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目的合成Eg95T-B联合表位肽段,通过噬菌体展示技术获得含不同联合表位的肽段,为细粒棘球蚴病的多抗原表位疫苗的研制奠定基础。方法采用DNAman软件设计3对相应引物,分别扩增各段联合表位的核苷酸序列,连接PMD18-T载体,PCR扩增测序正确的序列,然后用T4连接酶将各段外源基因重组到M13KE噬菌体载体上,构建M13KE/Eg95-1、M13KE/Eg95-2和M13KE/Eg95-3质粒,转入大肠埃希菌ER2738感受态细胞中,运用PCR方法对插入序列进行初步鉴定,测序确定序列正确且无基因突变。使各段基因所对应的外源肽融合表达于M13KE噬菌体PⅢ蛋白上,通过PEG/NaCl沉淀法提纯重组噬菌体。结果成功构建了M13KE/Eg95-1 M13KE/Eg95-2与M13KE/Eg95-3噬菌体展示系统,PCR扩增Eg95-1、Eg95-2、Eg95-3片段分别为192、168和222bp,与预期大小一致。结论成功构建了T细胞和B细胞联合表位的噬菌体展示系统,为抗原表位的筛选奠定了基础。
OBJECTIVE: To synthesize a peptide fragment of Eg95T-B epitope and to obtain peptides with different epitopes by phage display technique, which will lay the foundation for the development of multi epitope vaccine against Echinococcus granulosus. Methods DNAman software was used to design three pairs of corresponding primers. The nucleotide sequences of each epitope were amplified and linked to the PMD18-T vector. The correct sequence was sequenced by PCR. Then the foreign gene was recombined with T4 ligase M13KE / Eg95-1, M13KE / Eg95-2 and M13KE / Eg95-3 plasmids were constructed on M13KE phage vector and transformed into Escherichia coli ER2738 competent cells. The inserted sequences were identified by PCR and sequenced Correct sequence and no gene mutation. The exogenous peptides corresponding to each gene were fused and expressed on the M13KE phage PⅢ protein, and the recombinant phage was purified by PEG / NaCl precipitation. Results The M13KE / Eg95-1 M13KE / Eg95-2 and M13KE / Eg95-3 phage display system were successfully constructed. The amplified fragments of Eg95-1, Eg95-2 and Eg95-3 were 192, 168 and 222 bp, respectively, Consistent. Conclusion The phage display system of T-cell and B-cell epitopes was successfully constructed, which laid the foundation for the screening of epitopes.