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当前位置: 首页 > 文章 > 毒害艾美耳球虫蔗糖非酵解解旋酶2基因片段的克隆与原核表达 畜牧与兽医 2017 (8) 72-75
Position: Home > Articles > Cloning and prokaryotic expression of partial sequence of SNF2 gene in Eimeria necatrix Animal Husbandry & Veterinary Medicine 2017 (8) 72-75

毒害艾美耳球虫蔗糖非酵解解旋酶2基因片段的克隆与原核表达

作  者：
马艺飞;杜彩娟;王乐乐;刘丹丹;许金俊;陶建平
单  位：
扬州大学兽医学院/江苏省动物重要疫病与人兽共患病防控协同创新中心
关键词：
毒害艾美耳球虫;蔗糖非酵解解旋酶2;克隆;表达
摘  要：
为了研究毒害艾美耳球虫蔗糖非酵解解旋酶2(SNF2)基因的功能,以提取的毒害艾美耳球虫子孢子总RNA为模板,应用RT-PCR技术扩增SNF2基因片段,克隆至pGEM-T-Easy载体,测序后构建pET30a-EnSNF2表达载体,转化大肠杆菌BL21,重组菌经测序鉴定后诱导表达,对表达产物进行SDS-PAGE分析和Western blot鉴定。结果显示:克隆的片段长822 bp,编码274个氨基酸;重组蛋白大小为36 ku左右,以包涵体形式为多,能被组氨酸(HIS)单抗特异性识别,表明该基因片段获得成功表达。研究结果为制备针对毒害艾美耳球虫SNF2的多抗和进一步研究该基因的功能奠定了基础。
译  名：
Cloning and prokaryotic expression of partial sequence of SNF2 gene in Eimeria necatrix
作  者：
MA Yifei;DU Caijuan;WANG Lele;LIU Dandan;XU Jinjun;TAO Jianping;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University;
关键词：
Eimeria necatrix;;SNF2;;cloning;;expression
摘  要：
In order to investigate the function of the sucrose non-feriorative helicase 2( SNF2) gene of Eimeria necatrix,the partial sequence of the gene was cloned from the total RNA of sporozoits of E. necatrix by RT-PCR and was inserted to pGEM-T-Easy vector by TA cloning. After sequencing analysis,EnSNF2 cDNA was subcloned to pET30 a vector to obtain a recombinant plasmid pET30a-EnSNF2. After transformed into E. coli BL21,the recombinant plasmid pET30a-EnSNF2 was induced to express by IPTG,which was verified by SDS-PAGE and Western blot analysis. The results showed that the partial cDNA of SNF2 gene was composed of 822 bp,coding 274 amino acids.SDS-PAGE and Western blot assay showed that the recombinant plasmid was expressed in E. coli BL21,and the fusion protein was mainly expressed as insoluble form with a molecular weight of 36 ku. This work lays a foundation for further study on function of E. necatrix SNF2.

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