甲型H5N1流感病毒神经氨酸酶生物信息学分析及其原核表达和免疫原性评价  

Bioinformatics analysis, prokaryotic expression and immunogenicity evaluation of neuraminidase of H5N1 influenza virus

作　　者：邓涛 马宁 刘京 吕传硕 周蓉 张国梅 乐洋 年悬悬 张家友 杨晓明 DENG Tao;MA Ning;LIU Jing;Lü Chuan-shuo;ZHOU Rong;ZHANG Guo-mei;LE Yang;NIAN Xuan-xuan;ZHANG Jia-you;YANG Xiao-ming(The Second Research Department of Viral Vaccine,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究二室国家联合疫苗工程技术研究中心,湖北武汉430207 [2]中国生物技术股份有限公司,北京100029

出　　处：《微生物学免疫学进展》2022年第2期1-8,共8页Progress In Microbiology and Immunology

基　　金：国家“重大新药创制”科技重大专项资助项目(2016ZX09106003-008)。

摘　　要：目的 分析甲型H5N1流感病毒(简称H5N1)神经氨酸酶(neuraminidase, NA)生物信息学特征,原核表达获得H5N1 NA重组蛋白,并在动物模型中评价其免疫原性。方法 利用生物信息学软件分析H5N1 NA的理化性质、跨膜区、信号肽以及二级结构和高级结构。将H5N1 NA的编码区进行密码子优化后基因合成,将合成目的基因片段与pET-22b(+)连接,构建重组原核表达质粒pET-22b(+)-H5N1 NA,将重组质粒转化到BL21(DE3)感受态细胞并进行诱导表达;超声破碎菌体,使用Ni-NTA亲和层析纯化,梯度透析复性,SDS-PAGE分析纯度,Bradford法测定浓度;分别用5、10和50μg的NA加或不加MF59佐剂免疫BALB/c小鼠,检测其诱导产生NA特异性抗体滴度,评价免疫原性。结果 经一系列生物信息学软件分析得知,H5N1 NA为稳定的亲水性跨膜蛋白,无信号肽,二级结构以无规则卷曲和α螺旋为主,高级结构为四聚体结构,相对分子质量48 941.92,分子式C_(2148)H_(3293)N_(595)O_(666)S_(26),等电点6.13,不稳定系数31.84,总平均亲水性-0.268;经双酶切和测序验证,重组质粒pET-22b(+)-H5N1 NA构建成功,转化至E.coli BL21(DE3),经诱导表达、纯化、复性、浓缩获得重组NA纯度为94%,相对质量浓度为620.23μg/mL,具有酶活性;使用该原核表达的NA免疫BALB/c小鼠,能产生结合H5N1疫苗原液中NA的特异性抗体,50μg+MF59佐剂组平均效价达3 520。结论 原核表达的NA免疫小鼠诱导产生的抗体能够与甲型H5N1流感病毒天然的NA发生反应,为基于NA的流感重组亚单位疫苗研发提供了可行路径。Objective To analyse neuraminidase(NA) of influenza A virus(H5 N1) using bioinformatics, to express the recombinant H5 N1 NA protein in E. coli, and to evaluate the immunogenicity of H5 N1 NA protein in animal models. Methods The physical and chemical properties, transmembrane region, signal peptide and secondary and tertiary structure of influenza virus H5 N1 NA protein were analyzed by bioinformatics software. The coding region of H5 N1 NA was synthesized after codon optimization. The target gene was ligated with pET-22 b(+). The recombinant expression plasmid pET-22 b(+)-H5 N1 NA was constructed and transformed into E. coli BL21(DE3) competent cells. After sonication of bacteria, the supernatant was purified by Ni NTA affinity chromatography, and the purified protein was renatured by gradient dialysis. Purity and concentration of the purified NA protein were detected by SDS-PAGE and by Bradford method, respectively. BALB/c mice were immunized with different concentrations of NA protein with or without MF59 adjuvants, and the antibody titer was detected to evaluate the immunogenicity. Results The results of bioinformatics analysis showed that H5 N1 NA protein was a stable and hydrophilic transmembrane protein without signal peptide, and the secondary structure was dominated by alpha-helices and random coils, and the quaternary structure of NA protein contains four subunits. The relative molecular weight of NA protein is 48 941.92, and the molecular formula is C_(2148)H_(3293)N_(595)O_(666)S_(26). Furthermore, the isoelectric point, the instability coefficient, and the total average hydrophilicity of NA protein were 6.13, 31.84 and-0.268, respectively. The recombinant plasmid pET-22 b(+)-H5 N1 NA was successfully constructed and transformed into E. coli BL21(DE3). After expression, purification and renaturation, the purified(94%) recombinant NA protein with enzyme activity was obtained, and the concentration of NA protein was 620.23 μg/ml. BALB/c mice immunized with NA protein produced specific antibodies bindi

关 键 词：神经氨酸酶 生物信息学 原核表达 免疫原性 

分 类 号：R373.13[医药卫生—病原生物学]