Zhang Rui-Hua, Jin Mei-Lin, Wang Gui-Hua, Yu Zheng-Jun, Zhao Si-Ting, Li Hong-Chao, Tan Ya-Di, Chen Huan-Chun
Lab of Animal virus, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Mar;21(2):315-9.
In order to differently diagnose avian influenza virus (AIV) subtypes, the HA gene of AIV H9 subtype was cloned, expressed and utilized in an enzyme-linked immunoad sorbent assay (ELISA). HA gene (1683bp) of H9N2 AIV was amplified by RT-PCR from a strain of field isolated H9N2 AIV, and its identity was confirmed by sequencing. The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed. The expressed HA-GST fusion protein in E. coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90kD protein with immunogenicity. The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The assay has 91.57% specificity to H9 AIV, 92.31% sensitivity and excellent reduplication. It could be used to differently detect antibodies to H9 AIV.
为了鉴别诊断禽流感病毒（AIV）亚型，克隆、表达了AIV H9亚型的血凝素（HA）基因，并将其应用于酶联免疫吸附测定（ELISA）。通过RT-PCR从一株田间分离的H9N2 AIV毒株中扩增出H9N2 AIV的HA基因（1683bp），并通过测序确认其一致性。将HA基因亚克隆到原核表达载体pGEX-KG中，去除其分泌信号序列。通过SDS-PAGE和western blotting分析鉴定，在大肠杆菌BL21中表达的HA-GST融合蛋白为具有免疫原性的90kD蛋白。融合蛋白主要存在于包涵体中，通过变性和复性进行纯化。利用HA-GST融合蛋白建立了间接ELISA法检测AIV H9亚型抗体。该方法对H9 AIV的特异性为91.57%，敏感性为92.31%，重复性良好。可用于鉴别检测H9 AIV抗体。
