【摘要】 目的 构建抗衰老Klotho分泌表达载体,鉴定表达产物。方法 采用高保真PCR扩增Klotho基因,将其克隆到穿梭载体pMG36e中,经鉴定后电击转化乳酸菌。通过RT-PCR和Western blot检测目的基因表达情况。结果 特异PCR、酶切和测序鉴定均证实Klotho基因克隆入表达载体pMG36e;将其转化至乳酸菌后,该乳酸菌能有效分泌表达Klotho。结论 成功构建能有效分泌表达Klotho的基因工程菌,为进一步口服重组乳酸菌进行抗衰老研究奠定了基础。更多还原

【Abstract】 Objective To construct a secretory-expression vector of anti-aging Klotho,and identify the secretory-expression product in L lactis MG1363.Methods The Klotho gene were amplified by PCR and cloned into shuttle vector pMG36 e to construct pMG36e-Klotho vector,and pMG36e-Klotho was transformed into L lactis MG1363 by electrophoration.RT-PCR and Western blot assays were applied to investigate the expression of the Klotho gene in L lactis.Results The Klotho gene could be amplified from the recombinant expression plasmid pMG36e-Klotho by PCR.The restriction analysis and sequence analysis also could prove Klotho gene were cloned into expression vector pMG36 e.The secretory-expressed Klotho in L lactis MG1363 harboring pMG36e-Klotho was identified by Western blot.Conclusion Successfully constructed of Klotho efficient expression and secretion of gene engineering bacteria,and laid a foundation for the oral administration of recombinant lactic acid bacteria for anti-aging research.更多还原