H9N2亚型禽流感病毒NP蛋白原核表达及多克隆抗体制备

Prokaryotic Expression of NP Protein of H9N2 Subtype Avian Influenza Virus and Preparation of Polyclonal Antibody

【目的】对华南地区分离到的1株H9N2亚型禽流感病毒(Avian influenza virus,AIV)流行株NP蛋白进行原核表达,制备H9N2亚型AIV NP蛋白多克隆抗体。【方法】将1株H9N2亚型AIV的NP基因克隆至pET-32a(+)原核表达载体,构建NP蛋白原核表达质粒。将重组质粒同时转化大肠杆菌BL21(DE3)和Rosetta(DE3)感受态细胞,经IPTG诱导表达重组蛋白。通过考马斯亮蓝染色和Western blotting分析并比较NP蛋白在两种表达菌中的表达量,进一步优化诱导剂浓度、诱导时间及诱导温度等条件,提高蛋白的表达效率。采用镍柱亲和层析法对NP蛋白进行纯化,用BCA法测定蛋白浓度。以纯化的NP蛋白免疫新西兰大白兔获得多克隆抗体,通过Western blotting和间接免疫荧光对所制备的多克隆抗体进行鉴定,通过间接ELISA测定抗体效价。【结果】试验成功构建pET-32a-H9N2-NP原核表达质粒并表达重组NP蛋白。考马斯亮蓝染色和Western blotting结果均表明,重组NP蛋白在大肠杆菌Rosetta(DE3)感受态细胞中表达水平远高于大肠杆菌BL21(DE3)感受态细胞,其大小约73 ku。诱导条件优化结果显示,重组NP蛋白在37℃、IPTG终浓度为1 mmol/L诱导7 h时的表达量最大,且通过镍柱纯化后得到纯度较高的重组蛋白。Western blotting和间接免疫荧光结果均表明,所制备的多克隆抗体能高效地与H9N2亚型AIV发生特异性结合。间接ELISA测得多克隆抗体的效价为1∶409600。【结论】本研究制备的兔抗NP蛋白多克隆抗体效价较高,表明NP蛋白具有良好的免疫原性,为今后NP蛋白单克隆抗体的制备和ELISA检测方法的建立奠定了基础。

【Objective】 This study was aimed to express NP protein of an epidemic H9 N2 subtype Avian influenza virus(AIV) strain isolated from South China using prokaryotic expression system, and prepare its polyclonal antibody.【Method】 The NP gene of a H9 N2 strain was cloned into pET-32 a(+) vector to construct the prokaryotic expression plasmid of NP protein.The recombinant plasmid was transformed into both the Escherichia coli(E.coli) BL21(DE3) and Rosetta(DE3) strains for expression.The recombinant protein was induced by IPTG.Coomassie bright blue staining and Western blotting were used to analyze and compare the expression of NP protein in the two kinds of expressing bacteria.Furthermore, the concentration of IPTG,induction time, and induction temperature were optimized to improve the protein production.By affinity chromatography, the recombinant protein was purified using a Ni-NTA column, and the concentration was determined using BCA assay.The polyclonal antibody was obtained from New Zealand, which had been inoculated with the purified protein.Western blotting and indirect immunofluorescence assay were used to identify the polyclonal antibody, the antibody titer was determined using indirect ELISA.【Result】 The prokaryotic expression plasmid pET-32 a-H9 N2-NP was constructed and the combinant NP protein was expressed successfully.Coomassie bright blue staining and Western blotting results revealed that the recombinant protein was expressed at a much higher level in E.coli Rosetta(DE3) than that in E.coli BL21(DE3),and its size was about 73 ku.The optimal induction conditions showed that the expression of recombinant NP protein reached the maximum at 37 ℃ and IPTG concentration of 1 mmol/L for 7 h.The results of Western blotting and indirect immunofluorescence assay demonstrated that the prepared polyclonal antibody could bind specifically to H9 N2 subtype AIV at a high titer.By indirect ELISA detection, the titer was about 1∶409 600.【Conclusion】 The polyclonal antibody against NP protein prepared from rabbits had a higher titer, indicating that NP protein had good immunogenicity.The results laid the foundation for the preparation of monoclonal antibodies against NP protein and establishing the ELISA detection method.