摘要
目的:利用RT-PCR方法扩增日本裂体吸虫HMGB1基因,构建原核表达载体。方法:日本裂体吸虫尾蚴感染纯种新西兰兔后8周,检获肠系膜下静脉血管中的成虫;匀浆成虫,提取总RNA,经RT-PCR扩增出含SjHMGB1的目标片段,克隆于pMD-19T载体,转化DH5α大肠杆菌增殖克隆,测序鉴定。以pMD-19T-SjHMGB1为模板进行二次PCR扩增,分别获得SjHMGB1、A-box和B-box片段,经BamHⅠ+XhoⅠ双酶切消化PCR产物,分别亚克隆至高效蛋白表达载体pET-26b(+)。结果:用RT-PCR从日本裂体吸虫成虫扩增出含SjHMGB1、A-box和B-box的基因片段,构建成功含目标基因的pMD-19T克隆载体,测序表明序列正确,进一步亚克隆,分别构建成功相应的融合蛋白表达载体pET-26b(+)-SjHMGB1、pET-26b(+)-SjHMGB1-Abox和pET-26b(+)-SjHMGB1-Bbox,经酶切电泳鉴定证实表达载体的构建正确。结论:克隆日本裂体吸虫HMGB1基因,并构建成功SjHMGB1的基因克隆载体和SjHMGB1、A-box、B-box的蛋白表达载体,为进一步获得目标蛋白及其相关研究奠定了基础。
Objective:The HMGB1 gene of Schistosorna japonicum is amplified by RT-PCR method. The prokaryotic expression vectors of Schistosoma japonicum HMGB1 gene are constructed. Methods: The eight weeks after Schistosoma japonicurn infected New Zealand rabbits, adults of Schistosorna japonicurn are collected in mesenteric veins of the rabbit. Total RNA is extracted after adults homogenate. Containing Sj HMGB1 target fragment was amplified by RT-PCR,and was cloned into p MD-19T. Then recombinant vector transformed into DHSa E. coli, and proliferation cloning, sequencing. PMD- 19T- Sj H MGB1 be used as a template for the secondary PCR amplification, respectively,obtained Sj H MGB1, A-box,and B- box fragment,via the Bam H I + Xho I double restriction enzyme digestion, PCR products were respectively subcloned into pET-26b(+), a efficient protein expression vector. Results: SjHMGB1 gene fragment is amplified by RT-PCR from Schistosoma japonicurn adult. The pMD-19T containing the target gene is build successfully by sequencing showed that the correct sequence. Further,the target gene are respectively subcloned into pET-26b(+), a fusion protein expression vector. Three recombinant vectors, pET-26b( + ) Sj HMGB1, pET-26b ( + )- Sj HMGB1-Abox and pET-26b ( + )- Sj HMGB1-Bbox are constructed successfully. The enzyme digesting product electrophoresis confirmed that the sequences are correct. Conclusion:The protein expression vector of Schistosorna japonicurn HMGB1 gene, within Sj HMGB1, Sj HMGB1 A-box, and B-box, are build successfully, which laid the foundation for getting the target protein and researching its associated function.
出处
《长治医学院学报》
2013年第2期81-84,共4页
Journal of Changzhi Medical College
基金
国家科技支撑计划项目资助(2009BA178805)
山西省自然基金项目(2008011073-4)
山西省教育厅科技项目(200611036)