Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker
Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker作者机构：College of Food Science and Technology Agricultural University of Hebei College of F ood Science and Nutritional Engineering China Agricultural University Institute of Microbiology Chinese Academy of Sciences
出 版 物：《Journal of Integrative Agriculture》 (农业科学学报（英文版）)
年 卷 期：2014年第13卷第8期
页 面：1802-1808页
核心收录：
学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种]
基 金：supported by the National High-Tech R&D Program of China (863 Program 2006AA10Z317)
主 题：food-grade expression vector Lactococcus lactis α-galactosidase gene amylase gene pMG36e
摘 要：Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
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