禽流感病毒H7N2血凝素HA1基因在大肠杆菌中的表达

目的　表达H7N2亚型禽流感病毒 (AIV)HA1基因 ,用于感染H7亚型禽流感病毒抗体的检测和HA1蛋白功能研究。方法　采用RT PCR方法对H7N2亚型AIVHA1基因进行扩增 ,将PCR产物克隆于pGEM T Easy载体 ,将该基因插入pGEX 4T 2中构建HA1基因原核表达载体 ,转化BL2 1大肠杆菌后 ,在IPTG诱导下表达HA1蛋白 ,Westernblot鉴定表达HA1蛋白。电洗脱方法纯化表达HA1蛋白 ,建立间接ELISA方法 ,对感染AIVH7、H9、H5亚型AIV阳性血清进行检测。结果　成功克隆H7N2亚型AIV的HA1基因 ,其核苷酸序列长度 96 6bp ,编码 32 2个氨基酸残基。构建HA1基因原核表达载体在大肠杆菌内表达出约 6 1× 10 3的HA1融合蛋白。Westernblot和ELISA方法鉴定表明 :表达HA1蛋白与感染H7亚型AIV鸡血清有反应 ,与H5、H9亚型AIV阳性血清没有反应。结论　本研究在大肠杆菌中成功表达了H7N2亚型AIVHA1基因蛋白 ,具有与感染H7亚型AIV阳性血清反应原性 ,不与H5和H9亚型AIV感染阳性血清发生反应。

Expression of HA1 Gene of H7N2 Subtype Avian Influenza Virus in E.coli

Objective To investigate the expression of HA1 gene of avian influe nza virus (AIV) subtype H7N2, which can be used for detection of antibodies to H 7 subtype AIV, and for further study of HA1 protein function. Method The gene of H7N2 subtype AIV was amplified by RT-PCR and cloned into pGEM -T-easy vecto r. The HA1 gene expressing plasmids was constructed by inserting the target g ene fragments into pCEX-4T-2 vectors. The proteins expression was induced by IPTG and analyzed by Western blot. An indirect ELISA method was established as to de tect the expression of HA1 fusion protein. Positive sera of H5, H7, H9 subtype A IV were detected by this indirect ELISA. Results HA1 gene of H7 N2 subtype AIV was s uccessfully cloned and sequenced. This gene encoded 322 amino acids. The constru cted HA1 gene expressing plasmids was transformed into E. Coli BL21 competen t ce lls, and the HA1 fusion protein with molecular weight of 61kD was expressed. The expressed HA1 fusion protein showed a positive reaction with H7N2 subtype AIV p ositive serum, and a negative reaction with H5 and H9 subtype AIV positive serum . Conclusion The HA1 gene of H7N2 subtype AIV was expressed in E. Coli BL21. T he expressed HA1 fusion protein shared a positive reaction with H7 subtype AIV p ositive serum, and a negative reaction with H5 and H9 subtype AIV positive serum .