CAJ | 학술논문

目的：构建六邻体嵌入1型登革病毒（ DENV1）抗原表位的人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pBRAdΔE3GFP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pBRAdΔE3GFP在大肠杆菌BJ5183同源重组，获得阳性重组腺病毒质粒pBRAdΔE3GFP-DENV1。线性化后转染AD293细胞拯救重组腺病毒rAdΔE3GFP-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抗原表位并包装出重组腺病毒，ELISA和Western blot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。
목적：구건륙린체감입1형등혁병독（ DENV1）항원표위적인3형중조선병독，감정기항원성。방법이인3형선병독골가질립pBRAdΔE3GFP위모판，overlap PCR재륙린체고변구HVR1삽입DENV1적항원표위，돌변적륙린체편단극륭도천사재체，매절후여선성화적3형선병독골가질립pBRAdΔE3GFP재대장간균BJ5183동원중조，획득양성중조선병독질립pBRAdΔE3GFP-DENV1。선성화후전염AD293세포증구중조선병독rAdΔE3GFP-DENV1병대량배양。순화후선병독면역BALB／c소서，통과ELISA화Western blot검측소서적체액면역응답。결과재인3형선병독륙린체성공삽입DENV1항원표위병포장출중조선병독，ELISA화Western blot결과현시소서면역후능산생혈청식별DENV1。결론성공구건륙린체HVR1감입DENV1항원표위적중조선병독，위다개등혁병독역묘적연구전정료기출。
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 AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-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.