结核分枝杆菌echA1蛋白的结构特征和抗原性分析  

Structural characteristics and antigenicity analysis of echA1 protein from Mycobacterium tuberculosis

作　　者：张娇 马国荣 朱娜 杨玉荣[1] ZHANG Jiao;MA Guo-rong;ZHU Na;YANG Yu-rong(Department of Pathogenic Biology and Medical Immunology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院病原生物与免疫学系,银川750004

出　　处：《中国人兽共患病学报》2023年第6期556-562,569,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81960604,81460311)。

摘　　要：目的对结核分枝杆菌Rv0222基因及其编码蛋白echA1进行生物信息学分析,制备该蛋白的多克隆抗体,为进一步研究其功能、筛选候选治疗靶点和免疫诊断提供基础。方法利用TMHMM、SignalP6.0、Net Phos3.1、Protpapram、Protscale和sopma等在线分析软件分析echA1蛋白的理化性质等信息。抗原表位则利用IEDB网站中的在线分析软件和NetCTL-1.2进行分析。利用分子克隆技术构建pET-30a-Rv0222表达载体并转化入E.coli BL21(DE3)中,IPTG诱导echA1蛋白表达后经His亲和层析柱纯化并免疫BALB/c小鼠制备多克隆抗体。收集结核病患者及健康人血清,ELISA法检测样本中特异性抗体水平。结果Rv0222基因编码的echA1蛋白由262个氨基酸组成,为一种亲脂性蛋白。echA1蛋白二级结构包括α-螺旋(43.51%)、β-折叠(13.74%)、β-转角(9.92%)和无规则卷曲(32.82%)。该蛋白无跨膜区和信号肽,含16个磷酸化位点,并可与fadB、fadB2、fadE1、和accD1等10多种蛋白发生相互作用。表位预测分析显示该蛋白含有多个B细胞表位和T细胞表位。纯化的echA1蛋白可有效引起小鼠的免疫应答,获得的多克隆抗体具有良好的抗原特异性,效价在102400和204800之间。结核病患者抗体检测敏感度为64.6%,健康人抗体检测特异度为94.6%,结核病患者与健康者相比,差异具有统计学意义(t=10.66,P<0.0001)。结论EchA1蛋白为一种稳定性的亲脂性蛋白,含有多个潜在优势B和T细胞表位,并可以与多个脂肪代谢相关蛋白相互作用;制备的多克隆抗体具有很高的抗原特异性;该蛋白在结核病血清学诊断方面具有一定应用价值。In order to provide a basis for further study of echA1’s function,screen candidate therapeutic targets and immunodiagnosis,TMHMM,SignalP6.0,Net Phos3.1,Protpapram,Protscale and sopma were used to analyze the physical and chemical properties of echA1 protein.Epitopes were analyzed using the IEDB online analysis software and NetCTL-1.2.The pET-30a-Rv0222 expression vector was constructed by molecular cloning technique and transformed into E.coli BL21(DE3),purified echA1 protein was immunized BALB/c mice to prepare polyclonal antibody.The specific antibody levels in serum of TB patients and healthy people were detected by ELISA.EchA1 protein is a lipophilic protein composed of 262 amino acids,and its secondary structure mainly includesα-helix(43.51%)、β-sheet(13.74%)、β-turn(9.92%)and random coil(32.82%).It contains 16 phosphorylation sites and has no transmembrane region and signal peptide which can interact with more than 10 proteins,such as fadB,fadB2 fadE1 and accD1.In addition,epitope prediction analysis showed that the protein contained multiple B and T cell epitopes.Purified echA1 protein can effectively induce immune response to mice which indicated that the polyclonal antibody obtained has good antigenic specificity,with titer between 102400 and 204800.The sensitivity of TB patients and specificity of healthy people was 64.6%and 94.6%respectively and the difference was statistically significant(t=10.66,P<0.0001).Result revealed that echA1 protein is a stable lipophilic protein,containing multiple potentially dominant B and T cell epitopes,and can interact with multiple proteins related to fat metabolism.The protein has high antigen specificity and immunogenicity,which might have great application value in the diagnosis of tuberculosis.

关 键 词：结核分枝杆菌 生物信息学 原核表达 多克隆抗体 echA1 

分 类 号：R378.91[医药卫生—病原生物学]