摘要
目的 :为了寻找日本血吸虫病新的诊断和候选疫苗分子 ,克隆日本血吸虫 14 -3 -3蛋白质编码基因 ,并进行表达。方法 :提取日本血吸虫成虫RNA ,设计合成引物 ,用RT -PCR法扩增出日本血吸虫 14 -3 -3抗原 (Sj14 -3 -3 )基因编码序列 ,将其克隆入pGEM -T载体 ,然后在真核表达载体 pBKCMV中亚克隆 ,用IPTG诱导表达 ,SDS -PAGE观察表达结果。结果 :RT -PCR法扩增出一条大小约 765bp的特异性片断 ,克隆质粒pGEM -Sj14 -3 -3和真核表达质粒pBKCMV经双酶切和以重组质粒为模板进行PCR扩增 ,均可获得一条与PCR产物一致的DNA片段 ,诱导表达后 ,经SDS -PAGE可见一条约 3 2 5kDa大小的融合蛋白条带。结论 :本实验成功地克隆了日本血吸虫 14 -3 -3抗原的编码基因 ,并在原核细胞中进行表达 ,为进一步的免疫诊断和核酸疫苗研究奠定了基础。
Objective To discover new diagnosis and candidate vaccine moleculae for Schistosomiasis japonica,the 14-3-3 protein gene of Schistosoma japonicum (Sj) were cloned and expressed in the Prokaryotic cell.Methods The total RNA were extract from adult worms of Schistosoma Japonicum.Primers were designed and synthesized.The cDNA encoding 14-3-3 antigen was amplified by RT-PCR.The product from PCR was clonid into pGEM-T vector,Then was subcloned into the eukaryocyte expression vector.After induced by IPTG,the cells were collected to be analyzed by SDS-PAGE.Results For RT-PCR,a specific band of around 765 bp was amplified.The same band was obtained by double restriction of recombinant plasmids and PCR using recombinant plasmids(pGEM-Sj14-3-3 and pBK-Sj14-3-3)as template After induced by IPTG,The results of SDS-PAGE revealed that the molecular weight of fusion protein was approximately 32.5kDa. Conclusion The cDNA encoding 14-3-3 protein of Schistosoma Japonicum was cloned and expressed in the prokaryotic cell.It provided the basis for the further study on value of immunological diagnosis and nuclear vaccine
出处
《安徽医学》
2001年第2期5-7,共3页
Anhui Medical Journal
基金
安徽省自然科学基金!(编号 9741 0 0 4 2 )
安徽省教育委员会委自然科学基金!(编号 98JL0 62 )
安徽省财政厅人才基金资助课题!(未编
