The Role And Mechanism Of RNS1/2 On Mouse Beta Defensin 4 Induced By Influenza A Virus Through NF-κB Signaling Pathway

Objective: By the genetic engineering methods obtained the nonstructural protein 1 and 2 of Influenza virus A, to further study the role and mechanism of r NS1/2 on the mice beta defense 4 induced by influenza virus A based on NF-κB signaling pathways.Methods: ①NS1 and NS2 gene of human influenza virus A strain A/PR/8/34(H1N1) was amplified by RT-PCR and then the prokaryotic expression plasmid p ET22b(+)/NS1 and p ET22b(+)/NS2 were constructed by clone of prokaryotic expression vector p ET22b(+).The p ET22b(+)/NS1 and p ET22b(+)/NS2 were identified by enzyme digestion analysis, PCR and sequencing analysis.The correct recombinant plasmids were transformed into E. coli Rosetta-gami(2) for expression under induction of IPTG. The recombinant proteins were obtained through Ni affinity chromatography and ultra-filtration, and were identified by SDS-PAGE electrophoresis and Western Blot. ② Fifty-four BALB/c mice were divided into six groups, each group six mice: NS1 group(100 μg/kg), NS1 + Viral RNA group;NS1 + Viral group、RNA group(50 μg/kg), Viral group(0.2 × LD50), normal control, NS2+ Viral group, NS2+ Viral RNA group, NS2 group(200 μg/kg). Three mice of each group random Ly selected were intraperitoneally injected PDTC(50 mg/kg), then all mice were intranasally given corresponding reagents. After 12 h, the genic and proteinic expression of Mbd, IFN-γ, IκB, p IκB, P50 and P65 in trachea and lung tissue were detected using RT-PCR, HE staining, immunohistochemical method and Western Blot. Results: ①The prokaryotic expressing plasmids p ET22b(+)/NS1 and p ET22b(+)/NS2 were verified by enzyme digestion analysis, there were two fragment with size of about 2000 bp and 2000 bp.Two recombinant plasmid RCR identification,the size of fragment was about 693 bp and 366 bp respectively as expected.sequencing analysis and comparison of the p ET22b(+)/NS1, p ET22b(+)/NS2 prokaryotic expression plasmid insertion sequence without the mutation, the correct reading frame. ② The expression of recombinant protein r NS1 and r NS2 system, Rosetta-gami(2)-p ET22b(+)/NS1 and Rosetta-gami(2)-p ET22b(+)/NS2 were verified with SDS-PAGE electrophoresis after expression induced by IPTG,thus gained about 23 KD and 12 KD protein expression. The result of Western bolt showed that r NS1 and r NS2 can bind with rabbit anti- r NS1 and rabbit anti- r NS2 polyclone antibody and the protein bands on NC membrane can be seen. ③ Through purification, r NS1 and r NS2 protein by SDS-PAGE electrophoresis gained a single protein r NS1 or r NS2. ④Intranasal give mice after 12 hours, the results of HE staining demonstrated that mice trachea and lung tissue appeared a large number of inflammatory infiltrate cells. ⑤ The results of RT-PCR, immunohistochemistry and Western blot showed that the Virus induced IFN-γ and MBD4 in mice, and r NS1 and r NS2 inhibited the induction of IAV. The expression of MBD4 decreased after blocking the NF-κB signaling pathways. Conclusion: ① The prokaryotic expression plasmid p ET22b(+)/NS1 and p ET22b(+)/NS1 were successfully constructed and recombinant NS1 and NS2 protein were obtained. ② Influenza A virus can induce β defense 4 in mice respiratory tissue through activating the NF-κB signaling pathways. ③ The recombinant NS1 and NS2 can inhibit the induction of influenza A virus to the β defense 4 in mice respiratory tissue, and the inhibital effect is related with the NF-κB signaling pathway.