大肠杆菌-链霉菌穿梭型BAC载体的构建及功能验证

目的构建可由大肠杆菌接合转移至链霉菌并整合至其基因组的穿梭型BAC载体,以实现大片段基因簇在大肠杆菌中完成遗传操作后,转移至链霉菌中进行异源表达。方法从原始B载体pBeloBAC11出发,利用pKD46介导的Red同源重组技术,将一段包含接合转移元件oriT、定点整合元件attp-int和安普霉素抗性基因Am的DNA片段替换其上的氯霉素抗性基因,该DNA片段通过PCR扩增得到,两端为待替换氯霉素抗性基因上下游55 bp的同源臂,替换后的载体通过酶切连入链霉菌组成型启动子ermEP1P2和绿色荧光蛋白（GFP）基因以验证其接合和整合功能。结果原载体pBeloBAC11的氯霉素抗性基因被成功替换,得到载体pBTIBAC1,利用该载体可在变铅青链霉菌（Streptomyces lividans）TK24和1326中表达GFP,在pBTIBAC1上插入新的多克隆位点（MCS）后得到载体pBTIBAC11。结论 pBTIB AC11载体可通过接合转移携带外源基因片段进入链霉菌并整合至其基因组中,同时保留了原有BAC载体的功能元件,为大片段在链霉菌中的异源表达奠定了基础。

Construction of an E. coli-Streptomyces shuttle BAC vector and demonstration of its functions

Objective To construct a shuttle BAC vector which can be conjugated from E.coli to Streptomyces and integrated into its genome so that the large fragment of gene clusters can be transferred to Streptomyces for heterologous expression after genetic manipulation in E.coli.Methods With the help of pKD46 vector which mediated the Red homologous recombination,the chloramphenicol resistance gene of the original vector called pBeloBAC11 was replaced with a DNA fragment containing conjugal transfer element oriT,integration element attp-int and apramycin resistance gene Am.The DNA fragment,both sides of which were 55 bp homologous arm upstream and downstream of the chloramphenicol resistance gene in the original pBeloBAC11 vector,was obtained by PCR.The conjugation and integration functions of the vector after replacement were demonstrated by inserting the constructional promotor of Streptomyces ermEP 1 P 2 and gene of green fluorescence protein（GFP）.Results The chloramphenicol resistance gene of the original plasmid pBeloBAC11 was successfully replaced,generating the BAC vector named pBTIBAC1.The gene of GFP could be expressed after being transformed into Streptomyces lividans TK24 and 1326 by this vector.The vector with new MCS was named pBTIBAC11.Conclusion The pBTIBAC11 vector can take exogenous gene clusters to Streptomyces through conjugal transfer to be integrated into its genome while retaining the element of a BAC vector.