摘要
目的:从短短芽孢杆菌GZDF3菌株中克隆嗜铁素合成酶基因Gene5693,构建其原核表达载体并进行重组表达。方法:利用antiSMASH软件对GZDF3全基因组序列进行次级代谢物预测分析,然后采用ExPASy在线分析预测嗜铁素合成酶基因Gene5693的基本理化性质并进行重组表达。结果:生物信息学分析结果显示,短短芽孢杆菌GZDF3全基因组序列与Petrobactin嗜铁素基因簇的相似性为83%,Gene5693基因编码的蛋白与Petrobactin嗜铁素编码的蛋白AsbE的相似性为51.06%;Gene5693编码蛋白氨基酸长度为327个氨基酸,分子量为39.05kDa,等电点(pI)为4.94,为酸性亲水性蛋白;Gene5693基因的PCR扩增成功获得预测的Gene5693基因片段,菌落PCR及重组DNA分子的双酶切电泳结果显示成功构建重组质粒;SDS-PAGE结果表明Gene5693编码的蛋白大小与生物信息学预测的结果一致。结论:成功克隆了嗜铁素合成基因Gene5693,并进行了重组表达。
Objective: To clone the ferritin synthase Gene5693 from Bacillus brevis strain GZDF3 and construct its prokaryotic expression vector. Methods: AntiSMASH software was used to analyze GZDF3 genome sequence to predict secondary metabolites,then basic physicochemical properties of the ferromycin synthase Gene5693 were predicted using ExPASy online analysis. Results: Bioinformatics analysis revealed that the similarity between the whole genome sequence of Bacillus brevis GZDF3 and the Petrobactin ferritin gene cluster was 83%,and the similarity between Gene5693-encoded protein and Petrobactin ferritin-encoded AsbE protein was 51.06%.Gene5693-encoded protein contains 327 amino acids,and its molecular weight is 39.05 KDa,and its isoelectric point (pI) is 4.94,so it is acidic hydrophobic.Predicted Gene5693 gene fragment was successfully amplified by PCR.The size of amplified Gene5693 fragment was verified using colony PCR.Double enzyme digestion showed the cloning of the amplified Gene5693 fragment into prokaryotic expression vector was correct.SDS-PAGE analysis showed that the amplified Gene5693 fragment was expressed and its size was the same as predicted one. Conclusion: Gene5693 was successfully amplified,cloned and expressed in prokaryotes.
作者
袁林
贾化可
盛淼淼
王兵
曾丽娜
刘红美
隆耀航
YUAN Lin;JIA Huake;SHENG Miaomiao;WANG Bing;ZENG Lina;LIU Hongmei;LONG Yaohang(Research Center of Medical Biotechnology, Guizhou Medical University, Guiyang 550025, Guizhou, China;Key Laboratory of Biology and Medical Engineering, Guizhou Medical University, Guiyang 550025, Guizhou, China;Research Center of Immunocyte and Antibody Engineering, Department of Education, Guizhou Province, Guiyang 550025, Guizhou, China;Biological and Engineering School, Guizhou Medical University, Guiyang 550025, Guizhou, China)
出处
《贵州医科大学学报》
CAS
2019年第8期916-920,共5页
Journal of Guizhou Medical University
基金
贵州省科技支撑计划[黔科合支撑(2017)2833]
贵州医科大学医药生物技术工程研究中心(2016002)
贵阳市联合基金[(2017)5-33]
贵州省科技计划项目[黔科合基础(2019)1253]
