重组大肠杆菌制备副溶血弧菌噬菌体内溶素Lys qdvp001 CHAP域及诱导条件初步优化

Preparation of recombinant endolysin from Vibrio parahaemolyticsinfecting bacteriophage Lys qdvp001 CHAP domain portein in Escherichia coli and preliminary optimization of inducing conditions

目的:为了利用噬菌体内溶素防控副溶血弧菌,通过克隆表达工程菌pET30a-CHAP制备目的蛋白,并将以包涵体存在的目的蛋白复性得到有活性的噬菌体内溶素蛋白。方法:首先克隆工程菌pET30a-CHAP,经IPTG的诱导表达后,检测菌液上清中内溶素含量判断表达形式,再进行诱导条件初步优化。通过将表达的pET30a-CHAP包涵体蛋白先用洗涤剂洗涤去除杂蛋白,然后用尿素变性剂溶解,并用Ni^(2+)Sepharose^(TM)6 Fast Flow亲和层析柱进行层析纯化,用EDTA、氧化型谷胱甘肽、还原型谷胱甘肽等为折叠复性促进剂,经过透析制备可溶性的pET30a-CHAP蛋白,再检测目的蛋白的抑菌活性。结果:本实验确定了重组菌内溶素的表达形式为没有活性的包涵体;初步确定最佳诱导条件为诱导温度为16℃,IPTG终浓度0.5 mmol/L,诱导时间为7 h;复性后的内溶素具有抑菌活性。结论:本研究为利用内溶素防控副溶血弧菌以及其他革兰氏阴性细菌提供了制备方法。

Objective：In order aimed to use E.Coli BI21 （DE3）to over express Lys qdvp001 CHAP recombinant and produce soluble pET30a-CHAP protein using renaturation.Methods：PET30a-CHAP expression vector was constructed, and to express the recombinant protein, IPTG was added. Then detect the protein of supernatant in order to know the form of protein expressing.After that,the optimal expression condition was studied.PET30a-CHAP inclusion body protein was washed once by using detergent to get rid of protein impurity and then dissolved by Uera. After purification by Ni2＋ SepharoseTM 6 Fast Flow affinity chromatography column,the purified protein was dialyzed by renaturation accelerator（ EDTA, oxidized glutathione and glutathione asxpression）to obtain soluble pET30a-CHAP protein.Finally, detect the activity of the protein.Results ：The results shows recombinant protein is in the inclusion body.The optimal expression condition was Incubation with IPTG（0.5 mmol/I,）at 16 ~C for 7 h. And the protein can slower the growth of Vibrio parahaemolyticus. Conclusion： This research would provide a method for the proparation of endolysin to prevent the infection of Vibrio parahaemolyticus and other gram negative bacteria.