C2株蓝氏贾第鞭毛虫SUMO特异性蛋白酶基因的克隆、生物信息学分析及其催化活性区的原核表达  

Cloning and bioinformatics analysis of Giardia lamblia C2 strain SENP gene and prokaryotic expression of SENP catalytic domain

作　　者：李少东[1] 周英斌 刘晓莉[1] 禇晗 李思瑾[1] 田喜凤[1] 王洋[1] 

机构地区：[1]河北联合大学生命科学学院,唐山063000

出　　处：《中国人兽共患病学报》2015年第3期203-207,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31471954);河北省青年科学基金(No.C2012401039)联合资助~~

摘　　要：目的克隆C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的SUMO-Specific Protease(SENP)基因,并对其序列进行生物信息学分析,原核表达贾第虫SENP的催化活性区。方法提取C2株贾第虫基因组DNA,以基因组DNA为模板获得SENP编码区全长片段,连入克隆载体pGM-T,测序后进行生物信息学分析;根据分析结果克隆SENP的催化活性区,构建其原核表达载体pET-28a(+)-SENPc,在E.coli Rosetta(DE3)中诱导表达,SDS-PAGE及Western blot观察表达结果。结果成功克隆了C2株贾第虫SENP编码区全长序列,生物信息学分析显示C2株贾第虫SENP蛋白序列与WB株相同,二级结构以无规则卷曲为主,其催化活性区位于126-497aa,被一段插入序列分割成两个部分;构建了SENP催化活性区原核表达载体并在大肠杆菌中高效表达,在相对分子量约43kD的位置出现目的蛋白条带,与理论值相符。结论成功克隆了贾第虫SENP基因并原核表达了其催化活性区,为贾第虫SENP蛋白功能的研究提供了基础。SUMOylation is a post‐translational modification involved in various cellular processes .SUMO‐specific protease （SENP） regulates SUMOylation by removing SUMO from conjugated substrates （deSUMOylation） and promoting maturation of SUMO precursor .In order to express Giardia lambia （C2 strain） SENP catalytic domain in E .coli ,the full‐length open reading frame of SENP was amplified by PCR from Giardia lamblia genome DNA .The PCR product about 1 620 bp was cloned into cloning vector pGM‐T .Sequencing result showed the sequence of SENP in C2 strain was identical with that in Gi‐ardia WB strain .Bioinformatics analysis showed that SENP protein possessed a 372 aa discontinuous ULP catalytic domain at C‐terminal .The catalytic domain of SENP was cloned into prokaryotic expression vector pET‐28a（＋ ） .The recombinant vector pET‐28a（＋ ）‐SENPc was transformed into E .coli Rosetta（DE3） ,then the recombinant SENPc protein was expressed by IPTG induction .SDS‐PAGE and Western blot using anti‐His Tag antibody showed that the expression product of SENPc was a fusion protein with a molecular weight of 43 kD .The successful prokaryotic expression and bioinformatics analysis of Giardia lamblia SENP protein provide basis for further functional study of SENP .

关 键 词：蓝氏贾第鞭毛虫 SUMO特异性蛋白酶 原核表达 生物信息学 

分 类 号：R382.21[医药卫生—医学寄生虫学]