中国畜牧兽医2017，Vol.44Issue(7)：1947-1953,7.DOI:10.16431/j.cnki.1671-7236.2017.07.007
羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析
Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis
摘要
Abstract
This study was aimed to clone and express dhbC gene of Brucella melitensis, and analyze the bioinformatics of its expressed protein.A pair of primers were designed by referring to dhbC gene sequence information of Brucella melitensis M5-90 strain in GenBank, and the dhbC gene fragment was amplified by PCR method.The obtained dhbC gene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5α competent cells.The plasmid was identified by restriction enzyme digestion.The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21 (DE3) competent cells.The expression was induced by IPTG.The expressed product was analyzed by SDS-PAGE and Western blotting.Bioinformatics analysis of the amino acid sequence encoded by dhbC gene was carried out using bioinformatics software DNAMAN and related online sites ProtParam, SOPMA and Protscale.The results showed that dhbC gene was cloned with the length of 1 093 bp, and protein expression was expressed.The expressed fusion protein was about 47 ku, and was mainly in the form of inclusion body.The molecular weight of the dhbC protein was C1866H2968N544O562S15, the molecular mass was 42 496.3 u, the theoretical isoelectric point (pI) was 5.81, the extinction coefficient was 33 835, the instability coefficient was 36.76, the hydrophobic index was 86.19, the total average hydrophobicity (GRAVY) was -0.215.The half-life of reticulocytes in mammals was predicted to be 30 h, and the secondary structure was dominated by α-helix (41.94%) and random coil (31.46%).关键词
布鲁氏菌/dhbC基因/克隆/原核表达/生物信息学分析Key words
Brucella melitensis/dhbC gene/cloning/prokaryotic expression/bioinformatics analysis分类
生物科学引用本文复制引用
李宝宝,李亚颖,王凤阳,杜丽,聂鑫,杨小健,曹瑞勇,朱姝,黄海峰,张振兴,彭冬梅,李国华..羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953,7.基金项目
国家自然科学基金(31460670) （31460670）
海南省重大科技计划项目(ZDKJ2016017-01) （ZDKJ2016017-01）
