河南猪流感病毒H3N2分离株NP基因表达及抗原性分析

Expression and its antigenicity of NP gene of H3N2 swine influenza virus isolated in He'nan province

目的表达猪流感病毒(swine influenza virus,SIV)H3N2分离株NP基因,获得具有良好抗原性的NP蛋白。方法 PCR扩增猪流感病毒A/swine/Henan/2/2008(H3N2)NP基因,经EcoR I和Xho I双酶切后插入p ET32a(+)载体,并转化大肠杆菌Rosset(DE3)菌株;PCR、EcoR I和Xho I双酶切及DNA测序鉴定重组质粒p ET32a-NP;IPTG诱导,表达、纯化NP并免疫家兔制备免疫血清,SDS-PAGE、WB测其反应原性;同时构建pc DNA3.1-NP并转染293T细胞,免疫荧光检测其免疫源性。结果成功构建重组载体p ET32a-NP和pc DNA3.1-NP,SDS-PAGE和WB显示融合蛋白约80 k D,能与感染SIV的猪血清特异性反应,并且纯化蛋白制备的多克隆血清能识别293T细胞中表达的NP蛋白。结论正确表达SIV H3N2 NP蛋白,并有良好的抗原性,为研究猪流感病毒NP诊断抗原和亚单位疫苗奠定基础。

Objective To clone and express nucleoprotein（NP） gene of swine influenza virus（ SIV） and to identify NP protein with good antigenicity. Methods The NP gene of A/swine/Henart/2/2008 （ H3N2 ） was amplified with PCR. After digested by EcoR I and Xho I,the PCR products were cloned into prokaryotic expression vector pET32a and transformed into Rosetta strain. The recombinant vector, named pET32a-NP, was identified with PCR, EcoR I/Xho I di- gestion and DNA sequencing. After sopropyt-beta-D-thiogalactopyranoside （IPTG） inducing, the recombinant NP was ex- pressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. Serum antibody against NP was prepared by immunizing the rabbits with the purified NP. The recombinant pcDNA3, l- NP was constructed and transfected into 293T cells simultaneously, and the immunogenicity of NP was detected with im- munofluorescence. Results The recombinant plasmid pET32a-NP and pcDNA3.1-NP were successfully construc- ted. SDS-PAGE and Western blot showed that the expressed protein was about 80 kDa and reacted specifically with sera from pig infected with SIV. Immunofluorescence indicated that NP protein immunized sera could be used in the detection of NP protein expression 293T cells. Conclusion The NP protein of SIV H3N2 was successfully expressed in Rosetta strain and the expressed NP had good antigenicity and could be used as diagnostic antigen and in the development of subunit vaccine of SIV.