基因重组乳链球菌防龋疫苗的研究 Ⅴ.重组乳链球菌中RNA的提取纯化及RNA点印迹杂交

Study on the Vaccine Against Dental Caries of Recombinant Streptococcus lactis and Artificial Active Immunization V. Extraction, Purification and Dot Blot Analysis of RNA in the Recombinant S.lactis

对参与蛋白质生物合成的转录产物ＲＮＡ进行异硫氰酸胍提取，氯化铯超速离心纯化以及ＲＮＡ点印迹杂交，测定重组乳链球菌中特定的ＲＮＡ，分析影响重组基因表达的因素。紫外分光法测定获得高浓度（５～８ｍｇ／ｍｌ）、纯度（ＯＤ２６０／ＯＤ２８０＞２．０）ＲＮＡ。以切口平移生物素标记ＤＮＡ探针并进行ＤＮＡ－ＲＮＡ点印迹杂交，光基因核酸系统检测结果提示，乳链球菌ＨＬ１０７，ＨＬ４５均含有与变形链球菌相同的特定ＰＡｃ－ｍＲＮＡ靶序列及密度扫描影像。提示ＰＡｃ－ｍＲＮＡ作为指导蛋白质合成的中介物存在于重组乳链球菌中，使重组乳链球菌完成了克隆ｐａｃ基因的表达。

To investigate the transcription level of the pac gene in the recombinant S.lactis, the amount of PAcspecific mRNA was measured by RNA dot blot analysis with the biotinlabeled 1.5 kb PstI fragment of the pac gene. RNA were extracted with guanidinium thiocyanate followed by centrifugation in cesium chloride solutions. These results indicated the sequence of PAcmRNA in S.lactis HL107 or HL45 was the same as that in S.mutans Ingbritt and showed similar density photograph. The PAcspecific mRNA plated an intermediary role of protein synthesis in recombinant S.lactis and made it complete the expression of cloned pac gene.