靶向不可分型流感嗜血杆菌外膜蛋白P6重组M13噬菌体疫苗的构建及其免疫效果评价  

Construction and immune effect evaluation of recombinant M13 phage vaccine targeting outer membrane protein P6 of nontypeable Haemophilus influenzae

作　　者：王财 常月立[1] 胡楠 李唯峰 赵子红 安晶 张玉妥[1] WANG Cai;CHANG Yueli;HU Nan;LI Weifeng;ZHAO Zihong;AN Jing;ZHANG Yutuo(Institute of Pathogenic Biology and Immunology,Hebei North University,Zhangjiakou 075000,Hebei Province,China)

机构地区：[1]河北北方学院病原生物学与免疫研究所,河北张家口075000

出　　处：《中国生物制品学杂志》2025年第2期129-136,142,共9页Chinese Journal of Biologicals

基　　金：河北省高校科学技术研究项目(Z2018030);河北省研究生创新项目(CXZZSS2022144,CXZZSS2022146,CXZZSS2022152);河北北方学院创新项目(2023YJSCX01)。

摘　　要：目的构建靶向不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae,NTHi)外膜蛋白P6的重组M13噬菌体疫苗,并评价其免疫原性,以期为NTHi疫苗的进一步研发提供新思路。方法通过基因重组技术将NTHi外模蛋白P6基因整合至载体pMECS Phagemid,获得重组P6-M13噬菌体单克隆,经包装及纯化后,制备为重组P6-M13噬菌体疫苗。Western blot法检测疫苗中P6-M13 PⅢ融合蛋白的表达情况,双层琼脂平板法测定疫苗滴度,透射电镜下观察重组P6-M13噬菌体形态。将90只5周龄BALB/c雌性小鼠随机分为PBS、M13噬菌体、重组P6-M13噬菌体疫苗组,每组30只,分别于0、14、28 d经腹腔注射PBS(500μL/只)、M13噬菌体[1×10^(12)pfu/(500μL·只)]、重组P6-M13噬菌体疫苗[1×10^(12)pfu/(500μL·只)]。末次免疫2周后,ELISA法检测小鼠血清中特异性IgG水平及脾淋巴细胞培养上清中IFNγ、IL-2、IL-4、IL-5和IL-17A水平,CCK-8法分析脾淋巴细胞的增殖情况;末次免疫3周后,用1.5×108cfu/mL NTHi经小鼠鼻腔进行攻毒,攻毒1周后,HE染色法观察小鼠鼻黏膜和肺组织病理变化;末次免疫4周后,称量小鼠心脏、肝脏、脾脏、肺脏、肾脏质量,计算脏器系数,并制备组织病理切片,进行病理学观察。结果制备的重组P6-M13噬菌体疫苗可正确表达P6-M13 PⅢ融合蛋白,滴度为5.5×10^(14)pfu/mL,镜下观察可见形态规则的重组P6-M13噬菌体。与M13噬菌体及PBS组比较,重组P6-M13噬菌体疫苗组小鼠血清特异性抗体IgG水平显著升高(F=71.489,P<0.05);脾细胞分泌IFNγ、IL-2、IL-4、IL-5水平明显降低(F分别为8.315、16.986、39.204、6.291,P均<0.05),3组间IL-17水平差异无统计学意义(F=0.863,P>0.05);脾细胞刺激指数明显升高(F=22.952,P<0.05)。攻毒后,PBS和M13噬菌体组小鼠鼻黏膜及肺组织结构严重受损,炎性细胞增多;重组P6-M13噬菌体疫苗组小鼠鼻黏膜及肺组织结构正常,少见炎性细胞。各组小鼠心脏、肝脏、脾脏、�Objective To construct a recombinant M13 phage vaccine targeting the outer membrane protein P6 of nontypeable Haemophilus influenzae(NTHi)and evaluate its immunogenicity in order to provide new ideas for further development of NTHi vaccines.Methods The NTHi P6 gene was fused with the vector pMECS Phagemid by gene recombination technique.After packaging and purification,the obtained recombinant P6-M13 phage was prepared into recombinant P6-M13 phage vaccine.The expression of P6-M13 PⅢfusion protein in the vaccine was detected by Western blot,the vaccine titer was determined by double-layer agar plate method,and the recombinant P6-M13 phage morphology was observed under transmission electron microscope.Ninety 5-week-old BALB/c female mice were randomly divided into PBS group,M13 phage group and recombinant P6-M13 phage vaccine group,30 for each,and intraperitoneally injected with PBS(500μL/mouse),M13 phage[1×10^(12)pfu/(500μL·mouse)]and recombinant P6-M13 phage vaccine[1×10^(12)pfu/(500μL·mouse)]on the 0,14th and28th day,separately.Two weeks after the last immunization,the levels of specific IgG in serum and IFNγ,IL-2,IL-4,IL-5and IL-17A in spleen lymphocyte culture supernatant were detected by ELISA,and the proliferation of spleen lymphocytes was analyzed by CCK-8.Three weeks after the last immunization,the mice were challenged with 1.5×108cfu/mL NTHi through the nasal cavity.After one week of challenge,the pathological changes of nasal mucosa and lung tissue were observed by HE staining.Four weeks after the last immunization,the heart,liver,spleen,lung and kidney of mice were weighed,the organ coefficients were calculated,and histopathological sections were prepared for pathological observation.Results The recombinant P6-M13 phage could correctly express P6-M13 PⅢfusion protein with a titer of 5.5×10^(14)pfu/mL,and the recombinant P6-M13 phage with regular morphology was observed under microscope.Compared with M13 phage group and PBS group,the level of serum specific antibody IgG in mice of recombin

关 键 词：不可分型流感嗜血杆菌 外膜蛋白P6 噬菌体展示技术 M13噬菌体疫苗 免疫原性 

分 类 号：R186.3[医药卫生—流行病学]