【摘要】 人工合成4条两两互补的DNA序列,经磷酸化和退火后与经BglⅡ酶切的pET-E2载体连接,得到pET-E2HA重组质粒。该质粒除表达E2蛋白A/D抗原区外,还表达3个串联的人流感病毒(Human influeozavirus)血凝素表位。重组质粒转化到大肠杆菌(Escherichia coli)BL21(DE3),用IPTG诱导表达。利用纯化后的表达产物与流感病毒血凝素单抗及乳胶建立了诊断猪瘟抗体水平的乳胶凝集试验。利用盐酸胍溶解包涵体并过His·Bind柱,获得纯化的重组蛋白。研究结果表明,乳胶凝集方法具有操作简便、快速、敏感性高、特异性强、价格低廉且可用于现场检测等优点,是一种适合基层兽医单位用于猪瘟病毒(Classical swine fever virus,CSFV)血清抗体检测的新方法。更多还原

【Abstract】 Three linked HA gene of Human influenza virus was obtained by four primers which could pair with each other after phosphorylation and anneling, and then it was ligated into pET-E2 digested with BglⅡ to get the recombinant plasmid pET-E2HA, which could express Classical swine fever virus(CSFV) E2 A/D antigenic domains as well as three HA epitopes. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cell and highly expressed in inclusion bodies with the induction of IPTG. The recombinant protein was purified with His·Bind chromatography column. Western blotting analysis proved that the recombinant protein had good reactivity against CSFV positive antibodies. The latex agglutination test (LAT) was established for the detection of antibody against CSFV with this recombinant protein. The result indicated that the LAT method was a simple, rapid, sensitive, specific and inexpensive method which was suitable for detecting antibody against CSFV in serum and serological survey of CSFV.更多还原