德氏乳杆菌保加利亚亚种β-半乳糖苷酶基因在大肠杆菌中的非融合表达  

β-Galactosidase Gene from Lactobacillus delbrueckii subsp.bulgaricus Gets Non-fusion Expression in Escherichia coli

作　　者：汪川[1] 张朝武[1] 余倩[1] 刘衡川[1] 裴晓方[1] 

机构地区：[1]四川大学华西公共卫生学院医学检验教研室,成都610041

出　　处：《四川大学学报（医学版）》2008年第4期544-546,共3页Journal of Sichuan University(Medical Sciences)

摘　　要：目的在大肠杆菌中非融合表达德氏乳杆菌保加利亚亚种β-半乳糖苷酶,为构建能高效表达β-半乳糖苷酶的食品级益生菌株奠定基础。方法选择德氏乳杆菌保加利亚亚种(1.1480和wch9901)β-半乳糖苷酶基因(lacZ)起始密码ATG上游-18bp到ATG下游1bp的一段包含SD位点和ATGA位点的序列为上游引物,PCR扩增lacZ,插入表达质粒pMG36e构建重组表达质粒。转化大肠杆菌,筛选阳性克隆,提取重组质粒进行酶切鉴定和测序,并测定转化菌β-半乳糖苷酶活性。结果重组质粒酶切鉴定合格;其中插入的外源片段序列与德氏乳杆菌保加利亚亚种lacZ标准序列符合率达99%以上;携带重组质粒pMG36e-lacZ1.1480的大肠杆菌DH5α酶活性为3.074U/mL,酶比活性为6.939U/mgpro;携带重组质粒pMG36e-lacZwch9901的大肠杆菌DH5α酶活性为4.755U/mL,酶比活性为8.537U/mgpro。结论成功在大肠杆菌中非融合表达了德氏乳杆菌保加利亚亚种β-半乳糖苷酶;本研究选择的SD位点和ATGA位点能在大肠杆菌中有效地引导蛋白的非融合表达。Objective To construct the food-grade recombinant probiotic strain with high activity β-galactosidase,the β-galactosidase gene（lacZ）from Lactobacillus delbrueckii subsp.bulgaricus was in non-fusion expressed in Escherichia coli.Methods 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 β-galactosidase activities of recombinant strains were determined.Results 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 DH5α 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 DH5α carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively.Conclusion 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.

关 键 词：Β-半乳糖苷酶基因 非融合 表达 重组 德氏乳杆菌保加利亚亚种 

分 类 号：Q78[生物学—分子生物学] R151[医药卫生—营养与食品卫生学]