一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

Construction of a Novel E.coli-Bifidobacterium Shuttle Vector

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.

At present, the efficient transformation of Bifidobacterium with plasmid vector is a technical bottleneck. It isn＇ t so easy for Bifidobacterium to be transformed with any ordinary plasmid as for E. coll. In this article, we hypothesized that transformation of Bifidobacterium required species-specific plasmid replicon so we utilized Bifidobacterium＇ s own replicon DNA to construct a novel E. coli- Bifidobacterium shuttle vector. The DNA sequence information of Bifidobacterium plasmid pMB1 was obtained from GenBank. The whole plasmid pMB1, as an own plasmid replicon of Bifidobacterium itself, was amplified by overlap-PCR and was recombined with the fragment of pMK4 plasmid containing original E. coli replicon pUC and anti-chloramphenicol gene Cat to construct the shuttle vector. The recombinants were transformed into Bifidobacterium with electroporation. The optimal electroporational conditions for transforming Bdobacterium were selected by observing the transformation efficiencies under different electroporational parameters. The results were as follows： the replicon pMB1, whole long 1 899 bp, was successfully obtained. The recombinant shuttle vector, named pCMB1, was successfully constructed and was proved to be efficient for transforming two Bifidobacterium strains： B. longum NCC2705 and B. longum NQ-1501. However, the other three Bifidobacterium strains could not be transformed with pCMB1. The plasmids extracted from the transformed Bifidobacterium could be transformed once again into the E. coli, indicating that the shuttle vector was constructed successfully. Based on these results, we concluded that it was essential for transforming Bifidobacterium with plasmids containing a species- specific replicon. Even if containing a species-specific replicon, the plasmids can only be transformed into quantity-limited species or quantity-limited strains of Bifidobacterium. The transformation efficiency can be obviously raised by using the optimal electroporational parameters.