SARS冠状病毒多聚酶表位区的原核表达及应用

Erokaryotic expression and application of RNA polymerase epitope domain of SARS coronavirus

目的原核表达SARS冠状病毒非结构蛋白RNA多聚酶表位区,了解其在SARS病毒感染诊断及作为病毒复制指标的意义。方法通过PCR扩增获得RNA多聚酶表位区基因,与原核表达载体Pet32a+连接,转化E.coliBL-21表达获得重组融合蛋白。经切胶透析纯化后,应用ELISA检测SARS患者及动物模型血清标本的IgG抗体。结果获得原核表达的RNA多聚酶表位区。ELISA检测正常人血清均阴性,SARS患者血清阳性率为95%;检测SARS病毒感染实验动物血清阳性率100%,灭活纯化病毒接种恒河猴均为阴性。结论RNA多聚酶表位区具有很好的抗原性,可用于感染诊断的检测及作为病毒复制性指标。

Objective To study the antigenicity of the RNA polymerse epitope domain and the relationship between the protein and the SARS-CoV replicaction. Methods The fragment was amplified by PCR, ligated with the prokaryotic expression vector pet32a+ and transformed into E. coli BL-21. The expressed fusion protein identified by WB and ELISA was used to detect the anti-SARS CoV IgG in different sera. Results The fusion protein was expressed successfully in E. col i BL-21. Detected by ELISA, the positive rates of the anti-SARS IgG in the health donor, patients, infected animals and the rhesus administrated with the inactivated-virus were 0%, 95%, 100%, 0% respectively. Conclusion The expressed RNA polymerase epitope domain is antigenic. It can be used to diagnose the infection and demonstrate the replication of SARS-CoV.