蓝氏贾第鞭毛虫α-7．1、α-11贾第素的原核表达及抗原活性鉴定  

Prokaryotic expression and antigenicity analysis of α-7.1 and α-11 giardin of Giardia lamblia

作　　者：王洋[1] 杨志宏[2] 王沂[3] 曹蕾[1] 余源[1] 陈阳[1] 王卫亮[1] 慈雅丽[1] 田喜凤[1] 

机构地区：[1]河北联合大学生命科学学院,唐山063000 [2]唐山市职业技术学校,唐山063000 [3]中国医学科学院北京协和医学院放射医学研究所天津市分子核医学重点实验室,天津300192

出　　处：《中国人兽共患病学报》2012年第5期474-478,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.30970313);河北省科技厅项目(No.07276101D-68)联合资助

摘　　要：目的原核表达蓝氏贾第鞭毛虫的α-7.1、α-11贾第素蛋白。方法经RT-PCR获得α-7.1、α-11贾第素基因片段,经双酶切连入原核表达载体pET-28a(+),构建成为重组表达载体pET-28a(+)-α-7.1及pET-28a(+)-α-11,并转化大肠杆菌Rosetta(DE3)。IPTG诱导后,收集菌体,裂解后进行SDS-PAGE及Western blot检测。结果成功构建了原核表达载体pET-28a(+)-α-7.1和pET-28a(+)-α-11,经IPTG诱导后,在大肠杆菌中高效表达,SDS-PAGE及Western blot分析显示,在相对分子量约43kD和35kD的位置分别出现目的蛋白条带,与理论值相符;通过相应α-7.1、α-11贾第素抗血清的Westernblot证实两种贾第素重组蛋白的抗原活性良好;经Ni-NTA亲和层析柱纯化获得了高纯度的重组的α-7.1、α-11贾第素融合蛋白。结论成功地克隆、表达并纯化了具有良好抗原活性的α-7.1、α-11贾第素蛋白。In order to express protein in E. coli, the full-length cDNA encoding the 2 giardins were amplified by RT- PCR. The PCR products about 900bp and 1 100bp in length were respectivelycloned into prokaryotic expression vector pET-28a （＋） with restriction enzymes Nco I and Xho I . Sequencing results showed the sequences of the 2 giardins were identical with those in GenBank. The recombinant vector pET-28a（＋）-α-7.1 and pET-28a（＋）-α-11 were transformed into E. coli Rosetta （DE3） ,then the 2 fusion proteins were expressed by IPTG induction and the expression conditions were optimized. SDS-PAGE and western blot by using anti-His Tag antibody showed that the expressed products ofa-7.1 and a-ll giardins were fusion pro- teins about 43kD and 35kD respectively. The antigenic activities of two recombinant proteins were proved by western blot using corresponding antiserum. Highly purified recombinant α-7.1 and α-11 giardins were obtained by Ni-affinity chromatography. The present study might provide the foundation for the study of α -7. I and α-11 giardins.

关 键 词：蓝氏贾第鞭毛虫 α-7.1贾第素 α-11贾第素 原核表达 

分 类 号：R382[医药卫生—医学寄生虫学]