抗衰老Klotho基因在乳酸菌的表达与鉴定

Expression and Identification of Klotho in Lactococcus Lactis

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.

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 pMG36e 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 pMG36e. 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.