摘要
目的 以克隆小肠结肠炎耶尔森菌强毒力岛可变区首尾区域的双独立片段为例 ,探讨克隆入同一载体的基因数多于一个时可采取的构建策略。方法 在一个基因A正义引物与另一基因B负义引物的 5′端分别设计与克隆载体相匹配的酶切位点 ,在B基因正义引物与A基因负义引物的 5′端设计相同的酶切位点。分别进行PCR扩增 ,再通过引物的 5′端相同的酶切位点进行两PCR产物的酶切与连接 ,以连接产物为模板 ,应用A基因正义引物和B基因负义引物进行PCR扩增 ,将此次PCR产物经常规方法克隆入载体。
A construction strategy for clonging several independent PCR products into the same vector was reported here with the example of cloning two fragments in the beginning and the end of the variable of Yersinia enterocolitica HPI(High pathogenicity island). Restriction enzyme sites that complemented with the cloning vector were respectively designed at the 5′end of A gene's sense primer and B genes antisense primer. The same restriction enzyme site was adopted in the design of the 5′end of B gene's sense primer and A gene's antisense primer. Then the two genes were amplified,digested with the same enzyme for the 5′ end of primer and ligated.The ligated product was PCR amplified with A gene's sense primer and B gene's antisense primer. The PCR product was then cloned into the vetor by routine methods.
出处
《热带医学杂志》
CAS
2001年第2期133-135,共3页
Journal of Tropical Medicine
基金
国家重点基础研究发展规划项目资助(No.G19990 5 410 1)
