汉坦病毒CTL多表位重组腺病毒的构建及免疫学分析

Construction and Immunologenic Study of Recombinant Adenovirus Containing CTL Epitopes of Hantaan Virus

构建含汉坦病毒M基因(编码糖蛋白G1)和部分S基因(编码核蛋白主要抗原区段)以及S基因的多个CTL表位的多种重组腺病毒表达载体,并对这些重组腺病毒的免疫学特性进行研究。用构建含目的基因的重组腺病毒载体,并对其表达产物进行鉴定。利用纯化后的重组腺病毒免疫BALB/C小鼠,通过多种免疫学方法检测其免疫反应。成功地表达出可被汉坦病毒核蛋白特异性单抗(mAb)及糖蛋白G1的特异性单抗所识别的融合蛋白;重组的腺病毒可以有效刺激针对汉坦病毒NP及GP的免疫应答;同时结果表明在CTL多表位间加入间隔序列AAY的重组腺病毒免疫小鼠能产生更高的细胞免疫应答。说明构建的含CTL多表位的重组腺病毒可以有效地提高嵌合基因刺激细胞免疫应答的能力。间隔序列AAY对于各CTL表位的分隔有效地提高了重组腺病毒的免疫效果。

To express G1 fragment of M segment and 0.7 kb fragment of S segment and several CTL epitopes of S segment in adenovirus expression system and investigate the immunological properties of hantaan virus chimeric gene,the recombinant adenovirus was constructed and the recombinant adenovirus was obtained after transfecting HEK293 cells. The titer of it was determined and the expressed product was detected by IFA and ELISA. Further,BALB/c mice were vaccinated by the recombinant adenovirus and the immune response was tested by ELISA ,microcell-culture neutralizing experiment,T lymphocyte proliferation test （MTT assay） and cell-mediated cytotoxicity assay.The resulare are that recombinant adenovirus AG1S0.7CTL1,AG1S0.7CTL2 were constructed successfully and the titer of it was about （1010—1011） pfu/ mL. The expressed protein could be recognized by the hantaan virus NP-specific mAb and glycoprotein G1-specific mAb. The recombinant adenovirus containing CTL epitopes could elicit effectually the cellular immune response aimed to the NP and GP of hantaan virus in BALB/c mice.It is conclusioned that recombinant adenovirus containing CTL epitopes could induce the higher cellullar immune response than the group that not containing CTL epitopes.The recombinant adenovirus group which CTL epitopes are spaced by intervening sequence AYY （CTL1） could induce higher cellular immune response than the group which CTL epitopes are not spaced by intervening sequence （CTL2）.