SARS冠状病毒多聚酶表位区的原核表达及应用

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。

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

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.