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。
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.
出处
《畜牧与兽医》
北大核心
2017年第8期72-75,共4页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31472181)
江苏省高校优势学科建设工程项目
