广州管圆线虫Asp-1基因的克隆表达及各虫期转录水平研究  

Cloning and expression of Asp-1 gene from different stages of larvae of Angiostrongylus cantonensis

作　　者：王琳[1,2] 杨潇[1,2] 曲振宇[1,2] 刘静[1,2] 刘茜[1,2] 何蔼[1,2] 陈婧[1,2] 李卓雅[1,2] 詹希美[1,2] 

机构地区：[1]中山大学中山医学院寄生虫学教研室,广东广州510080 [2]中山大学热带病防治研究教育部重点实验室,广东广州510080

出　　处：《热带医学杂志》2011年第5期512-515,共4页Journal of Tropical Medicine

基　　金：国家973基金(2010CB530004);国家自然科学基金重点项目(2005U0632003)

摘　　要：目的构建广州管圆线虫天冬氨酰蛋白酶(Asp-1)基因的原核表达系统,研究该基因在各虫期的转录水平。方法构建重组质粒pET-30a(+)-Asp-1,并转化至大肠杆菌BL21(DE3)表达载体,经IPTG诱导表达后,Ni-IDA亲和层析纯化表达产物。通过以β-肌动蛋白为内参、以各期虫cDNA为模板的实时荧光定量PCR,研究该基因在各虫期的表达水平。结果获得纯化的重组蛋白,相对分子质量约为43000,与生物信息学分析预测结果一致。qRT-PCR显示该基因在三期幼虫中表达丰度最高,四/五期幼虫、成虫雄虫、雌虫依次降低。结论 Asp-1基因的表达丰度在三期幼虫中最高的实验数据与三期幼虫感染性最强的事实相符,提示天冬氨酰蛋白酶有可能是幼虫入侵宿主的关键酶,为后续广州管圆线虫虫体侵入宿主的机制以及宿主保护机制研究打下基础。Objective To establish a prokaryotic expression system for the Asp-1 gene and to evaluate the transcriptional level of Asp-1 at different stages of the larval development.Methods The coding region of aspartyl protease（Asp-1） gene from Angiostrongylus cantonensis was firstly amplified by PCR,then the PCR product was cloned into the prokaryotic expression vector pET-30a（＋） and transformed into E.coli BL21（DE3）.After transformation,IPTG-induced product was purified by Ni-IDA affinity chromatography.Results The recombinant plasmid pET-30a（＋）-Asp-1 was constructed and the purified recombination protein was obtained successfully.Results from qRT-PCR indicated that the relative expression level of Asp-1 mRNA was highest at the 3rd larval stage.The expression level was gradually decreased in the order of 4/5th larval stage,male adult,and female adult.Conclusion Results obtained from qRT-PCR are correlated with the infectivity of the 3rd stage larvae development.Our results laid the foundation for the future study on the molecular mechanism of infection of mammal host and the mechanism of protection by the host.

关 键 词：广州管圆线虫 天冬氨酰蛋白酶 原核表达 实时荧光定量PCR 

分 类 号：R383.1[医药卫生—医学寄生虫学]