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变形链球菌(简称变链菌)是公认的主要致龋细菌。葡糖基转移酶(GTF)是变链菌致龋的主要因子,也是主要的保护性抗原。目前认为应用抗龋齿菌苗是控制龋齿的有效手段。为研制安全有效的基因工程龋齿菌苗,我们采用PCR技术,应用变链菌In-gbritt株(血清C型),提取染色体DNA,经EcoRl酶切后作为模板。参照文献,设计合成两个引物。在两个引物5＇端,分别加入EcoRl和BamHl酶切位点。经变性、退火、延伸,反复进行30个循环,以1.5％琼脂糖凝胶电泳鉴定,约有1.46kb的扩增产物带出现,与预期的变链菌GTF基因大小相符。经内切酶谱分析,酶切后片段的数量和大小均符合设计要求。将此产物和大肠杆菌pBV220载体DNA,分别用EcoRl和BamHl双酶切后连接,再转化到大肠杆菌
Streptococcus mutans (Streptococcus mutans) is recognized as the main cariogenic bacteria. Glucosyltransferase (GTF) is a major factor that Streptococcus mutans cause caries and is also a major protective antigen. At present, the application of anti-caries vaccine is an effective tool to control dental caries. To develop safe and effective genetic engineering dental caries vaccine, we use PCR technology, the use of Streptomyces In-gbritt strain (serum C type), extracted chromosomal DNA, EcoRl digested as a template. Reference literature, design and synthesis of two primers. At the 5 ’end of both primers, EcoRl and BamHl restriction sites were added, respectively. After denaturation, annealing, extension and repeated cycles of 30 cycles, 1.56% agarose gel electrophoresis was performed to identify the presence of an amplified product of about 1.46kb, which was consistent with the expected GTF gene size of S. mutans. After the endonuclease analysis, the number and size of the fragments after digestion met the design requirements. This product and E. coli pBV220 vector DNA were digested with EcoRl and BamHl double after ligation, and then transformed into E. coli