摘要
为评价猪链球菌病-副猪嗜血杆菌病表面展示二联亚单位疫苗对小鼠的保护效果,本研究在应用融合PCR构建副猪嗜血杆菌(HPS)保护性抗原AfuA-OppA2和CdtB-OppA基因的串联序列基础上,将其分别插入pMD-28a-INP-His表面展示质粒,并将猪链球菌(SS)保护性抗原MRP、SLY基因也分别克隆至该表面展示质粒,构建pMD-INP-CdtB-OppA、pMD-INP-AfuA-OppA2、pMD-INP-MRP、pMD-INP-SLY 4种重组质粒,分别转入E.coli BL21(DE3)中诱导表达,使重组蛋白AfuA-OppA2、CdtB-OppA、MRP、SLY分别展示于E.coli BL21(DE3)的菌体表面。将灭活后的4种重组大肠杆菌按等质量比混合并添加ISA 201佐剂乳化制成表面展示二联亚单位疫苗,间隔14 d两次免疫KM小鼠,另设含rAfuA、rOppA2、rCdtB、rOppA、rMRP、rSLY的纯化蛋白亚单位疫苗免疫组、副猪嗜血杆菌-猪链球菌(HPS-SS)灭活疫苗免疫组和PBS对照组,二次免疫后的小鼠血清通过ELISA检测相应抗体和细胞因子水平。随后用HPS血清5型HN10株、血清13型ZD12株和SS血清2型M126株和血清9型GZ2株进行攻毒。结果显示:表面展示二联亚单位疫苗刺激小鼠产生的抗体和细胞因子水平与纯化蛋白亚单位疫苗组相当。用副猪嗜血杆菌5型和13型菌株分别攻毒后,表面展示二联亚单位疫苗对小鼠的保护率均为80%,其对HPS 5型菌株攻毒的小鼠保护率低于纯化蛋白亚单位疫苗(90%),但对HPS 13型菌株攻毒小鼠的保护率高于纯化蛋白亚单位疫苗(60%),且均优于HPS-SS灭活苗(80%和60%)。用SS 2型和9型菌株分别攻毒后,表面展示二联亚单位疫苗对小鼠的保护率为50%和80%,低于纯化蛋白亚单位疫苗的保护效果(100%和80%),优于HPS-SS灭活苗(50%和60%)。以上结果表明,基于表面展示技术的猪链球菌病-副猪嗜血杆菌病二联亚单位疫苗能刺激机体产生相应的抗体,其对HPS血清5型、13型和SS血清2型、9型菌株的攻击均能提供良好的交叉保护,保护效果优于HPS-SS灭活苗。本研究首次为基于表面展示技术的亚单位疫苗的研制提供实验依据,为细菌疫苗的研发提供新思路。
To evaluate immune protection of the HPS-SS binary subunit vaccine based on surface display technology to KM mice,surface display plasmids pMD-INP-Cdt B-OppA,pMD-INP-AfuA-Opp A2,pMD-INP-MRP,and pMD-INP-SLY were constructed and transformed into E.coli BL21(DE3).The fusion proteins AfuA-OppA2,CdtB-OppA,MRP,and SLY were displayed on the surface of E.coli BL21(DE3)certificated by immunoeletron microscopy.Different inactivated bacteria were proportionally mixed according to the proteins concentration,then the bacteria liquid was emulsified with equal quantity of ISA201 VG,which is named surface display subunit vaccine.Antibodies and cytokins were examined by enzyme-linked immunosorbent assay(ELISA)after the KM mice were respectively immunized twice with surface display subunit vaccine,purified protein subunit vaccine and inactivated vaccine as well as PBS as the negative control with an interval of 14 days.The KM mice were challenged with multiple strains of HN10,ZD12,M126,and GZ2,respectively belonging to HPS serotype 5,HPS serotype 13,SS serotype 2,and SS serotype 9 after 14 days of the second immunization.The results showed that the antibody and cytokine levels stimulated by the surface display vaccine were comparable to those of purified protein subunit vaccine.The immunoprotective efficacy of the surface display vaccine against both HPS serotype 5 strain and serotype 13 strain was 80%,which was better than that of the HPS-SS inactivated vaccine(80%and60%,respectively).Compared with the purified protein subunit vaccine,the immune protection of surface display vaccine was lower than that of against HPS serotype 5 strain(90%),but higher than that of against HPS serotype 13 strain(60%).The immunoprotective efficacies of surface display vaccine against SS serotype 2 strain and serotype 9 strain were respectively 50%and 80%,lower than those of the purified protein subunit vaccine(100%and 80%,respectively),but higher than those of HPS-SS inactivated vaccine(50%and 60%,respectively).These results demonstrated that the HPS-SS binary subunit vaccine based on the surface display technology can stimulate body to produce the corresponding antibody and hasa better cross-protection when the mice were challenged with HPS serotype 5 and serotype 13,as well as SS serotype 2 and serotype 9 strains compared with the protective efficacy of HPS-SS inactivated vaccine.This study provides experimental basis for the development of subunit vaccine based on surface display,which also gives new ideas for the development of bacterial vaccine.
作者
赵谦
龚俊
栾天
徐盛奎
李刚
王春来
ZHAO Qian;GONG Jun;LUAN Tian;XU Sheng-kui;LI Gang;WANG Chun-lai(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,ChineseAcademy ofAgricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第1期58-64,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
十三五重点研发计划--猪未知病原诊断与检测新技术(2016YFD0500600)
哈尔滨市科技创新人才研究专项(2015RQQYJ073)
黑龙江省自然科学基金面上项目(C2017078、C2016067)
国家自然科学基金面上项目(31672575).
