摘要
目的 观察裸DNA重组质粒pCD -SjFABPc在体外转染HepG2细胞以及质粒DNA免疫鼠肌组织中的表达 ,为进一步应用该质粒进行DNA免疫实验奠定基础。方法 用脂质体介导DNA转染法将pCD -SjFABPc转染贴壁细胞HepG2 ,G418加压筛选获得阳性克隆细胞并传代培养 ,SDS -PAGE及Western -blot鉴定目的基因在HepG2细胞中的表达 ;将pCD -SjFABPc质粒肌注免疫BALB/c小鼠 ,于 6 5d后取注射部位的肌组织约 0 3cm3 大小 ,经固定、包埋、切片 ,免疫酶组织化学染色 ,显微照相记录。结果 在被转染的HepG2细胞裂解样品中 ,呈现一分子量约 14kDa大小且能被感染兔血清特异识别的条带 ;免疫组化结果显示免疫组切片标本中有特异性阳性反应。结论 pCD
Aim\ In order to further use recombinant plasmid pCD-SjFABPc in DNA-based immunization against Schistosoma japonicum, determining expression of the gene of interest inserted into the eukaryotic expression plasmid pCD-SjFABPc being transfected into HepG2 cells in vitro and injected in mouse muscular cells in vivo . Methods\ DNA recombinant plasmids pCD-SjFABPc were introduced into HepG2 cells by liposome-mediated transfection method, and positive cell clones were obtained using G418 selected-screening approach; expression of the gene of interest was determined by SDS-PAGE and Western blot techniques; the muscular cells from the injected location of the mice immunized with recombinant plasmid were detected by indirect immune-histochemistry assay to confirm produce of the protein of interest in vivo. Results\ The results demonstrated that a strip of 14KDa was present in the lysates of transfected positive cells in SDS-PAGE and recognized by immune sera from infected rabbits in Western blotting; a specific positive reaction of indirect immunohistochemistry assay also present in the specimens of muscular cells from mice immunized with pCD-SjFABPc plasmid. Conclusion\ The recombinant plasmid pCD-SjFABPc could be expressed to produce the protein of interest in HepG2 and mouse muscular cells.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2000年第5期39-42,共4页
Chinese Journal of Zoonoses
基金
中山医科大学"211"工程重点学科建设课题资助
关键词
日本血吸虫
脂肪酸结合蛋白
重组质粒
DNA
Schistosoma japonicum
Fatty acid-binding protein
Recombinant plasmid
Expression
