The Cloning And Expression Of Hantavirus S Segment In Escherichia Coli

Objective: Hantaviruses (HV) cause two human infections: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrom (HPS). The nucleocapsid protein (NP) is one of its main structural proteins. This study is to clone and express the HV S segment which encode the NP in Escherichia coli (Ecoli) BL21(DE3), and identify its product.Methods: The ORF of the S gene of hantavirus strain L99 and Z10 was obtained by PCR from pGEM-T-L99S and pGEM-T-Z10S, then the former was cutted by EcoRI and XhoI, the latter was cutted by SacI and XhoI. After purifed, they were linked with the longer segment of pET32a and pET28a which were cutted by corresponding enzemies. Then they were transformed into the competent cell of E. coli Top10. After screened with antibiotics, the positive recombinant was select. After identified by PCR, double enzymes cutting and sequence analysis, the correct recombinant plasmids were transformed into the competent cell of E. coli BL21(DE3). Then the target protein was induced with IPTG, and it's antigenicity was identified with HFRS patients' serum. Moreover the optimal concentration of IPTG and tune at which the maximal product of target protein obtained was tested in our study.Results: The S gene of strain L99 and Z10 was obtained by PCR and was linked with pET32a and pET28a. Then the positive recombinant were selected and were named as pET32a-L99S, pET32a-Z10S, pET28a-L99S and pET28a-Z10S respectively. After identified by PCR, double enzymes cutting and sequence analysis, the S gene was correctly combined with the vectors. And the ORF hasn't been changed. Then the recombinant plasimids was transformed into the competent cell of E. coli BL21(DE3). The positive ones were selected and namedas E.coli BL21(DE3)/ pET32a-L99S, E.coli BL21(DE3)/ pET32a-Z10S, E.coli BL21(DE3)/ pET28a-L99S and E.coli BL21(DE3)/ pET28a-Z10S respectively. The former two were induced with IPTG, and the yield of target protein reach 40% of the total protein. The latter two were induced with IPTG and the yield of target protein reach 32% and 27% respectively. We optimized the expression on the further. As the results demonstrated, the recombinant protein of pET32a and HV S gene was affected by concentration of inducer and the hours, they reach maximal yield at 0.5 mmoL and 1.0 mmoL IPTG after induced 5h. The recombinant protein of pET28a and HV S gene was affected by concentration only, they reach maximal yield at 0.5 mmoL IPTG But all of the recombinant NP reacted with positive serum of HFRS patients.Conclusion: In this study, we constructed Prokaryotic expression system of S gene of the HV L99 strain and Z10 strain successfully, and obtained recombinant NP. Moreover we optimized the target protein's expression so that the large scale expression and purification of target protein will go on smoothly. The cloning and expression of Hantanvirus S gene laid primary basis on further reaserches.