大鼠Endostatin在乳酸乳球菌中的克隆与表达大鼠Endostatin在乳酸乳球菌中的克隆与表达

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。

Cloning and Expression of Rat Endostation Gene in Lactococcus lactis

Objective To clone and express rat endostatin gene in Lactococcus lactis. Methods Extract total RNA from rat kidney tissue using RNA kit and amplify endostatin gene by RT-PCR.Clone the amplified gene into plasmid pUC 19 and transform to E.coli DH5α.Extract the recombinant plasmid and isolate endostatin gene.Digest the gene with restriction endonuclease and ligate with p lasmid pLA141 carrying nisin promoter of Lactococcus lactis.Transform the re combinant plasmid into Lactococcus lactis NZ9000 by electrophoration and ino culate the transformant into GM17 medium containing chloramphenicol.Induce the e xpression of endostatin by nisin and identify the expressed product by SDS-PAGE and Western blot.Results The relative molecular weight of expressed protein was about 14 000,and its expression level was about 10 mg/L. Conclusion Recombinant endostatin protein was successfully expr essed in Lactococcus lactis.It provided a basis for the determination of act ivity as well as clinical trial of recombinant endostatin protein.