鸡毒支原体HS株粘膜免疫抗原VlhA4.12的重组表达及其功能结构预测

Identification and functional structure predication of mucosal antigen VlhA4.12 from HS strain of Mycoplasma gallisepticum

为筛选参与呼吸道局部免疫反应的鸡毒支原体(MG)抗原,本研究收集MG-HS株感染鸡的血清和呼吸道冲洗液,并以其为一抗,与MG菌体裂解物进行western blot检测。结果筛选到一个大小约为75 ku的抗原,质谱分析显示与MGR株的血凝素基因家族成员VlhA4.12同源,生物信息学分析显示存在多个B细胞和T细胞抗原表位。重组VlhA4.12中4个色氨酸密码子"TGA"进行同义突变,并在大肠杆菌中进行表达,结果显示表达产物能够与MG感染鸡呼吸道冲洗液中特异性IgA和IgY反应。VlhA4.12的筛选工作为新型MG疫苗抗原筛选提供新思路,也为粘膜免疫亚单位疫苗研制奠定了基础。

To investigate the antigens of Mycoplasma gallisepticum （MG） involved in stimulation of local immune responses in the respiratory tract of MG-infected chicken, sera and tracheal washes with PBS （pH7.4） were collected from chicken inoculated with 100 folds concentrated MG strain HS for 2 weeks and used as 1st antibody in western blot to react with lysates of MG HS strain. As results, a 75 ku protein, homology to VlhA4.12 of MG strain R indentified by Mass spectrometry analysis, was screened to react with both IgA and IgY in respiratory tract washes. Several antigenic epitopes of B cell and T cell were predicated in the protein, indicating a candidate antigen of mucosal immunity. To prepare recombinant VlhA4.12 （rVlhA4.12）, 4 TGAs encoding Sers in vlhA4.12 gene were sense-muted as TGGs and then expressed in Ecoli BL21（DE3） after cloning into pET-32a. The recombinant VlhA4.12 showed immunity as antigen and reacted with specific antibodies including IgA and IgY in the respiratory tract washes and serum of MG HS infected chicken. Our results could provide new idea of investigating vaccine candidate antigen of MG and developing new mucosal vaccine.