谷氨酸脱羧酶65重组杆状病毒表达载体的构建及其在昆虫细胞中的表达

目的构建谷氨酸脱羧酶GAD65基因的杆状病毒重组供体质粒,包装重组GAD65的杆状病毒,感染昆虫细胞进行表达。方法先将已克隆的GAD65 cDNA与线性化的pFASTBACHTb进行连接,使pFASTBACHTb-GAD65与DH10BAC感受菌进行转座重组,利用抗性及蓝白斑筛选重组Bacmid/GAD65克隆,提取Bacmid/GAD65转染昆虫细胞Sf 9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GAD65的杆状病毒,细胞能表达出与GAD65单抗结合的蛋白,相对分子质量为65kDa左右,细胞可直接分泌GAD65蛋白至上清。结论GAD65能在昆虫细胞中获得良好表达为研究GAD65的作用及免疫治疗奠定基础,。

Construction of glutamic acid decarboxylase-65 (GAD-65) baculovirus expression plasmid and its expression in insect cells

Objective To construct the recombinant glutamic acid decarboxylase-65(GAD65)baculovirus expression plasmid and express GAD65 in Sf9 cell line.Methods The GAD65 cDNA was cloned into a pFASTBACHTb 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/GAD65.The recombinant baculoviruses were obtained after the transfection of Bacmid/GAD65 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 GAD65 protein was expressed in Sf9 insect cells and detected by IFA and Western-blot using monoclonal antibody CS1-4.The MW of the recombinant GAD65 protein was 65kDa,and could be detected in the supernatant of Sf9 cell culture.Conclusion Recombinant GAD65 could be expressed in Sf9 lines using the baculovirus expression system,and thus provided the basic material for studying of the function of GAD65 and its application in immunotherapy.