Cloning, Expression And Substrate Preparation Of Nsp14 Gene Of SARS Coronavirus

The purpose of experiment is to clone and express the Nsp14 gene of SARS coronavirus(SARS-CoV),to get the Nsp14 protein of SARS-CoV by prokaryotic expression . It was a promising candidate protein for further studies.Experiment I: The cloning of Nsp14 gene of SARS-CoV. On the basis of the reported nucleotides sequence of Nsp14 gene of SARS-CoV,the primers were designed, then the gene was cloned from the cDNA segment of SARS-CoV by PCR. The target fragment was digested with BamHI and XhoI. Then the gene was inserted into the prokaryotic expression vector pET30a to construct recombinant vector pET30a-exo. The recombinant vector was tested by PCR and was digested with BamHI and XhoI, then the sequence was tested. Nsp14 gene was identification with published genes. The vector of prokaryotic expressions of Nsp14 gene was successfully constructed.Experiment II: The Nsp14 of SARS-CoV was expressed and purified. the competent E.coli cells were trans-formed by the recombinant vector and induced by IPTG. The target protein was expressed in the form of inclusion body, and the conditions of expression and purification were probed. then the inclusion body was washed and refolded , Optimal refolding conditions were obtained by rapid dilution. The Nsp14 in the form fusion protein was obtained that was tested by SDS-PAGE.Experiment III: Preparation of substrate of Nsp14 of SARS-CoV. We obtained substrate of Nsp14 of SARS-CoV by transcribe RNA.It was a promising candidate protein for further studies.