Lactobacillus casei was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant LTB and porcine parvovirus (PPV) VP2 protein. The fusion protein gene encoding PPV VP2 protein and LTB, was cloned into the surface expression vector pPG, and then the recombinant expression vector pPG-VP2-LTB was electrotransformed into Lactobacillus casei 393, generating recombinant strain pPG-VP2-LTB/L. casei 393. After induced by 2% Lactose in MRS broth, an about 78 kD protein was detected in the recombinant Lactobacillus casei by SDS-PAGE. The result of Western blot indicated that the protein possessed the antigenic specificity same as the native virus protein. The result of the whole bacteria cell ELISA indicated that the LTB protein was expressed at the same time. The results of indirect immunofluorescence test and immuno-gold electron microscopy showed that the interest protein was expressed on the surface of L. casei 393. The results provide potential for the development of lactic acid bacteria oral vaccine of PPV, which used LTB as mucosal adjuvant.%将分别编码猪细小病毒(PPV)主要免疫保护性抗原VP2蛋白与大肠杆菌不耐热肠毒素B亚单位(LTB)基因插入乳酸杆菌细胞表面表达载体pPG中,成功构建了重组表达载体pPG-VP2-LTB,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了表达猪细小病毒VP2-LTB融合蛋白的重组乳酸菌表达系统,经2%乳糖诱导,SDS-PAGE和Western-blot检测表明,有大小约78 kD的蛋白得到了表达,具有与天然病毒蛋白一样的抗原特异性,全细胞ELISA结果表明,LTB同时获得了表达;间接免疫荧光实验及免疫胶体金定位试验结果表明,所表达的蛋白定位于干酪乳杆菌的菌体表面.本研究成果为猪细小病毒重组乳酸菌活菌口服疫苗的研制和LTB作为粘膜免疫佐剂作用的研究奠定了重要的物质基础.
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