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

doi:10.16431/j.cnki.1671-7236.2021.02.030

Abstract

Abstract: 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 2 000 μg/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 1:12 800.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 mainly α-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 was α-helix.The results provided references for further study on the function and molecular mechanism of BspD secreted by Brucella.

Key words: Brucella; BspD secreted protein; bioinformatics analysis; polyclonal antibody

CLC Number:

S852.6⁺14

LI Ruirui, MA Zhongchen, ZHANG Hongyang, WANG Zhen, NUSUFU·Nuersailike, WANG Yong, CHEN Chuangfu. Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 676-684.

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Polyclonal Antibody Preparation and Bioinformatics Analysis of Brucella Secreted Protein BspD

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