Establishemnt of prokaryotic expression system and polyclonal antibodies preparation of F1,F2 subunit gene of human metapneumovirus

Objective To establish a prokaryotic expression system of F1, F2 subunit gene of human metapneumovirus, prepare the corresponding polyclonal antibodies and provide evidence for the development of vaccine. Methods The F1, F2 gene was amplified with PCR,and cloned into vector of pET32a (+) after T-A cloning and double digests. The recombinant proteins were expressed in E.coli BL21(DE3) according to the optimized conditions,purified and tested by using Western Blot,and subsequently used to immunize BALB/c mouse.The resultant polyclonal antibodies were evaluated with ELISA assays.Immunofluorescence assays were used to test if the prepared antiserum could specifically reacted with the fusion protein. Results F1, F2 genes were correctly inserted pET32a (+), and had correct reading frames. A large number of recombinant proteins with His tag expressed after inducing by 0.5 mmol/L IPTG at 37 ℃ for 5 h, and the concentration of purified products reached 200 g/ml and 300 g/ml respectively. Western Blot results showed that the recombinant proteins could be identified by His tag antibody specifically; the highest valence of antibodies in immuned mouse were 1 : 640 (anti-F1 sera) and 1 : 40 960 (anti-F2 sera).The results of immunofluorescence assays showed that the prepared antiserum could specifically reacted with F protein. Conclusion The recombinant plasmids of pET32a-F1 and pET32a-F2 were successfully constructed and expressed efficiently in E.coli, the recombinants had good antigenicity and efficient polyclonal antibodies were attained by immunized BALB/c mouse. The results of the study can be used in the further research of MPV infection.