融合表达白细胞介素10与卵清蛋白重组腺病毒口服变应原疫苗的构建及鉴定

Construction and identification of oral recombinant adenovirus vaccine expressing fused IL-10 and ovalbumin

目的构建免疫调节因子白细胞介素(IL)-10与模式变应原卵清蛋白(OVA)融合表达的重组腺病毒载体口服疫苗。方法直接合成鸡IL-10及OVA的DNA序列后,采用重叠PCR法进行连接。连接序列插入穿梭质粒p HBAd-MCMV的多克隆区,获得重组穿梭质粒p Ad IL-10-OVA。经测序鉴定后,p Ad IL-10-OVA与腺病毒骨架质粒p HBAd-BHG共转染293细胞,包装获得重组腺病毒Ad IL-10-OVA原代病毒,将原代病毒在293细胞中扩增到合适滴度。采用ELISA法检测细胞上清液中融合蛋白的表达水平。结果重组穿梭质粒插入目标序列经测序鉴定正确,重组腺病毒载体疫苗r Ad IL-10-OVA在293细胞中成功表达。结论成功构建了融合表达IL-10与OVA的重组腺病毒载体疫苗Ad IL-10-OVA,为后续动物实验验证其对变态反应性疾病的防治效应奠定了基础。

Objective To construct and identify an oral recombinant adenovirus vector vaccine expressing fused interleukin-10（ IL-10） and ovalbumin（ OVA）. Methods Target gene sequences of mouse IL-10 and OVA were synthesized respectively and then linked together based on overlapping PCR technology. The fused sequence was inserted into the shuttle plasmid p HBAd-MCMV. Recombinant shuttle plasmid p Ad IL-10-OVA was co-transfected into 293 cells with adenovirus backbone plasmid p HBAd-BHG after identification by DNA sequencing. Recombinant adenoviruse Ad IL-10-OVA was packaged and amplified in the 293 cells. IL-10 level in the supernatant was detected by ELISA assay. Results Target gene sequence was inserted into the shuttle plasmid correctly as DNA sequencing indicated. The recombinant Ad IL-10-OVA could express fused IL-10 and OVA effectively. Conclusion The recombinant adenovirus vaccine Ad IL-10-OVA is successfully constructed,which formes the basis for further investigation on the preventive and therapeutic effects of the vaccine.