布鲁氏菌分泌蛋白BspD多克隆抗体制备及其生物信息学分析

本研究旨在获得布鲁氏菌BspD蛋白，制备其多克隆抗体，并分析其潜在生物学功能。根据流产布鲁氏菌（Brucella abortus）2308的BspD基因序列（GenBank登录号：NC_007618.1）获得BspD基因序列，对布鲁氏菌BspD氨基酸序列进行生物信息学分析，然后将目的基因嵌入pMD19-T载体，使用限制性内切酶切下目的基因重组入pET-28a（+）载体，构建pET28a-BspD重组载体，经过双酶切、测序验证后，IPTG诱导表达菌株，SDS-PAGE法鉴定BspD的表达，His标签蛋白纯化柱纯化BspD蛋白，BCA试剂盒检测蛋白浓度。用纯化BspD蛋白免疫新西兰大白兔，Western blotting鉴定多克隆抗体的特异性，间接ELISA法检测抗体的效价及反应原性。结果表明，成功表达出37 ku BspD蛋白，BCA方法测得纯化BspD浓度为2 000 μg/mL；Western blotting结果显示制备的抗体具有良好的特异性，间接ELISA检测出BspD多克隆抗体的效价为1：12 800，成功制备出BspD多克隆抗体，但其反应原性较低。生物学信息分析得出BspD蛋白具有亲水性，存在跨膜区，无信号肽，有17个磷酸化位点和11个抗原决定簇，BspD蛋白二级结构以α-螺旋为主，达到86.80%，还存在少量延伸链、无规则卷曲、β-折叠等；在线软件Phyre 2构建BspD三级结构证实其多为α-螺旋。以上结果可为进一步研究布鲁氏菌分泌蛋白BspD的功能及分子机制提供参考。

Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD

The purpose of this study was to obtain Brucella BspD protein,analyze its potential biological functions,and prepare its polyclonal antibody.After the synthesis of BspD gene sequences and the bioinformatics analysis of the amino acid sequence of Brucella BspD,according to the BspD gene sequence of Brucella abortus 2308(GenBank accession No:NC_007618.1),the target gene was inserted into pMD19-T vector,and the target gene was digested by restriction endonuclease and recombined into pET-28a(+)vector to construct the pET28a-BspD recombinant vector.After double enzyme digestion and sequencing verification,IPTG was used to induce expression strain,SDS-PAGE was used to identify the expression of BspD,His label protein purification column was used to purify BspD protein,BCA kit was used to detecte the protein concentration,purified BspD protein was used to immunize New Zealand White rabbits,Western blotting was used to identify the specificity of polyclonal antibodies.Indirect ELISA was used to detect the titer and reactivity of antibody.The 37 ku BspD protein had been expressed successfully,and the purified BspD concentration was 2000μg/mL by BCA method.The Western blotting results showed that the prepared antibody had good specificity,and the titer of BspD polyclonal antibody detected by indirect ELISA was 1∶12800.The polyclonal antibody against BspD had been successfully prepared,but its reactivity was low.According to the analysis of biological information,BspD protein was hydrophilic,existed transmembrane region,had no signal peptide,possessed 17 phosphorylation sites and 11 antigenic epitopes.The secondary structure of BspD protein was mainlyα-helix,reaching 86.80%,and there were a few extended chains,random coil,β-folding and other structures.In addition,the tertiary structure of BspD was constructed by Phyre 2,an online software,also confirmed that it wasα-helix.The results provided references for further study on the function and molecular mechanism of BspD secreted by Brucella.