摘要
目的应用PCR方法从人血中克隆抗白色念珠菌抗体重链(VH)和轻链(VL,VK)可变区基因,重组到PGEM-llzf(+)载体中,为构建抗白色念珠菌抗体库奠定基础。方法设计7条VHbank primer(5')和6条For primers(3')引物,9条VLbank primer(5')和2条For primers(3')引物,4条VKbank primer(5')和4条For primers(3')引物。从45名念珠菌病恢复期患者的外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人抗体重链(VH)和轻链(VL,VK)可变区基因。通过于linker链两端引入酶切位点,经T4DNA连接酶连接成完整的基因片段并重组到pgem-llzf(+)载体中。结果经PCR方法扩增出了7条VH、9条VL和4条VK片断,经测序证实扩增片断全部为抗体可变区基因。经linker连接入pgem-llzf(+)载体中,获得完整的重组基因。结论获得了全人源抗白色念珠菌单链抗体基因,为构建抗白色念珠菌抗体库奠定基础,也为研发有效治疗白色念珠菌和耐药性白色念珠菌的感染的药物提供了新的手段。
Objective Amplification and clone of antibody variable region genes for the construction of antibody library for Candida albicans. Methods Total RNA was extracted from human peripheral blood of 45 recovering patient infected Candida albicans. Through reverse transcription and Polymerase Chian Reaction,the genes of light chain and weight chain of antibody variable region genes were amplified. A new light chain linker and weight chain linking and clone method was used in this research. Results Total 9 VL,4VK and 7VH chains of variable region genes were obtained. Conclusion Using new linking method increased linking and cloning efficiency. The result suggested that the antibody library could be constructed.
出处
《现代检验医学杂志》
CAS
2007年第2期14-17,共4页
Journal of Modern Laboratory Medicine
基金
西安市科技攻关项目(GG05076)
