively to the pGEX-4Tl expression vector with T4 ligase, which is digested by the same two enzymes. And we also further sequenced it and analyzed its autoploidy. After the cloning the Sl genes into the expression vector pGEX-4Tl, further digesting by BamH I and XhoI and identification,we finally got the positive expression vector pGEX-4Tl-S1.2.The host BL21 (DE3) was induced by IPTG with the concentration of 2 mmol/L. The amount of expression counts 36.363 percent of the whole proteins. By Western Blotting, the proteins expressed can be detected by SARS positive serum, which will contribute greatly to the establishment of SARS antibody detecting method.3.Using the SI protein expressed as antigen, we established the indirect method of detecting SARS virus and verified the conditions of ELISA. The results indicate that the optimal coating concentration is 50μg/ml, the optimal coating condition is 37°C, 1h, 4°C overnight, and working condition of HRP-coating antigen IgG is 1 to 400. The method is characteristic of its well specificity in which the serum of patient did not react with the regular serum. The indirect ELISA method provided a technology for the diagnosis and serology detection of the SARS virus in China. And it is also the basis of the product of kit.4.The PCR amplification product of AY274119 was cloned into vector pMD18-T. Then we recycled the target fragment after the agarose gel electrophoresis and cloned the S1 genes into the recombinant eukaryotic expression vector pVAX-1. Finally,we got the eukaryotic expression vector pVAX-S1 through the digestion and identification of EcoR V and Xho I,and verified that the recombinant vector can express successfully in the extraneous cells. The successful establishment of eukaryotic expression vector will make great contribution to the further study and produce of the SARS DNA bacterin.