| 摘要: |
| 为探究捻转血矛线虫GST基因的原核表达及其生物信息学特征，以捻转血矛线虫cDNA为模板，设计特异性引物扩增GST基因，构建原核表达载体pET30a(+)-GST转化至E. coil BL21(DE3)感受态细胞，将鉴定正确的重组菌进行表达条件优化后，再通过SDS-PAGE分析蛋白表达的特征，并利用在线软件对GST蛋白进行生物信息学分析。结果表明:捻转血矛线虫GST基因大小为618 bp；诱导后的pET30a(+)-GST重组菌最佳诱导条件为0.05 mmol/L的IPTG在37 ℃时诱导6 h，以包涵体的形式存在，大小约为29 ku。生物信息学分析表明:GST分子式为C1083H1657N277O294S6，属于不含信号肽和跨膜区的亲水性蛋白；二级结构主要由α螺旋形成；潜在的抗原表位主要位于25~48、55~63、92~106、113~124、139~147、170~189和195~205位氨基酸附近。本试验成功构建GST基因原核表达系统，优化表达条件下能稳定纯化GST重组蛋白，为进一步探索捻转血矛线虫丙硫咪唑耐药机制及GST蛋白的生物学特性提供研究基础。 |
| 关键词: 捻转血矛线虫 GST基因 原核表达 生物信息学 |
| DOI：10.11841/j.issn.1007-4333.2020.01.14 |
| 投稿时间：2019-06-09 |
| 基金项目:国家自然科学基金项目(31760731)；内蒙古自治区科技计划项目(201702074) |
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| Cloning, expression and bioinformatics analysis of drug resistance GST gene of Haemonchus contortus |
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ZHAO Xueliang,SUN Ke,SU Qian,Huhebateer,WANG Wenlong
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| (College of Veterinary Medicine/Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease ofMinistry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot 010018, China) |
| Abstract: |
| This study was aimed to investigate the prokaryotic expression and bioinformatics character of Haemonchus contortus GST gene. Using the cDNA of H. contortus as template, specific primers were designed to amplify GST gene. The prokaryotic expression vector pET-30a(+)-GST was constructed and transferred into E. coli BL21(DE3)competent cells, and the expression conditions for the correct recombinant plasmid were optimized. The characteristics of expression protein were analyzed by SDS-PAGE, and the GST protein was analyzed by bioinformatics software. The results showed that the GST fragment of H. contortus was 618 bp. The optimal induction condition of pET-30a(+)-GST recombinant plasmid was 0. 05 mmol/L IPTG at 37 ℃ for 6 h, and the expressed recombinant protein was about 29 ku in the form of inclusion bodies. Bioinformatics analysis showed that the GST formula was C1083H1657N277O294S6, which belonged to hydrophilic protein without signal peptide and transmembrane region. The secondary structure is mainly formed by α-helix, and the potential epitopes are mainly located near 25-48, 55-63, 92-106, 113-124, 139-147, 170-189 and 195-205 amino acids. In this study, the prokaryotic expression system of H. contortus GST gene was constructed, and the GST recombinant protein was stably purified after optimized induction conditions, which provided a theoretical basis for further exploring albendazole resistance mechanism of H. contortus and the biological characteristics of GST-encoded protein. |
| Key words: Haemonchus contortus GST genes prokaryotic expression bioinformatics |
