苏云金芽孢杆菌生产菌株溶原性噬菌体pep基因的克隆、表达及功能分析

Cloning, Prokaryotic Expression and Functional Analysis of pep Gene from Bacillus thuringiensis Lysogenic bacteriophage

溶原性噬菌体的随机爆发是微生物杀虫剂苏云金芽孢杆菌(Bacillus thuringiensis,B.t)发酵生产的首要危害。【目的】本文的目的就是通过研究B.t生产菌株溶原性噬菌体的遗传背景,以便从分子水平上控制其在生产中的随机爆发。【方法】通过对广东梅州某公司的生产菌株MZ1采用Mitomycin C进行诱导,提取噬菌体颗粒MZTP01的基因组DNA,克隆和表达该噬菌体的pep基因,并进行了功能分析。【结果】诱导获得的溶原性噬菌体MZTP01斑点清晰,直径约1mm,成斑时间12h;从噬菌体基因组DNA双酶切(HindⅢ/EcoRⅠ)片段中回收长度为2362bp的D片段(Genbank登录号:AY639599),又从D片段中克隆了长度为1101bp、编码367aa、分子量为47kDa的pep基因,表达载体M15(pQE30pep)在大肠杆菌(Escherichia coli,E.coli)中表达获得了47kDa的清晰表达带,在1h时开始产生蛋白并有逐步上升的趋势;Western blot也在47kDa处得到一条清晰的条带;可溶性分析表明PEP蛋白在重组菌株中是以不可溶的包含体形式存在的,该蛋白的产生明显地抑制了宿主的生长速度;噬菌体PEP氨基酸序列之间的同源性比较表明,噬菌体MZTP01PEP蛋白与来自E.coliK12噬菌体的PEP蛋白的同源性程度最大。【结论】我们获得了一种新的噬菌体MZTP01,并首次报道了该噬菌体D片段的核苷酸序列及pep基因的功能,试验证明PEP表达蛋白能够水解酪蛋白,其活力相当于0.3mg/mL的胰蛋白酶。

[Objective] Random outbreak of lysogenic bacteriophage from Bacillus thuringiensis was very harmful to the production of Bacillus thuringiensis insecticide. We clarified the background of the phage from Bacillus thuringiensis MZ1 at the molecular level to solve the problem of random outbreak. [Methods] After the strain MZ1 from a company in Meixian County of Guangdong Province was induced, we obtained phage particles. Then phage DNA was extracted and pep gene was cloned, expressed and analyzed. [Results] We obtained a lysogenic bacteriophage MZTP01 with clear plaque and I mm diameter. Fragment D with 2362bp （Genebank No. AY639599） was obtained after the phage DNA hydrolyzed by HindⅢ/EcoR Ⅰ. Among fragment D, the pep gene with molecular weight of 47 kDa and length of 1101bp was cloned and expressed. Recombinant M15 （pQE30pep） was built and overexpressed in Escherichia coli with a 47kDa clear band. At the same place a clear band was observed by Western blot. Judging from the time course expression, we could conclude that PEP protein produced at 1 hour after induction and then increased gradually. PEP protein was mainly in the form of inclusion body in the recombinant and slowed the growth speed of host. Homologous comparison of PEP protein from phage MZTP01 with other PEPs from BLAST were that phage MZTP01 PEP protein had 100% homologe with that of Escherichia coli K12, and most of others took the similarity in the range between 37%-84%. [Conclusion] PEP protein had ability to hydrolyze casein with the enzyme activity of 0.3mg/ml trypsin. PEP protein may be a kind of trypsin.