共表达新城疫病毒F基因和H9亚型禽流感病毒HA基因的重组鸡痘病毒  

Recombinant Fowlpox Virus Expressing F of Newcastle Disease Virus and HA of Subtype H9N2 of Avian Influenza Virus

作　　者：韦栋平[1] 刘玉良[1] 邵卫星[1] 卢建红[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,扬州225009

出　　处：《微生物学报》2004年第3期286-290,共5页Acta Microbiologica Sinica

基　　金：国家"8 63计划"( 2 0 0 1AA2 13 0 41)~~

摘　　要：应用RT PCR扩增出新城疫病毒F4 8E8株融合蛋白 (F)基因 ,将其克隆入pGEM Teasyvector构建重组质粒pGEM TF并进行测序确证。分别从pGEM T和pUCHA切下F基因和H9亚型禽流感病毒F株 (A chicken china F 1 998)血凝素 (HA)基因 ,通过一系列分子生物学操作步骤插入到质粒pFPV7S中的鸡痘病毒基因组复制非必需片段构建重组质粒p7SHF ,其中F基因和HA基因分别由鸡痘病毒启动子PE L和合成启动子PS调控。最后将P1 1 LacZ报告基因表达盒插入质粒p7SHF获得转移载体pFPVHF ,用以转染已预先感染鸡痘病毒 2 82E4疫苗株的鸡胚成纤维细胞 (CEF)。通过在含有X Gal的营养琼脂上连续挑选蓝色病毒蚀斑获得并纯化重组病毒。PCR和Southernblot检测证实了F基因和HA基因已插入鸡痘病毒的基因组 ;间接免疫荧光试验结果表明重组病毒能够同时正确表达HA和F蛋白。The fusion protein (F) gene of Newcastle disease virus strain F48E8 was obtained by PCR and its sequence was determined. To form recombinant plasmid p7SHF, the F gene in pGEM-TF and the hemagglutinin (HA) gene of AIV, A/Chicken/China/F/1998(H9N2) in pUCHA were cut and inserted into the randomly selected nonessential regions of the fowlpox virus (FPV) under the control of fowlpox virus promoter P E/L and the synthetic promoter Ps respectively. Then the P11-LacZ reporter gene from pppG18 was cloned into p7SHF resulting in recombinant transferring vector pFPVHF, which was transfected into chicken embryo fibroblast (CEF) monolayers pre-infected with Chinese vaccine strain 282E4 of FPV to generate recombinant fowlpox virus co-expressing HA and F (rFPV-HA-F). By selection of blue plaques on the CEF overlaid with agar containing X-gal, rFPV-HF-F was screened and purified. PCR and southern blot assays indicated that the F and HA genes had been inserted into the genomic DNA of FPV. Subsequently expression of F and HA in CEF infected with rFPV-HA-F was confirmed by indirect immunofluorescence assay.

关 键 词：新城疫病毒 H9亚型禽流感病毒 重组鸡痘病毒 融合蛋白 血凝素 

分 类 号：Q786[生物学—分子生物学]