【摘要】 目的:旨在构建一个将抗原靶向于乳酸乳球菌细胞表面的表达系统。方法:运用PCR技术从金黄色葡萄球菌基因组中克隆出蛋白A(SPA)C-末端544个碱基对的锚定域序列(Spax)。通过酶切、连接将Spax构建入分泌型质粒pAMJ399形成携有整合外源基因位点BglⅡ的pAMJ400质粒。将报告蛋白—绿色荧光蛋白的基因(Gfp)插入载体pAMJ400的整合位点产生模式质粒pAMJ401并电转化其于乳酸乳球菌MG1363。绘制转化子MG1363(pAMJ401)生长曲线确认诱导期。调节pH值(6.0~6.5)诱导转化子并在荧光显微镜下观察杂合蛋白(GFP:SPAX)的表达情况。结果:在395nm的蓝色激发光下,诱导后的细菌发出较明亮的绿色荧光,而未诱导的细菌几乎不产生荧光。结论:成功地构建了乳酸乳球菌表面展示表达系统,此系统可以作为口服活菌疫菌研究的可行性操作平台。更多还原

【Abstract】 Objective:To construct a novel expression system capable of locating antigen moleculers on the cell surface of Lactococcus lactis.Methods: The 544bp-length sequence situated within Protein A Carboxy-terminal region was amplified via PCR from genome extract of Staphylococcus aureus Cowan I and used as a cell wall anchor called Spax.This sequence was digested with SpeI and ligated into a secretion vector pAMJ399,and a cell surface display system called pAMJ400 was obtained with a restriction endonuclease recognition site BglⅡ qualifiing for intergation of exogenous genes.To characterize this expression system,a reporter gene encoding Green Fluorescent Protein was amplified from pYX112 and intergated into the above described intergation site.Subsequently,the obtained plasmid pAMJ401,who can be regarded as a target model,was electroporated into a recipient cell of Lactococcus lactis MG1363.For getting an exact inducement period,the identified transformants,Lactococcus lactis MG1363(pAMJ401) ,were incubated in GM17 medium supplemented with 5mg/L Erythromycin.And the growth curve of their culture was protracted.In terms of the obtained inducement period,inducement of this recombinant strain was performed by modulating pH value of its culture between 6.0 and 6.5.And fluorescent microscopy was used to observe presentation of hybrid protein GFP:SPAX.Results: Under 395nm blue light,bacteria after inducement were excitateded to generate less intense green fluorescence,but non-induced bacteria hardly to generate any fluorescence.Conclusion: A cell surface display system was constucted with success.And it can be used as an applicable platform for research on oral live-bacterial-vaccines.更多还原