表达H5亚型AIV HA和NA基因重组鸡痘病毒的构建及在高母源抗体商品鸡的免疫效力试验

Development of recombinant fowlpox virus coexpressing HA and NA gene from subtype H5N1 of avain influenza virus and it's protective efficacy in commercial chickens of high maternal antibodies

母源抗体的干扰是重组鸡痘病毒(FPV)活载体基因工程疫苗至今未能得到推广应用的主要原因,本试验使用FPV新的复制非必需区构建的载体pP12-18构建在高母源抗体商品鸡具有较高免疫力的基因工程疫苗。将H5亚型禽流感病毒分离株的血凝素(HA)基因和神经氨酸酶(NA)基因定向插入鸡痘病毒转移载体pP12-18中,H5A和NA基因的启动子分别为PS和PE/L,获得用不同的启动子启动不同的外源基因且两基因盒方向为背向串联的重组转移载体p12LSH5 HANA。将p12LSH5 HANA转染至已感染鸡痘病毒282E4疫苗株(wt-FPV)的鸡胚成纤维细胞(CEF)中。p12LSH5 HANA与wt-FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV-12LSH5 HANA。通过在含X-Gal的营养琼脂上连续挑选蓝色病毒蚀斑,获得纯化的重组病毒。经传代证实该重组病毒具有良好的遗传稳定性。用105PFU的rFPV-12LSH5 HANA免疫无特定病原体(SPF)鸡,能激发机体产生有效的血凝抑制(HI)抗体。初步的动物试验表明,该重组病毒能使经滴鼻点眼攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为100%。在高母源抗体的商品鸡上,rFPV-12LSH5 HANA与原有载体构建的重组疫苗rF-PV-11SH5 HANA的免疫效力有显著差异,保护率分别为81.4%和45.4%。结果表明,选择FPV合适的复制非必需区构建载体是提高重组FPV在高母源抗体商品鸡免疫效力的有效策略之一。

The rFPV based vaccines have not been widely used in the field because maternal antibodies present in commercial chickens usually exert a substantial interference to the efficacy of rFPVs. We constructed new transferrion vector pP12-18 of selecting suitable FPV nonessential regions for insertion of foreign gene and generation of recombinant viruses might be an effective way to solve this problem. The hemagglutinin （HA） and neuraminidase （NA） gene from subtype H5N1 avain influenza virus were directly inserted into the transferring vector pP12-18,constructing a recombinant transferring vector p12LSH5HANA. Then p12LSH5HANA was transfected into the chicken embryo fibroblasts（CEF） pre-infected with wild type fowlpox virus, to generate the recombinant fowlpox virus coexpressing HA and NA （rFPV-12LSH5HANA）. By selection of blue plaques on the CEF,rFPV-12LSH5HANA was obtained and purified. Experiments on SPF chickens demonstrated that rFPV-12LSH5 HANA had the same protective efficacy as the H5 AI killed vaccine against lethal challenge with homologous subtype HSN1 avain influenza virus. In positive maternal antibody commercial chickens, the PIs induced by rFPVs with insertion vector p12LS were slightly higher than those induced by rFPVs with pllS, there were significant difference between two groups. The results showed that the rFPV-12LSH5HANA was a safe and highly efficient gene engineering vaccine candidate for preventing HPAI.