【摘要】 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。更多还原

【Abstract】 To construct a secretory-expression vector of antimicrobial peptide Bactenecin 7(Bac7),and identify the secretory-expression product in L.lactis MG1363 and its bioactivity.The splicing primers of regulation elements and Bac7 gene,which designed according to codon usage preferences of L.lactis MG1363,were chemically synthesized,and the overlap-extension PCR method was used to splice the full length of Bac7 gene.Then the Bac7 gene was linked to expression vector pMG36e to construct pMG36e/Bac7 vector,and pMG36e/Bac7 was transformed into L.lactis MG1363 by electrophoration.RT-PCR and Western blot assays were applied to investigate the expression of the Bac7 gene in L.lactis,and bioactivity of Bac7 in culture supernatant of L.lactis was tested with plate-diffusion method.The results showed that the Bac7 gene and its regulation elements was amplified and cloned in the vector pMG36e successfully,The secretory-expressed Bac7 in L.lactis MG1363 harboring pMG36e/Bac7 was identified by Western blot,and it had high bacteriostatic activity against E.coli.These results indicate that the recombinant L.lactis MG1363 could express bioactive Bac7,which lays a foundation for further study of oral administration of a Bac7-secreting L.lactis to treat intestinal bacteria infection.更多还原