共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力

为了构建更为安全有效地抵抗高致病性H5亚型禽流感病毒的基因工程疫苗,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p11S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p11SH5ANA。将p11SH5ANA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。p11SH5ANA与wt-FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV-11SH5ANA。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-11SH5NA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,该重组病毒能使经肌肉注射攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为100%,显示出一定的应用前景。

Recombinant fowlpox virus coexpressing HA and NA gene from subtype H5N1 of avain influenza virus and its protective efficacy

The hemagglutinin(HA) and neuraminidase(NA)gene from subtype H5N1 avain influenza virus were directly inserted into the transferring vector p11S,resulting in the recombinant transferring vector p11SH5ANA.Then p11SH5ANA was transfected into the chicken embryo fibroblasts(CEF),which was pre-infected with wild type fowlpox virus,to generate the recombinant fowlpox virus coexpressing H5A and NA(rFPV-11SH5ANA).By selection of blue plaques on the CEF,rFPV-11SH5ANA was obtained and purified.Experiments on SPF chickens demonstrated that the HI antibody titers in chickens vaccinated with HA-NA coexpressed vaccine was higher than those with HA expressed monovalent vaccines,and all chickens receiving either rFPV-11SH5ANA or rFPV-11SH5A were completely protected from the virulent AIV(H5N1) challenge,while those receiving wt-FPV experienced 100% mortality. The results showed that the rFPV-11SH5ANA was a safe and highly efficient gene engineering vaccine candidate for preventing HPAI.