【作者】 贺松；

【导师】 张德纯；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2010， 硕士

【摘要】 目的:在乳酸乳球菌克隆表达嗜酸乳杆菌β-半乳糖苷酶基因(lacZ),并观察以lacZ基因作为食品级筛选标记的活性及筛选稳定性,为构建食品级表达载体奠定基础。方法:1.根据GenBank已报道的嗜酸乳杆菌ATCC4356-lacZ基因的DNA序列,利用PCR引物设计和分析软件,设计引物,扩增ATCC4356-lacZ基因,并将其克隆入乳酸菌表达质粒载体pMG36e中构建重组质粒pMG36e-lacZ,借助X-gal显色在大肠杆菌DH5α中筛选阳性克隆子。2.优化乳酸乳球菌MG1363的电转化条件,并在此条件下将重组质粒pMG36e-lacZ电转入MG1363中,借助X-gal显色筛选阳性转化子MG1363-lacZ以观察筛选活性。3. MG1363-lacZ在含乳糖的M17培养基中表达β-半乳糖苷酶,SDS-PAGE和native-PAGE分析蛋白表达和酶活性。通过X-gal显色筛选方法将重组菌株MG1363-lacZ传60代,并测定β-半乳糖苷酶酶活和比活力以观察X-gal筛选的稳定性。结果:1.成功构建了重组质粒pMG36e-lacZ,经PCR和酶切鉴定后与预期结果完全吻合。核酸序列测定显示插入的lacZ基因序列与预期结果亦相符。借助X-gal显色从大肠杆菌DH5α中成功筛选出阳性克隆子。2.优化了MG1363的电转化条件,确定了最佳参数:电压2kV,时间5ms。利用电转化成功地将重组质粒pMG36e-lacZ导入MG1363中,并借助X-gal显色成功筛选出阳性转化子MG1363-lacZ。3. SDS-PAGE结果显示,转化子MG1363-lacZ能表达约70kDa的蛋白,native-PAGE分析显示,该蛋白具有β-半乳糖苷酶活性。对借助X-gal传60代后的重组乳酸乳球菌进行β-半乳糖苷酶比活力检测,与X-gal筛选的第二代相比没有显著差异(P=0.596>0.05),与红霉素筛选比较也没有显著差异(P=0.882>0.05),表明lacZ基因作为筛选标记具有较好的活性和稳定性。结论:通过克隆表达ATCC4356-lacZ借助X-gal筛选方法从大肠杆菌DH5α和乳酸乳球菌MG1363中成功筛选出阳性克隆转化子。ATCC4356-lacZ基因作为筛选标记具有较好的活性和稳定性。本实验为以β-半乳糖苷酶基因作为筛选标记的食品级载体的研究奠定了基础。更多还原

【Abstract】 Objective:Cloning and expressingβ-galactosidase gene (lacZ) from Lactobacillus acidophilus in Lactococcus lactis, and observing the selective activity and stability of lacZ gene as a food-grade selection marker for the construction of a food-grade vector.Methods:1. According to the lacZ sequence of Lactobacillus acidophilus ATCC4356 in GenBank, a pair of primers was designed. And then the lacZ gene was amplified from L. acidophilus ATCC4356-DNA by PCR techniques. After purification, the lacZ gene fragment was digested with restriction endonucleases PstI and XbaI, and then linked with the lactic acid bacterial expression vector pMG36e digested with the same enzymes to construct the recombinant plasmid pMG36e-lacZ. The positive recombinant from Escherichia coli DH5αwas selected with blue colonies on X-gal-containing LB medium.2. Optimize the electroportion procedures for L. lactis MG1363, And the recombinant plasmid pMG36e-lacZ was transformed into MG1363 by electroportion. Selected the positive transformant MG1363-lacZ by selecting blue colonies on X-gal- and lactose-containing M17 medium.3. MG1363-lacZ was grown at 30℃on M17 medium containing lactose and induced for expressing ofβ-galactosidase. The proteins expression andβ-galactosidase activity was analysed with SDS-PAGE and native-PAGE respectively. The activity and specific activity ofβ-galactosidase were detected after a period of 60 generations with X-gal as the coloration for the stability observation.Results :1. Restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-lacZ was successful. The positive recombinant from Escherichia coli DH5αwas selected successfully with blue colonies on X-gal-containing LB medium.2. The electroportion procedures of L. lactis MG1363 was optimized, and the recombinant plasmid pMG36e-lacZ was transformed into MG1363 at 1.9 kv of voltage and 2.8 ms of duration. The positive transformant MG1363-lacZ was selected successfully by selecting blue colonies on X-gal- and lactose-containing M17 medium. And the PCR identification and restriction enzyme digestion also confirmed that the recombinant plasmid pMG36e-lacZ was electroporated into L. lactis MG1363 successfully. The recombinant L. lactis strain MG1363-lacZ was obtained.3. SDS-PAGE showed the genetic engineering L. lactis strain MG1363-lacZ could express a protein, about 70kDa, which was confirmed to be ofβ-galactosidase activity by native-PAGE analysis. Theβ-galactosidase specific activity of recombinant L. lactis MG1363-lacZ after a period of 60 generations selected with X-gal was similar to the 2nd generation (P=0.592>0.05), and also similar to the 61st generation selected with Erythromycin (P=0.882>0.05). These indicated that theβ-galactosidase lacZ gene had good activity and stability as a selection marker.Conclusion:The positive transformant DH5α-lacZ and MG1363-lacZ was selected successfully by expressing L. acidophilus ATCC4356β-galactosidase and selecting blue colonies on X-gal-containing medium. Theβ-galactosidase lacZ gene had good activity and stability as a selection marker, and it could be used as a selection marker for constructing of a food-grade vector.更多还原