瑞替普酶在乳酸乳球菌中的表达

摘要

To establish an expression system of reteplase(rPA)in lactic acid bacteria(LAB),rpa gene was amplified from pET22b-rpa,a recombinant plasmid constructed in our previous study.After that,the PCR product was ligated with Lacto-coccus lactis expression vector pCYT,which resulted in the recombinant plasmid pCYT-rpa. In order to improve the stability of rPA protein in L. lactis NZ9000,the rpa gene was also inserted into the downstream of thermostable Nuclease(Nuc)tag of Staphylococcal in the vector pCYT. The obtained recombinant plasmids pCYT-rpa and pCYT-nuc-rpa were respectively introduced into L. lactis NZ9000 through electroporation. After being induced by Nisin,the Western bolt was performed and the result showed that the Nuc did improve the stability of rPA protein in L. lactis,suggesting that Nuc-fusion could enhance the production of rPA in L. lactis. After being refolded,both recombinants rPA and Nuc-rPA exhibited thrombolysis activity, and the activities were 800,U/L and 1,000,U/L.%为建立溶栓药物瑞替普酶(rPA)的乳酸菌表达系统，从实验室前期构建的大肠杆菌表达质粒 pET22b-rpa 上扩增出目的基因 rpa，将该基因与乳酸菌表达质粒 pCYT 连接，构建了 pCYT-rpa 质粒．此外，为提高 rPA 在乳酸乳球菌NZ9000中的稳定性，同时构建了rpa位于葡萄球菌(Staphylococcal)耐热核酸酶基因nuc下游融合表达的重组质粒pCYT-nuc-rpa．将质粒pCYT-rpa和pCYT-nuc-rpa分别电转化至乳酸乳球菌NZ9000中，经Nisin诱导表达，Western blot结果显示Nuc可提高rPA在乳酸乳球菌中的稳定性，从而增加了rPA在乳酸菌中的表达量．重组rPA及Nuc-rPA在复性后均具有溶栓活性，活性分别为800,U/L和1,000,U/L．