表达H7N9亚型禽流感病毒HA蛋白的重组血清4型禽腺病毒构建及其免疫效果评估

Construction and Immune Efficacy of Recombinant Serotype 4 Fowl Adenovirus Expressing HA Protein of H7N9 Subtype Avian Influenza Virus

为构建H7N9亚型禽流感病毒和血清4型禽腺病毒(FAdV-4)的二联疫苗候选株,试验通过CRISPR/Cas9和Cre-Loxp系统,以课题组前期构建的表达EGFP蛋白的重组病毒FAdV4-EGFP为载体,构建能够稳定表达H7N9亚型禽流感病毒血凝素(HA)蛋白的重组血清4型腺病毒;并通过PCR、间接免疫荧光试验(IFA)、Westernblot试验以及SPF鸡保护性试验等方法,探究此重组病毒体外生物学特性并评估其为鸡提供的保护力。结果显示:研究成功构建能稳定表达H7N9禽流感亚型病毒HA蛋白的重组血清4型腺病毒,命名为FAdV4-H7;FAdV4-H7能在LMH细胞高效复制,并能在LMH细胞传代15次后仍能稳定表达HA蛋白;FAdV4-H7不仅高度致弱,而且能诱导机体产生高滴度针对H7N9亚型禽流感病毒HI抗体和FAdV-4的中和抗体,能为SPF鸡提供足够保护来抵抗致死性FAdV-4感染。研究表明,试验成功构建了重组病毒FAdV4-H7,为H7N9亚型禽流感病毒和FAdV-4的联防联控提供了二联苗候选疫苗株。

To generate the dual vaccine strain of H7N9 subtype avian influenza virus and serotype 4 fowl adenovirus(FAdV-4),a recombinant FAdV-4 expressing HA protein of H7N9 subtype avian influenza virus,was generated by using FAdV-4 expressing EGFP protein(FAdV4-EGFP)previously constructed by our research team as vector,through CRISPR/Cas9 and Cre-Loxp system in this study.The biological characteristics in vitro and the protective effect on SPF chickens were evaluated through PCR,immuofluorescence assay(IFA),Western blot and SPF chicken protective assay.The results showed that the recombinant FAdV-4 expressing HA proetein of H7N9 subtype avian influenza virus was successfully generated,named as FAdV4-H7.The FAdV4-H7 could replicate efficiently in LMH cells,and H7 protein could be stably expressed in FAdV4-H7 after at least 15 passages in LMH cells.The FAdV4-H7 was not only highly attenuated in pathogenicity to SPF chickens,but also induced both high HI titer antibodies against H7N9 subtype avian influenza virus and high-level neutralizing antibodies against FAdV-4,and provided fully protection against lethal challenge of FAdV-4.The results indicated that the recombinant virus FAdV4-H7 was constructed and could provide a candidate strain for a dual vaccine for the joint prevention and control H7N9 subtype avian influenza virus and FAdV-4 in the future.