乳酸菌同源重组载体的构建与鉴定

Construction and identification of lactic-acid bacteria homologous recombination vector

【目的】构建乳酸菌同源重组载体,实现外源基因在乳酸菌中的整合型表达。【方法】根据乳酸菌L.lactis MG1363全基因组中ThyA基因序列设计引物,PCR扩增乳酸菌基因组中ThyA基因起始密码上游及终止密码下游约1000bp的基因序列,将获得的目的基因依次克隆至载体pMUTIN4中,以琼脂糖凝胶电泳检测目的条带,以双酶切技术、DNA测序予以确证。【结果】琼脂糖凝胶电泳结果显示,目的基因成功扩增,双酶切及DNA测序结果证实,成功构建了同源重组载体pThyA-up-down。【结论】乳酸菌同源重组载体pThyA-up-down为实现外源基因在乳酸菌中的非抗性、整合型表达奠定了基础。

【Objective】 To construct homologous recombination vector of lactic-acid bacteria,and to implemente the ectogenic genome integratate into lactic-acid bacteria genome successful.【Methods】 Designing primer according to ThyA gene sequence of lactic-acid bacteria L.lactis MG1363 genome as a template,amplify the ATG upstream and TAA downstream 1000 base pair sequences of ThyA gene by PCR,then,the target genes were cloned to the multiple cloning site of pMUTIN4 vector in turns,the target genes was tested by Agarose gel electrophoresis,also,DNA sequencing and double enzyme digestion technique was employed to conformed it.【Results】 Agarose gel electrophoresis results showed that the target genes were amplified successfully,double enzyme digestion and DNA sequencing confirmed that homologous recombination vector pThyA-up-down was successfully constructed.【Conclusions】 The pThyA-up-down vector laid a foundation for integrated expression of foreign genes in lactic acid bacteria.