抗菌肽Bactenecin7重组质粒构建及其在乳酸菌的分泌表达和活性鉴定

构建抗菌肽Bactenecin7分泌表达载体，鉴定表达产物及检测其生物活性。采用重叠延伸PCR方法拼接合成Bactenecin7基因及其相关调控元件，将目的基因克隆到穿梭载体 pMG36e中，经鉴定后电击转化乳酸菌。通过RT PCR，Western blot检测目的基因表达情况，采用琼脂扩散法对表达产物的生物活性进行初步检测。结果显示成功构建了携Bactenecin7基因的重组质粒，将重组质粒转化至乳酸菌后，该乳酸菌能有效分泌表达Bactenecin7，经体外抑菌实验证实表达产物Bactenecin7具有抑菌活性。实验表明携Bactenecin7基因的乳酸菌能有效分泌表达具有生物活性的抗菌肽Bactenecin7，为进一步研究口服重组乳酸菌进行肠道细菌感染的治疗奠定了基础。

Construction of antimicrobial peptide Bactenecin 7 plasmid and its secretary

To construct a secretary-expression vector of antimicrobial peptide Bactenecin 7 (Bac7), transform it into Lactococcus lactis MG1363, and identify the expression product and its bioactivity. The DNA sequence of Bac7 and its regulation elements was synthesize with overlap-extension PCR. Then the interest gene was cloned into the shuttle-vector pMG36e after it was digested by Sac I and Hind III, the recombination vector was transformed into Lactococcus lactis MG1363 with electrophoration. RT-PCR and Western blot assays were applied to investigate the expressions of the Bac7 gene, and the bioactivity of the expression products Bac7 in culture supernatant was tested with plate-diffusion method. According to the DNA sequencing and double enzyme digestion results, the recombinant vector was successfully constructed and transformed into Lactococcus lactis MG1363, Lactococcus lactis MG1363 carried the recombination vector could secrete bioactive antimicrobial peptide Bac7. All these results indicate the recombination lactic acid bacteria can expression and secrete bioactive Bac7 efficiently, which lay a foundation for further study of oral administration of a Bac7-secreting lactic acid bacteria to treat intestinal bacteria infection.