CAJ | 학술논문

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．
위건립용전약물서체보매(rPA)적유산균표체계통，종실험실전기구건적대장간균표체질립 pET22b-rpa 상확증출목적기인 rpa，장해기인여유산균표체질립 pCYT 련접，구건료 pCYT-rpa 질립．차외，위제고 rPA 재유산유구균NZ9000중적은정성，동시구건료rpa위우포도구균(Staphylococcal)내열핵산매기인nuc하유융합표체적중조질립pCYT-nuc-rpa．장질립pCYT-rpa화pCYT-nuc-rpa분별전전화지유산유구균NZ9000중，경Nisin유도표체，Western blot결과현시Nuc가제고rPA재유산유구균중적은정성，종이증가료rPA재유산균중적표체량．중조rPA급Nuc-rPA재복성후균구유용전활성，활성분별위800,U/L화1,000,U/L．
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.