Wang Chuan, Zhang Chao-wu, Yu Qian, Liu Heng-chuan, Pei Xiao-fang
Department of Medical Technology, West China School of Public Health, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Jul;39(4):544-6.
To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli.
From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined.
The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively.
lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.
构建具有高活性β-半乳糖苷酶的食品级重组益生菌菌株，将德氏乳杆菌保加利亚亚种的β-半乳糖苷酶基因（lacZ）在大肠杆菌中进行非融合表达。
以德氏乳杆菌保加利亚亚种lacZ基因起始密码子ATG上游18 bp至下游1 bp之间包含Shine-Dalgarno（SD）和ATGA序列的DNA序列为上游引物，PCR扩增lacZ基因。然后将lacZ cDNA插入表达质粒pMG36e中构建重组表达质粒。将重组质粒导入大肠杆菌，并筛选阳性克隆。为鉴定基因重组，用限制性内切酶切割重组质粒并测序。为鉴定蛋白表达，测定重组菌株的β-半乳糖苷酶活性。
重组质粒的酶切图谱合格。插入重组质粒的基因与德氏乳杆菌保加利亚亚种的lacZ基因同源性超过99%。携带pMG36e-lacZ 1.1480的大肠杆菌DH5α的酶活性和酶活性比分别为3.074 U/mL和6.939 U/mg蛋白。携带pMG36e-lacZ wch9901的大肠杆菌DH5α的酶活性和酶活性比分别为4.755 U/mL和8.537 U/mg蛋白。
德氏乳杆菌保加利亚亚种的lacZ在大肠杆菌中实现了非融合表达。我们选择的SD和ATGA序列可使lacZ在大肠杆菌中进行非融合表达。
