日本血吸虫(中国大陆株)14-3-3信号蛋白的真核表达  

Eukaryotic Expression of 14-3-3 Signal Protein from Schistosoma Japonicum (Chinese Mainland Strain)

作　　者：胡元生[1] 沈继龙[1] 

机构地区：[1]安徽医科大学病原生物学教研室安徽省基因研究重点实验室,合肥230032

出　　处：《临床输血与检验》2005年第1期4-7,共4页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：国家自然科学基金 (编号 :3 0 170 841);安徽省自然科学基金 (编号 :0 0 44 5 47;9843 63 2 9)资助

摘　　要：目的　从日本血吸虫成虫 c DNA中扩增出信号蛋白 Sj1 4-3 -3编码基因 ,并亚克隆至真核表达载体 pc DNA3 .1( +) ,旨在制备重组诊断抗原和分子疫苗。方法　提取日本血吸虫成虫总 RNA,分离m RNA,RT-PCR法制备c DNA,并扩增出 Sj1 4-3 -3编码基因 ,通过线性 TA克隆载体 p GEM-T连接 ,亚克隆至真核表达载体 pc DNA3 .1 ( +) ,经转染至 COS-7细胞中表达 ,Western blotting鉴定。结果　RT-PCR产物、p GEM-T-Sj1 4-3 -3及 pc DNA3 .1 ( +) -Sj1 4-3 -3分别经双酶切后均获得一特异性基因片段 ,经 SDS-PAGE及 Western blotting鉴定后的分子量为 2 9k D。结论　真核表达重组质粒 pc DNA3 .1( +) -Sj1 4-3 -3在 COS-7细胞中的表达产物是一分子量为 2 9k D的蛋白 ,并能被抗 Sj1 4-3Objective Sj14-3-3 signal protein gene from cDNA of Schistosoma Japonicum adult worms was amplified and subcloned into eukaryotic expression vector pcDNA3.1(+) for a new vaccine candidate of S. Japonicum. Methods The total RNA of S. Japonicum was extracted to prepare for cDNA by RT-PCR, from which the Sj14-3-3 encoding gene was amplified. The DNA fragment was subcloned into eukaryotic expression vectors pcDNA3.1(+) following the insertion and amplification in pGEM-T. The recombinant plasmid was transfected into COS-7 cells and identified by Western blotting. Results The size of RT-PCR product was approximately 760 bp and the inserts of pcDNA3.1(+)-Sj14-3-3 and pGEM-T-Sj14-3-3 digested with BamH I and Xho I were all the same in length as RT-PCR product. The expression was confirmed by monoclonal antibody against Sj14-3-3.Conclusion The recombinant DNA with Sj14-3-3 could be expressed in COS-7 cells with a molecular weight of 29 kD.

关 键 词：日本血吸虫 14-3-3信号蛋白 真核表达 

分 类 号：R383.24[医药卫生—医学寄生虫学] R392.11[医药卫生—基础医学]