摘要
J亚群禽白血病病毒(ALV-J)在中国普遍存在,严重危害养禽业,暂时尚无有效预防、治疗禽白血病的措施,只能通过净化控制。因此,研究一种低成本、易推广的检测方法对防控此病非常重要。用DF-1细胞接种病毒,以细胞基因组为模板,通过巢式PCR方法扩增出941bp的含酶切位点的ALV-J特异性基因gp85基因序列,连接于pET32a(+)载体上,实现gp85蛋白在宿主菌BL21的表达,以切胶回收方式纯化蛋白,成功获得约52ku的重组融合蛋白,免疫新西兰兔,制备gp85单因子抗血清,Dot-ELISA检测抗体效价。结果显示,纯化所得蛋白质具有良好抗原性,制备的抗血清效价高于1.024×105。本试验通过较简便、低耗的方法获得高效单因子血清,为抗血清的制备提供参考,为ALV-J gp85蛋白的研究、ALV-J特异性检测方法的研制打下基础,对ALV-J的净化有重要意义。
Avian leukosis virus subgroup J（ALV-J） is widespread in China,causes great damage to Chinese poultry industry.There is no effective methods of prevention and therapy of avian leukosis,but purification control.So,researching a low-cost,easy-to-promote way for specific detection of ALV-J is important.In this study,the ALV-J ZH-08 strain was inoculated into DF1 cells,an ALV-J gp85 DNA fragment of 941 bp was amplified from infected cells and inserted into pET32a（＋） plasmid at the location between restriction endonucleases Nco Ⅰ and Hind Ⅲ sites.The recombinant plasmid pET32a-gp85 was transformed into E coli.BL21 for gp85 gene expression,and separated by using SDS-PAGE.Target protein was isolated by cutting the gel slices that contain the right band.With purified recombinant gp85 protein as antigen,mono-specific serum against gp85 would be produced afterward.The results demonstrated that good antigenic protein was made,and high titer serum（above 1.024×105） was prepared by this method.It showed a reference for preparation of mono-specific antiserum,laid the basis for gp85 protein researching and ALV-J specific detection method establishment.This study has high value for ALV-J purification.
出处
《中国畜牧兽医》
CAS
北大核心
2011年第10期76-81,共6页
China Animal Husbandry & Veterinary Medicine
基金
广东省科技计划项目(2008B02070009)
NSFC-广东联合基金(U0831002)
广东省科技攻关项目(2009A020101006)
国家肉鸡产业技术体系(nycytx-42-43-03)
