【摘 要】
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Construction of food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system.An a-galactosidase (aga) gene encoding an enzyme degrading mel
【机 构】
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College of Food Science and Technology, Agricultural University of Hebei, Banding, China, 071001
【出 处】
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2011全国转基因新品种培育及产业化应用交流研讨会
论文部分内容阅读
Construction of food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system.An a-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000.The aga gene was introduced into pMG36e to substitute the primary antibiotic selection marker of pMG36e, resulting in the 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 a-amylase (amy) gene from Bacillus licheniformis 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.The selection efficiency ofaga was 8.71 X 103 CFU with a standard deviation of 9.1 X 102 CFU/μg DNA ofpMG36-aga.Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of a-amylase.The starch plate assay also indicated that Lactococcus lactis ML23 displayed high activity of a-amylase by expressing of amy gene of pMG36-aga-amy.
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