摘要
为了获得高纯度牛布鲁氏菌VirB12蛋白及其抗原表位蛋白VB-BW,试验采用生物信息学方法对牛布鲁氏菌VirB12蛋白的理化性质、空间结构、信号肽、结构域、跨膜结构、磷酸化位点、抗原表位等进行预测,在此基础上设计抗原表位蛋白VB-BW;VirB12与VB-BW基因经PCR扩增后与pGEX4T-1载体连接,并转化至E.coli BL21(DE3)感受态细胞中诱导表达蛋白;经His-Trap HP亲和纯化后对VirB12与VB-BW蛋白进行SDS-PAGE分析与Western-blot鉴定。结果表明:VirB12蛋白由172个氨基酸组成,理论等电点为10.28,脂肪族氨基酸占比较高,半衰期大于10 h;α-螺旋、β-转角、β-折叠与无规则卷曲结构占总蛋白的比例分别为38.95%、4.07%、13.95%与43.02%;含有15个氨基酸组成LIPO型信号肽与一个OmpA同源区域;无跨膜结构;有13个磷酸化位点;含有7个优势B淋巴细胞抗原表位与4个优势T淋巴细胞抗原表位;设计出的抗原表位蛋白VB-BW共有79个氨基酸,由VirB12蛋白的第67~97位与第130~172位氨基酸经刚性连接子连接组成。扩增出的VirB12与VB-BW基因大小为561 bp与270 bp;重组表达载体BL21-VB和BL21-BW在诱导剂IPTG浓度为0.2 mmol/L、诱导时间为10小时时表达出的VirB12与VB-BW蛋白含量最高,经纯化后最终获得大小为47.7 ku与36.3 ku的VirB12与VB-BW蛋白。说明试验构建出表达VirB12与VB-BW蛋白的原核表达体系。
In order to obtain the high-purity Brucella abortus VirB12 protein and its epitope protein VB-BW,in this experiment,bioinformatics methods were used to predict the physicochemical properties,spatial structure,signal peptide,domains,transmembrane structure,phosphorylation sites,and epitopes of Brucella abortus VirB12 protein;on this basis,the epitope protein VB-BW was designed.VirB12 and VB-BW genes were amplified by PCR and linked to pGEX4T-1 vectors and transformed into E.coli BL21(DE3)competent cells to induce the expression of proteins.After His-Trap HP affinity purification,VirB12 and VB-BW proteins were analyzed by SDS-PAGE and identified by Western-blot.The results showed that VirB12 protein was composed of 172 amino acids;the theoretical pI was 10.28;the proportion of aliphatic amino acids was relatively high,and the half-life was greater than 10 h.The proportions ofα-helix,β-corner,β-fold and irregular coil structures accounted for 38.95%,4.07%,13.95%and 43.02%of the total protein,respectively,which contained LIPO-type signal peptide composed of 15 amino acids,and an OmpA homologous region;it had no transmembrane structure;there were 13 phosphorylation sites,and 7 dominant B lymphocyte epitopes and 4 dominant T lymphocyte epitopes.The designed VB-BW protein had a total of 79 amino acids,which were composed of amino acids in positions 67-97 and 130-172 of VirB12 protein connected by rigid linkers.The amplified VirB12 and VB-BW genes were 561 bp and 270 bp.An inducer IPTG concentration was 0.2 mmol/L in the expression of recombinant E.coli BL21-VB and BL21-BW;higher levels of VirB12 and VB-BW proteins were obtained at an induction time of 10 hours.After purification,VirB12 and VB-BW proteins with the sizes of 47.7 ku and 36.3 ku were obtained.The results suggested that the prokaryotic expression system expressing VirB12 and VB-BW proteins was constructed.
作者
徐赵玉
任立松
岳海涛
杨洁
XU Zhaoyu;REN Lisong;YUE Haitao;YANG Jie(College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;TECON Biotechnology Co.,Ltd.,Urumqi 830011,China)
出处
《黑龙江畜牧兽医》
北大核心
2023年第19期72-81,140,共11页
Heilongjiang Animal Science And veterinary Medicine
基金
天康生物股份有限公司项目“布鲁氏菌B12蛋白表达与纯化”(201801)。
关键词
VirB12蛋白
生物信息学
抗原表位
原核表达
亲和层析
VirB12 protein
bioinformatics
antigen epitope
prokaryotic expression
affinity chromatography
