犬恶丝虫酪蛋白激酶2β亚基基因片段的克隆和原核表达  

Cloning and Prokaryotic Expression of Casein Kinase Ⅱ Subunit Beta Gene Fragment of Dirofilaria immitis

作　　者：高珺珊[1] 吴威[1] 侯洪烈[1] 宫鹏涛[1] 李建华[1] 李赫[1] 张国才[1] 张西臣[1] 杨举[1] 

机构地区：[1]吉林大学动物医学学院,长春130062

出　　处：《中国寄生虫学与寄生虫病杂志》2013年第4期290-292,共3页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家科技重大专项(No.2008ZX10401-B)~~

摘　　要：目的对犬恶丝虫酪蛋白激酶2β亚基(Csnk2b)部分基因片段进行克隆、原核表达及免疫反应性分析。方法根据犬恶丝虫cDNA文库中筛选出的Csnk2b部分基因片段设计引物,以含有插入Csnk2b部分基因片段的噬菌体DNA为模板,进行PCR扩增。产物亚克隆至原核表达载体,构建重组载体pGEX-4T-1-Csnk2b,双酶切鉴定。将其转入大肠埃希菌(E.coli)Rosetta(DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达的重组蛋白,蛋白质印迹(Western blotting)以小鼠抗犬恶丝虫阳性血清为一抗,分析重组蛋白免疫反应性。结果Csnk2b部分基因片段PCR扩增产物约为700 bp,测序结果与cDNA文库中筛选得到的序列一致。重组表达载体pGEX-4T-1-Csnk2b双酶切鉴定正确。SDS-PAGE结果显示,IPTG诱导后重组蛋白GST-Csnk2b表达,相对分子质量(M r)约为45 000,与预期大小一致。Western blotting分析表明,重组蛋白能被小鼠抗犬恶丝虫阳性血清识别。结论克隆并表达了犬恶丝虫Csnk2b重组蛋白,且该蛋白具有免疫反应性。Objective To clone and express the partial fragment of Csnk2b gene of Dirofilaria immitis in prokaryotic cells, and analyze the immunoreactivity. Methods The partial fragment of Csnk2b gene was amplified by PCR with a pair of specific primers. The PCR product was cloned into pMD18-T, and then sub-cloned to pGEX-4T-1 expression vector. The constructed plasmid pGEX-4T-1-Csnk2b was transformed into E. coli Rosetta （DE3） and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SI）S-PAGE and identified by Western blotting. Results The PCR product was about 700 bp. Enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1-Csnk2b was constructed. SDS-PAGE results showed that the relative molecular weight （Mr） of the fusion protein （GST-Csnk2b） was about 45 000. GST-Csnk2b reacted positively with mouse anti-D, immitis serum. Conclusion The partial Csnk2b gene has been expressed in prokaryotic expression system and shows immunoreactivity.

关 键 词：犬恶丝虫 酪蛋白激酶2β亚基 原核表达 

分 类 号：R383.16[医药卫生—医学寄生虫学]