减毒沙门菌运送的H9N2亚型禽流感病毒DNA疫苗的研制及其免疫试验

DNA Vaccine Against Avian influenza virus H9N2 Subtype Delivered by Attenuated Salmonella and Their Immune Efficacy

根据GenBank中已发表的H9N2亚型AIV HA基因序列设计并合成一对PCR引物,通过RT-PCR扩增A/Chicken/Shanghai/3/2002(H9N2)AIV毒株的HA基因,测序确认后,将其克隆入真核表达载体pVAX1和asd-pVAX1得到重组表达质粒pVAX1-HA和asd-pVAX1-HA。将重组质粒转染COS-7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菌SL7207和X4550,得到两种运送DNA疫苗的重组沙门菌SL7207(pVAX1-HA)和X4550(asd-pVAX1-HA)。以5×109cfu/只的剂量两次口服接种1日龄的商品代伊莎褐蛋鸡,免疫鸡既能检测到HA特异性的血清抗体应答,又能检测到黏膜免疫应答,采用A/Chicken/Shanghai/3/2002(H9N2)AIV毒株经滴鼻和口服同时攻毒免疫鸡后,这两种疫苗抑制免疫鸡排毒的效果与灭活疫苗有显著差异。

A pair of primers specific for hemagglutinin（HA）gene of Avian influenza virus（AIV） H9 subtype were designed according to published sequences. The HA gene was amplified by reverse transcription-polymerase chain reaction（RT-PCR）from A/Chicken/Shanghai/3/2002. The RT-PCR products 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 COS-7 cells. It was shown that transfected COS-7 cells transiently expressed the HA gene by indirect immunofluorescent assay. Furthermore, the recombinants were transformed into attenuated Salmonella typhimurium SL7207 and X4550, the recombinants were screened and designated as SL7207（pVAX1-HA） and X4550 （asd-pVAX1-HA）, respectively. After administered into 1 day old commercial ISA brown chicks orally at a dose of 5 X 109 CFU per chicken, both SL7207 （pVAX1-HA） and X4550 （asd-pVAX1-HA） induced HA-specific mucosal immune response and systemic humoral immune response. The efficiency of two DNA vaccines to restrain immunized chickens from delivering AIV virus differed significantly from that of inactivated oil-emulsified vaccine after the chicks were challenged by homologous HgN2 subtype.