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目的构建基于4E10表位的免疫原,评价其在豚鼠体内诱导中和抗体的能力。方法构建3种免疫原,将3个4E10表位基因串联后连接至GST基因上融合表达蛋白GST-3EP;将4E10表位基因连接柔性连接子GGSGG,再将4个该肽串联后连接至CKS基因融合表达蛋白CKS-4EP;将合成的4E10表位多肽通过Sulfo-SMCC连接至P64K载体蛋白构建结合型免疫原PKEP。用SDS-PAGE电泳的方法检测各种免疫原的分子质量和纯度。用假病毒中和试验评价豚鼠体内中和抗体水平。结果蛋白电泳显示所有免疫原的分子量大小正确,纯度在95%以上。GST-3EP能在豚鼠体内诱导一定水平的中和抗体,3次免疫后5只豚鼠4只阳转,阳转的豚鼠中2只中和抗体水平IC50>200,另外两只抗体水平在100左右。对各组血清50倍稀释后中和抑制率进行统计分析,3EP组与佐剂对照组比较差异有统计学意义(P<0.05)。结论构建的融合蛋白GST-3EP 3次免疫后能在豚鼠体内诱导一定水平的中和抗体。
Objective To construct an immunogen based on the 4E10 epitope and evaluate its ability to induce neutralizing antibodies in guinea pigs. Methods Three kinds of immunogens were constructed. Three 4E10 epitopes were ligated in series to GST-3 fusion protein GST-3EP. Four E10 epitopes were linked to GGSGG, and four peptides were ligated to CKS The gene fusion expression protein CKS-4EP was synthesized. The synthesized 4E10 epitope polypeptide was linked to P64K vector protein by Sulfo-SMCC to construct the binding immunogen PKEP. The molecular mass and purity of various immunogens were determined by SDS-PAGE electrophoresis. The levels of neutralizing antibodies in guinea pigs were evaluated using a pseudovirus neutralization assay. Results Protein electrophoresis showed that all immunogens had the correct size and purity above 95%. GST-3EP induced certain levels of neutralizing antibodies in guinea pigs. After three immunizations, only four guinea pigs were positive, and two of the guinea pigs were positive for IC50> 200 and the other two were about 100 . The 50-fold dilution of serum in each group was statistically analyzed. The difference between 3EP group and adjuvant control group was statistically significant (P <0.05). Conclusion The constructed fusion protein GST-3EP can induce certain level of neutralizing antibodies in guinea pigs after three immunizations.