C1009del突变质粒载体的构建及MYOC基因的有效干扰  

Construction of C1009del mutation plasmid vector and intervention of gene MYOC

作　　者：匡多秀[1] 李萍[1] 周新[2] 曲喜英[2] 朱昕力[2] 谢小兵[1] 

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]武汉大学中南医院,湖北武汉430071

出　　处：《国际检验医学杂志》2011年第5期529-530,共2页International Journal of Laboratory Medicine

基　　金：湖南省自然科学基金资助项目(07JJ6047);湖南省科技厅科技计划资助项目(2007SK3096)

摘　　要：目的在前续课题基础上构建针对MYOC基因的RNAi慢病毒载体及C1009del突变质粒载体,并筛选出小梁网细胞作为后续转染阳性质粒合适的药物G418浓度,为下一步将突变质粒载体导入小梁网细胞使其表达突变蛋白,研究突变蛋白的功能、分泌特点等,进而研究原发性开角型青光眼的致病机制提供技术支持。方法采用基因定向克隆方法构建目的基因过表达载体及4种RNAi慢病毒载体、凝胶电泳及基因测序验证;采用Westernblot及基因测序方法筛选有效的RNA干扰靶点;应用基因定点突变技术合成C1009del突变序列,构建突变质粒载体;培养小梁网细胞并用G418筛选。结果经酶切和基因测序证实MYOC基因过表达载体、RNAi慢病毒载体及C1009del突变质粒载体构建成功,并筛选出PSC413为最有效的RNA干扰靶点;G418终浓度为400μL/mL及更高浓度的孔细胞凋亡。结论本实验构建的RNAi慢病毒载体能成功将正常MYOC基因干扰掉,并将G418为400μL/mL作为后续小梁网细胞转染成功的最适筛选浓度,为本课题下一步将突变基因导入小梁网细胞进行突变蛋白的功能研究奠定基础。Objective To study the functions and the secreting features of mutein by constructing RNAi lentivirus vector aimed at gene MY.OC and C1009del mutation plasmid vector,screening suitable G418 concentration of rabecular reticulum cells for follow ing successful positive plasmid transfection,which provided technical support for investigating pathogenesis of primary open-angle glaucoma. Methods Constructing hype^expression vector of objective gene and four sorts of lentivirus vectors by applying tech nique of gene directional cloning; Verificating vectors above mentioned by using agarose gel electrophoresis and gene sequencing; Screening an effective RNA interference target by using Western blot and gene sequencing; Synthesising C1009del mutation se quence by using site-specific mutagenesis technique and constructing mutation plasmid vector;Cultivating rabecular reticulum cells and screening the cells by using G418. Results It certificated by using techniques of enzyme cutting and gene sequencing that hy per-expression vector of MYOC gene,RNAi lentivirus vector and C1009del mutation plasmid vector were constructed correctly,and PSC413 was the most effective target. Cells at G418 400μl/ml or higher final concentration apoptosised. Conclusion The normal gene was interfered successfully by RNAi lentivirus vector,and G418 final concentration 400μl/ml was conduced as screening positive transfection of rabecular reticulum cells,which provided technical supports for investigating the functions of mutein by impor ting mutation gene into rabecular reticulum cells.

关 键 词：开角型 青光眼 基因 突变 

分 类 号：R775[医药卫生—眼科]