蓝氏贾第鞭毛虫核苷二磷酸激酶基因的克隆与表达  

Cloning and expression of the nucleoside diphosphate kinase gene of Giardia lamblia

作　　者：袁义雪 郑国侠[2] 王丹丹 专行 杜萌 王云华[1] YUAN Yi-xue;ZHENG Guo-xia;WANG Dan-dan;ZHUAN Hang;DU Meng;WANG Yun-hua(Medical School;Environmental and Chemical Technology School, Dalian University, Dalian 116622, China)

机构地区：[1]大连大学医学院,大连116622 [2]大连大学环境与化学工程学院,大连116622

出　　处：《中国寄生虫学与寄生虫病杂志》2018年第2期190-192,195,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81471807;No.41476085);辽宁省高等学校杰出青年基金(No.LJQ2015005);大连市高层次人才创新创业项目(No.2015R087)~~

摘　　要：以蓝氏贾第鞭毛虫中国C2株基因组DNA为模板,PCR扩增蓝氏贾第鞭虫核苷二磷酸激酶(Gl NDPK)基因编码序列,连接至p ET28a质粒,转化至大肠埃希菌(Escherichia coli)JM109,挑取阳性克隆进行鉴定与测序。将p ET28a-Gl NDPK转化至E.coli BL21(DE3),用异丙基-β-D-硫代半乳糖苷诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白表达情况。用8 mol/L尿素溶解包涵体并收集上清,镍离子亲和层析进行纯化,蛋白质印迹(Western blotting)分析重组蛋白的抗原性。结果显示,PCR扩增获得了长度约1 200 bp的蓝氏贾第鞭毛虫中国C2株Gl NDPK基因编码序列。构建的重组质粒p ET28a-Gl NDPK经PCR鉴定、单酶切鉴定和测序,结果均与预期相符。SDS-PAGE结果显示,重组蛋白以包涵体的形式表达,相对分子质量(Mr)约为40 000,与预期结果大致一致。Western blotting结果显示,该重组蛋白可被His标签抗体识别。提示获得了蓝氏贾第鞭毛虫中国C2株Gl NDPK基因编码序列及其重组表达蛋白。The nucleoside diphosphate kinase of Giardia lamblia（ Gl NDPK）（Chinese strain C2） coding sequence was amplified by PCR using the genomic DNA as the template. The PCR product was inserted into the p ET28 a plasmid and transformed into E. coli JM109. The positive clones were picked up for identification and sequencing. The p ET28 a-Gl NDPK recombinant plasmid was transformed into E. coli BL21（DE3）, and expressed under IPTG induction. The proteins expressed were analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea and the supernatant was collected and purified by Ni2 ＋affinity chromatography. The recombinant protein was verified by Western blotting using anti-His6 tag antibody. The results showed amplification of the Gl NDPK coding sequence of Giardia lamblia（Chinese strain C2） by PCR, resulting in a product of 1 200 bp. The recombinant plasmid p ET28 a-Gl NDPK was verified by PCR, single enzyme digestion, and sequencing. As expected, SDS-PAGE showed that the recombinant Gl NDPK protein was expressed as an inclusion body with a Mrof 40 000. Western blotting showed that the recombinant Gl NDPK could be recognized by anti-His6 tag antibody. Therefore, the coding sequence of Gl NDPK gene and its recombinant protein have been obtained.

关 键 词：蓝氏贾第鞭毛虫 核苷二磷酸激酶 克隆 表达 纯化 

分 类 号：R382.213[医药卫生—医学寄生虫学]