日本血吸虫(中国大陆株)14-3-3信号蛋白的真核表达

目的从日本血吸虫成虫cDNA中扩增出信号蛋白Sj14-3-3编码基因，并亚克隆至真核表达载体pcDNA3．1( )，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mRNA，RT-PCR法制备cDNA．并扩增出Sj-4-3-3编码基因，通过线性TA克隆载体pGEM-T连接，亚克隆至真核表达载体pcDNA3．1( )，经转染至COS-7细胞中表达，Western blotting鉴定。结果RT-PCR产物、pGEM-T-Sj14-3-3及pcDNA3．1( )-Sj14-3-3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29kD。结论真核表达重组质粒pcDNA3．1( )-Sj14-3-3在COS-7细胞中的表达产物是一分子量为29kD的蛋白，并能被抗Sj14-3-3单克隆抗体识别。

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

Objective 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.