摘要
目的 构建EBV LMP1基因的杆状病毒重组供体质粒 ,包装重组LMP1的杆状病毒 ,感染昆虫细胞进行表达。方法 先将已克隆的LMP1cDNA与线性化的pFASTBACHTb进行连接 ,使pFASTBACHTb LMP1与DH1 0BAC感受菌进行转座重组 ,利用抗性及蓝白斑筛选重组Bacmid/LMP1克隆 ,提取Bacmid/LMP1转染昆虫细胞Sf9包装杆状病毒 ,利用病毒感染Sf9细胞进行蛋白表达 ,通过免疫荧光、SDS PAGE和Western blot检测表达情况。结果 获得了重组LMP1的杆状病毒 ,细胞能表达出与LMP1单抗结合的蛋白 ,分子量为 6 3kDa左右 ,细胞可直接分泌LMP1蛋白至上清。结论 LMP1能在昆虫细胞中获得良好表达 。
Objective To construct the recombinant EBV LMP1 baculovirus expression plasmid and express LMP1 in Sf9 cell line.Methods The EBV LMP1 cDNA was cloned into a pF AST B AC HTb donor plasmid, and then the recombinant plasmid was transformed into DH10BAC competent cells. The transformant containing the recombinant bacmid was selected and named as Bacmid/LMP1. The recombinant baculoviruses were obtained after the transfection of Bacmid/LMP1 into Sf9 lines. The techniques of IFA, SDS PAGE and Western blot were used to detect and identify the products expressed in Sf9 lines.Results The recombinant baculavirus was obtained. The recombinant LMP1 protein was expressed in Sf9 insect cells and detected by IFA and Western blot using monoclonal antibody CS1 4.The MW of the recombinant LMP1 protein was 63kDa, and could be detected in the supernatant of Sf9 cell culture.Conclusions Recombinant LMP1 could be expressed in Sf9 lines using the baculovirus expression system, and thus provided the basic material for studying of the function of LMP1 in the cacinogenesis of EBV and its application in immunotherapy.
出处
《热带医学杂志》
CAS
2003年第2期142-145,F002,共5页
Journal of Tropical Medicine
基金
国家自然科学基金 (No .39970 2 92 )
卫生部科研基金 (No.98 2 346 )
广东省自然科学基金 (No .990 1 83)研究项目资助
