H5亚型禽流感病毒HA1基因的克隆表达及其生物学活性的初步分析.pdfVIP

业生物技术学报 Journal of Agricultural Biotechnology 2005,13 (6): 739~742 ·研究论文· H5亚型禽流感病毒 基因的克隆、表达及其生物学活性的初步分析 山 陈 晨 雷 霆 曹 红 陈福勇* (中国农业大学动物医学院，北京 100094) 摘要：参考GenBank 上发表的H5 亚型禽流感病毒( , AIV)血凝素( )基因序列设计引物，经RT-PCR 扩增 基因的 片段，再将其克隆到原核表达载体PET-28a 中，用大肠杆菌( ) BL21(DE3)原核表达， SDS和Western blot 对表达蛋白进行鉴 。Western blot 结果表明，表达蛋白能与H5 亚型AIV 单特异性血清特异性反 应。交叉反应试验表明，此蛋白具有良好的抗原性，可被H5 亚型AIV 抗体特异性识别且无交叉反应。用变性纯化后的蛋白建 立了对AIV 抗体检测的间接ELISA ，并对236 份鸡血清样本进行了实验室检测。检测结果与血凝抑制试验检测结果符合率达 到 100%。综合研究表明，表达蛋白具有良好抗原性。 关键词：禽流感病毒；H5 亚型；血凝素(HA) ；原核表达；ELISA Expression of Hemagglutinin Gene of Subtype H5 in and the Preliminary Analysis of Bioactivity WU Shan CHEN Chen LEI Ting CAO Hong CHEN Fu-Yong* The hemagglutinin(HA) gene of (AIV) subtype H5 was amplified by RT-PCR according to the sequence published on GenBank. It was cloned into the expression vector PET-28a. The protein encoded by this gene was ex- pressed in strain BL2 1 (DE3) and identified by SDSand Western-blot. The results indicated that this protein could be specifically recognized by H5 subtype AIV antiserum. The cross-reactivity test results showed that this protein could only re- act with H5 subtype AIV antiserum but not with any other antiserum against NDV, IBV, etc. The results from detection of 236 serum samples by ELISA using the purified protein were 100% in agreement with that of HI test. All these results demonstrate that this ex- pressed protein is of good antigenicity and can hold great promise for