以减毒沙门氏菌运送的H5亚型禽流感病毒DNA疫苗的构建及其对小鼠的免疫原性

DNA Vaccines Against H5 Subtype of Avian Influenza Virus Delivered by Attenuated Salmonella and Their Immunogenicity in Mice

根据GenBank中已发表的H5亚型禽流感病毒HA基因序列 ,设计一对引物 ,通过RT PCR扩增鹅源H5亚型高致病力禽流感病毒HA基因 ,测序确认后 ,将其克隆入真核表达载体pVAX1和asd pVAX1得到重组表达载体pVAX1 HA和asd pVAX1 HA。将重组质粒转染P815细胞 ,经间接免疫荧光试验证实 ,HA基因在细胞内得到了瞬时表达。进一步将重组质粒转化减毒鼠伤寒沙门氏菌X4 5 5 0得到两种运送DNA疫苗的重组沙门氏菌X4 5 5 0 (pVAX1 HA)和X4 5 5 0 (asd pVAX1 HA) ,以 1× 10 9CFU 只的剂量两次口服免疫BALB c小鼠 ,免疫小鼠不仅可以检测到HA特异性的血清抗体应答 ,而且还能抵抗稳定表达H5亚型禽流感病毒HA基因的P815肥大细胞瘤的攻击 ,说明该运送DNA疫苗的减毒沙门氏菌系统在体内能够成功释放所携带的质粒 ,并且能够刺激机体产生保护性免疫应答。

A pair of primers specific for hemagglutinin (HA) gene of H5 subtype Avian influenza virus (AIV) were designed according to published sequences. The HA gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from a H5 subtype AIV strain G5 isolated from goose. The RT-PCR product was recovered and sequenced, and then inserted into eukaryotic expression vector pVAX1 and asd-pVAX1. The recombinant plasmid pVAX1-HA and asd-pVAX1-HA were transfected into mouse mastocytoma P815 cells. It was shown that transfected P815 cells transiently expressed the HA gene in indirect immunofluorescent assay. Furthermore, the recombinant pVAX1-HA and asd-pVAX1-HA were transformed into an attenuated S.typhimurium strain X4550, the transformants were screened, identified and named as X4550(pVAX1-HA) and X4550(asd-pVAX1-HA), respectively. After administered into BALB/c mice orally, both X4550(pVAX1-HA) and X4550(asd-pVAX1-HA) induced HA-specific serum antibodies, and supplied effective protection against the challenge of transfected P815 cells stably expressing HA gene of AIV H5 subtype. These results demonstrated that two Salmonella-based delivering systems can release harboured DNA vaccines in vivo, and elicit protective immune responses.