Construction and verification of gene cloning vector pBACS by Red/ET homologous recombination
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.HP-F1001.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.
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万方数据
维普
