1型登革病毒抗原表位嵌合人3型腺病毒六邻体重组病毒的构建及免疫学鉴定

Construction and characterization of a hexon-chimeric human adenovirus type 3 vector expressing one major epitope of dengue virus type 1

目的构建六邻体嵌入1型登革病毒（DENVI）抗坂表位的人3型重组腺病毒，鉴定其抗原性。方法以人3型腺病毒骨架质粒pBRAd△E3GFP为模板，overlap PCR在六邻体高变区HVR1插入DENV1的抗原表位，突变的六邻体片段克隆到穿梭载体，酶切后与线性化的3型腺病毒骨架质粒pBRAd△E3GFP在大肠杆菌BJ5183同源重组，获得阳性重组腺病毒质粒pBRAd△E3GFP-DENV1。线性化后转染AD293细胞拯救重组腺病毒rAd△E3GFP-DENV1并大量培养。纯化后腺病毒免疫BALB／c小鼠，通过ELISA和Western blot检测小鼠的体液免疫应答。结果在人3型腺病毒六邻体成功插入DENV1抗坂表位并包装出重组腺病毒，ELISA和Westernblot结果显示小鼠免疫后能产生血清识别DENV1。结论成功构建六邻体HVR1嵌入DENV1抗原表位的重组腺病毒，为多价登革病毒疫苗的研究奠定了基础。

Objective To construct a recombinant human adenovirus type 3 （HAd3） vector ex- pressing one major epitope of dengue virus type 1. Methods The gene encoding the envelope protein （304- 314 aa） of dengue virus type 1 was inserted into the hypervariable region 1 （HVR1） of HAd3 hexon by using overlap PCR. The recombinant gene was cloned into the shuttle plasmid, then linearized with Asis I restriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas- mid for homologous reemnbination. The recombinant plasmid pBRAd△E3GFP-DENV1 was transfeeted into AD293 cells to rescue recombinant adenovirus strains （rAd△E3GFP-DENV1）. ELISA and Western blot as- say were performed to evaluate the bumoral responses induced in BALB/c mice after the immunization with rAd△E3GFP-DENV1 strains. Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains. Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed, This study provided evidence for the development of multivalent vaccines against dengue virus.