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目的 在乳酸乳球菌中克隆与表达大鼠endostatin。方法 采用RNA分离试剂盒从大鼠肾脏组织中提取总RNA ,用RT PCR方法扩增其endostatin基因 ,将该基因克隆进pUC19质粒中 ,转化大肠杆菌DH5a ,提取质粒 ,分离基因 ,酶切后与含有乳酸乳球菌启动子nisin的pLA14 1质粒连接 ,经电击转化 ,将重组质粒转入乳酸乳球菌NZ90 0 0中 ,转化子在含有氯霉素的GM17培养基上培养。用nisin诱导endostatin表达 ,SDS PAGE和Westernblot鉴定表达产物。结果 表达产物相对分子质量约为 14 0 0 0 ,表达量约为 10mg L。结论 在乳酸乳球菌中可以表达出正确的大鼠endostatin重组蛋白 ,为下一步进行重组蛋白的活性检测以及临床实验提供了依据。
Objective To clone and express rat endostatin in Lactococcus lactis. Methods Total RNA was extracted from rat kidney tissue by RNA isolation kit and its endostatin gene was amplified by RT PCR. The gene was cloned into pUC19 plasmid and transformed into E. coli DH5a. The plasmid was extracted and the gene was isolated. After digested with The pLA14 1 plasmid containing the nisin promoter of Lactococcus lactis was ligated and electroporated to transform the recombinant plasmid into Lactococcus lactis NZ90 0 0. Transformants were cultured on GM17 medium containing chloramphenicol. Endostatin was induced by nisin, and expressed by SDS PAGE and Western blot. Results The relative molecular mass of the expressed product was about 1400 and its expression was about 10 mg L. Conclusion The correct rat endostatin recombinant protein can be expressed in Lactococcus lactis, which provides a basis for the next step in the detection of recombinant protein activity and clinical trials.