基于鸡毒支原体黏附素蛋白的表位疫苗制备及免疫效果评价

Preparation and Evaluation of Subunit Vaccine based on Adhesin Protein of Mycoplasma gallisepticum

旨在使用鸡毒支原体(Mycoplasma gallisepticum,MG)的黏附蛋白设计一种更安全、有效的多表位候选疫苗。本研究运用生物信息学方法对鸡毒支原体的多个黏附素蛋白(CrmA、GapA、Mgc2、PvpA)的B、T细胞表位进行预测。将筛选的抗原表位合成新的表位基因肽段(命名为mEA)。通过生物学在线软件分析了mEA的二级结构、亲水性、抗原性、三级结构。构建重组质粒pET32a-MG-mEA,经限制性内切酶(BamHⅠ和HindⅢ)酶切鉴定及DNA测序鉴定后导入大肠杆菌BL21(DE3)进行诱导表达和纯化。Western blot验证重组蛋白与鸡MG阴性/阳性血清的反应性。纯化的MG-mEA重组蛋白与佐剂1∶1混匀,制备为多表位疫苗,通过SPF鸡免疫试验分析融合蛋白免疫原性。结果显示,mEA序列二级结构较稳定,抗原性良好。重组质粒经PCR扩增获得1680 bp大小的目的条带,与预计相符。SDS-PAGE检测表达蛋白相对分子质量约48 ku,主要以可溶性形式存在。Western blot试验证明该蛋白反应原性良好。多表位疫苗免疫SPF鸡,经ELISA检测显示:二免后10 d各免疫组抗体水平达到最高,3个重组蛋白免疫组与PBS组相比差异均具有显著统计学意义(P<0.05)。攻菌结果表明,MG感染明显损伤了气管黏膜结构,而重组蛋白+佐剂制备的疫苗能够有效保护MG的损伤。综上,本研究结果为研制安全有效的候选MG疫苗提供了新的途径。

The aim of this study was to design a safer and more effective multi-epitope vaccine candidate using the adhesion proteins of Mycoplasma gallisepticum(MG).In this study,bioin-formatics methods were used to predict B and T cell epitopes of several adhesin proteins(CrmA,GapA,Mgc2,PvpA)of MG.The screened antigenic epitopes were synthesized into a new epitope gene peptide(named mEA).The secondary structure,hydrophilicity,antigenicity,and tertiary structure of mEA were analyzed by Biology Online software.The recombinant plasmid pET32a-MG-mEA was constructed,identified by restriction endonucleases(BamHⅠand HindⅢ)and introduced into E.coli,BL21(DE3)for induction of expression and purification by DNA sequencing.Western blot was used to verify the reactivity of the recombinant protein with chicken negative/positive sera.The purified MG-mEA recombinant protein was mixed 1∶1 with adjuvant and prepared as a multi-epitope vaccine and the immunogenicity of the fusion protein was analysed by SPF chicken immunoassay.The results showed that the secondary structure of the mEA sequence was relatively stable and the antigenicity was good.The recombinant plasmid was amplified by PCR to obtain the target band with a size of 1680 bp,which was consistent with the prediction.SDS-PAGE detected that the relative molecular mass of the expressed pro-tein was about 48 ku,mainly in soluble form.The multi-epitope vaccine immunised SPF chickens showed the highest antibody levels in all immunised groups at 10 d after the second vaccination,with statistically significant differences between the three recombinant protein immunised groups compared to the PBS group(P<0.05).The results showed that MG infection significantly dam-aged the tracheal mucosa structure and that the recombinant protein+adjuvant vaccine was effec-tive in providing effective protection.In summary,the results of this study provide a new avenue for the development of a safe and effective MG vaccine candidate.