LTB抗原表位PT8A与重组轮状病毒VP8~*亚单位疫苗融合后的免疫效应研究

Study on the Immune Effect of LTB Epitope of PT8A Fusion with Recombinant Rotavirus VP8~* Subunit Vaccine

为评价LTB（大肠杆菌不耐热肠毒素B亚单位）抗原表位PT8A与人轮状病毒（rotavirus,RV）VP8＊基因融合后对VP8＊亚单位疫苗（VP8）免疫效果的影响,将PT8A分别融合到VP8＊基因的C-端和N-端,然后构建重组表达质粒p ET32a-PT8A-VP8和p ET32a-VP8-PT8A。表达的重组蛋白PT8A-VP8和VP8-T8A纯化后分别经鼻腔免疫BALB/c免疫小鼠,收集血清,间接ELISA法检测血清抗体水平。经测序鉴定,重组质粒p ET32a-PT8A-VP8和p ET32a-VP8-PT8A构建成功。6×His-VP8-PT8A、6×His-PT8A-VP8融合蛋白主要以包涵体形式表达,相对分子质量（Mr）约为34 k D。重组VP8-PT8A和PT8A-VP8表达成功,融合蛋白主要以包涵体形式表达。间接ELISA测量血清Ig G和肺s Ig A的滴度,与VP8组相比较,VP8＋LTB组和VP8-PT8A组OD值有显著性差异（p〈0.05）,PT8A-VP8组OD值没有显著性差异（p〉0.05）。即PT8A融合在VP8基因的C端后对重组轮状病毒VP8＊亚单位疫苗有显著性免疫佐剂活性。

To evaluate the immune adjuvant of T cell epitope of PT8A in LTB （heat-labile enterotoxin B subunit） to human recombinant rotavirus VP8＊ vaccine, the PT8A was fused with VP8＊ gene at N-terminus, named PT8A-VP8, and C-terminus, named VP8-PT8A, respectively. Then the fusion genes were cloned into plasmid pET32a, and the fusion proteins of 6xHis-PT8A-VP8 and 6xHis-VP8-PT8A were purified with magnetic nickel beads. BALB/c mice were random divided into 4 groups. The sera were collected and determined for antibody titer by ELISA. Recombinant plasmids of pET32a-PT8A-VP8 and pET32a-VP8-PT8A were constructed successfully. The fusion proteins of 6xHis-PT8A-VP8 and 6xHis-VP8-PT8A were expressed in E. coli with relative molecular mass of 34 kD in a form of inclusion body. The antibody titer was measured by ELISA. Compared with VP8 group, the titer of VP8 specific antibodies in groups of VP8＋LTB and VP8-PT8A were increased significantly （p〈0.05）, however, that of in PT8A-VP8 group has no significant difference （p〉0.05）. The results indicated that PT8A fused at the C terminal of VP8 can significantly enhanced the immunity of recombinant rotavirus VP8＊ subunit vaccine as adjuvant.