Red/ET同源重组介导的基因克隆载体pBACS的构建、鉴定与应用

Construction and verification of gene cloning vector pBACS by Red/ET homologous recombination

目的通过Red/ET同源重组技术构建pBACS基因克隆及测序质粒载体。方法以质粒pBAC2015、pET28a为模板设计四对引物,PCR扩增得到四段彼此间有50 bp同源区域的基因片段,分别命名为bac-sop、bac-ori、bac-kan和bac-T7。利用Red/ET同源重组技术,将四段基因片段电转至E.coli GB05-dir感受态中进行线线重组。经酶切、测序验证阳性克隆质粒。此外,对重组质粒pBACS进行稳定性鉴定;利用pBACS分别进行细菌16S rDNA和真菌ITS基因克隆和基因测序。结果酶切和测序的验证结果显示,质粒的大小、元件方向及序列正确;pBACS具有较强的稳定性,适用于作为基因工程的克隆载体;可高效用于细菌16S rDNA和真菌ITS序列的克隆,阳性率为100%。结论成功利用Red/ET技术构建克隆载体pBACS,质粒载体具有稳定性强、重组效率高的特点,在基因克隆和基因测序等方面具有良好的应用潜力。

Objective To construct the gene cloning and sequencing vector pBACS by Red/ET homologous recombination technology.Methods Four pairs of primers were designed using pBAC2015 and pET28a as templates.Four gene fragments with 50 bp homologous regions were amplified by PCR,named bac;sop,bac;ori,bac;kan,and bac;T7,respectively.Four gene fragments were co;electroporated into competent E.coli GB05;dir cells to carry on linear plus linear homologous recombination.The positive recombinant plasmid was verified by restriction enzyme digestion and gene sequencing.In addition,the recombinant plasmid pBACS was used for stability identification,as well as for cloning and sequencing of 16S rDNA from Streptomyces albus J1074 and ITS from Penicillium sp.HPF1001.Results The verification results of enzyme digestion and sequencing showed that the recombinant pBACS possessed the correct size,direction,and sequence,as well as with good stability,which indicated that pBACS could be used as a cloning vector for genetic engineering;The pBACS could be used for efficiently cloning bacterial 16S rDNA or fungal ITS sequences,with a positive rate of 100%.Conclusion The gene cloning vector pBACS is successfully constructed by Red/ET technology,identified as having the characteristics of strong stability and high recombination efficiency,and has potential for the application in gene cloning and sequencing.