肽抗生素hPAB-β制备工艺中基因工程菌的改建  

Reconstruction of engineered bacteria in preparing recombinant human peptide antibiotic β

作　　者：张椿[1] 丛延广[1] 胡福泉[1] 饶贤才[1] 

机构地区：[1]第三军医大学基础医学部微生物学教研室

出　　处：《第三军医大学学报》2006年第22期2243-2246,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30171119);全军医学科学技术研究"十五"计划基金重点课题(01Z070);国家"863"项目(2002AA214211)~~

摘　　要：目的通过重新构建新的3拷贝串联体基因工程菌phPAB-β(3)/M15,在原有肽抗生素hPAB-β制备工艺路线基础上,使发酵后目的肽的最终产量得到提高。方法重新构建含有重组质粒pQE31-hPAB-β(3)的基因工程菌M15,然后筛选出能够稳定而且高效表达目标蛋白的最佳工程菌株,进行传代稳定性分析,并明确新建工程菌中目标融合蛋白的表达形式和亲和层析的效果,最后通过相同条件下,在逐步放大的发酵规模(1L摇瓶,3.7L和10L发酵罐),与以往的3拷贝串联体工程菌phPAB-β(3)/JM109进行对比研究(湿菌产量、目标蛋白表达率),同时观察新建工程菌在10L发酵中目标蛋白的表达和遗传稳定性。结果筛选出的最佳工程菌株的目标蛋白的表达效率为34.8%,经LB培养传代10次后,点种LB(AMP、KAN)平板,生长率为100%,质粒保存率达100%。在1L摇瓶,3.7L和10L发酵罐的发酵规模下,phPAB-β(3)/M15与phPAB-β(3)/JM109一样,目标融合蛋白都以包涵体形式表达,亲和层析效果相近,目标蛋白表达率无显著性差异(P>0·05),但发酵产量大于后者,有非常显著性差异(P<0·01)。且新建工程菌在10L发酵中发酵产量达到55.15g/L,诱导表达后的第5小时,蛋白表达达到高峰,蛋白表达率为30.2%,重组质粒保存率为100%。结论新构建工程菌phPAB-β(3)/M15比起原来基因工程菌phPAB-β(3)/JM109,是更为理想且利于肽抗生素hPAB-β工业生产的基因工程菌。Objective To reconstruct the new engineered bacteria expressing hPAB-β triploids so as to improve the outputs of recombinant human peptide antibiotic β. Methods The recombinant plasmid pQE31- hPAB-β（3） was transformed into E. coll. M15 to screen the new engineered bacteria expressing hPAB-β triploids. The stabilities of phPAB-β（3）/M15 were observed in continuous cultures. The expression levels of the fusion peptides of interest and the bacterial yields of the new engineered bacteria phPAB-β（3）/M15 were compared with that ofphPAB-β（3）/JM109 in different fermentation scales. Results Genetic stability of the recombinant plasmid and phPAB-β（3）/M15 was 100% after 10 passages. Take bacterial yields into account, the new engineered bacteria phPAB-β （ 3 ）/M15 was better than phPAB-β （ 3 ）/JM109 at the similar expression levels of the target proteins by ＂t＂ test analysis （P 〈0.01 ）. The yields of βrmentation reached 55.15 g/L in 10 L fermenter, and the expression levels were up to 30.2% at 5th h after induction. During the fermentation, the recombinant plasmids were stable in culture. Conclusion The new engineered bacterium phPAB-β（3）/M15 successfully constructed was more consummate and effective on the basis of the original process.

关 键 词：肽抗生素 hPAB—β 发酵 M15 

分 类 号：Q819[生物学—生物工程] Q939.1