表达禽流感病毒M2基因的重组马立克氏病病毒的构建

将禽流感病毒M2基因克隆于真核表达质粒pIRES-EGFP中，使其位于pCMV启动子的调控下，并与绿色荧光蛋白基因（EGFP）串联后，将上述串联基因插入到含MDV CVI988的非必需区US基因的重组质粒pUS2中，构建带标记的重组质粒，然后将此重组质粒转染感染了MDV CVI988的鸡胚成纤维细胞，利用同源重组的方法，筛选了表达禽流感病毒M2基因的重组病毒MDV1。经PCR、Dot-blotting，Western-blotting等实验的结果表明，禽流感病毒M2基因的确插入到MDV1（CVI988）基因组中并获得表达。重组MDV1免疫1日龄SPF鸡21天后，用ELISA可检测到M2蛋白的特异性抗体。接种了重组病毒rMDV的鸡体内针对H9N2疫苗血凝素的抗体滴度(p<0.05)明显提高，以禽流感病毒AIV A/Chicken/Guangdong/00（H9N2）攻毒后进行病毒重分离试验的结果发现，重组病毒能有效地降低病毒的排出量(p<0.01)，说明该重组病毒可以用于防制禽流感的免疫。

Construction of Recombinant Marek's Disease Virus Expressing M2 Gene of Avian Influenza Virus

Avian influenza, caused by avian influenza virus (AIV), is a highly contiguous disease. AIV can infect almost all wild and domestic poultries and cause severe economic losses for the poultry industry. The M2 protein of AIV is a transmembrane protein. It has highly conserved antigenic epitopes and is a potential candidate antigen for avian influenza vaccine with cross-protection. Marek's disease virus(MDV), avian herpesvirus have mostly been used as virus vectors to express a foreign gene for the protection of chickens against poultry. Among MD vaccine viruses, attenuated MDV1 strains such as CVI988 are more suitable for be vaccine vectors because they have the closest antigenic similarity to the very virulent(vv) MDV1. To develop a recombinant herpesvirus -based AI vaccine, the M2 gene of Avian Influenza virus (AIV) ligated with green fluorescent protein (EGFP) was inserted into the US gene, which located in the nonessential region of MDV1 (CVI988) genome and an EGFP marked recombinant plasmid was obtained. The M2 gene of Avian Influenza virus was expressed under the control of a CMV immediately early promoter. Through homologous recombination, this construct was transfected into cells infected with Marek's disease virus CVI 988/Rispens and the recombinant MDV1expressing AIV M2 gene was screened out. The PCR, dot-blot and western-blot results demonstrated that AIV M2 gene was not only cloned into the genome of MDV1 (CVI988) but also expressed. A group of one day-old chickens was injected with the recombinant virus and the specific antibody against M2 protein could be detected by ELISA after 21 days. When chickens were inoculated with commercial H9N2 subtype AI vaccine, the titers of antibody against AIV hemagglutinin from chickens inoculated with the recombinant MDV1were higher significantly (P<0.05) than those of H9N2 subtype AI vaccine alone. In addition, the excretive amount of live AIV from throat and cloacae of chickens inoculated with recombinant MDV1 were less significantly (P<0.01) after challenged with Avian influenza virus A/Chicken/Guangdong/00（H9N2）. All these results indicated that the recombinant MDV1expressing AIV M2 gene could be responsible for the prophylactic of avian influenza.