| 摘要: |
| 【目的】获得表达H9亚型禽流感病毒血凝素(HA)基因的重组禽痘病毒。【方法】将含禽痘病毒启动子LP2EP2驱动的HA基因,插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSY681/HA。用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA的重组禽痘病毒rFPV-HA。在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组病毒进行多次蚀斑克隆,并用间接免疫荧光法对感染重组病毒鸡胚成纤维细胞中HA的表达产物进行鉴定。【结果】以重组禽痘病毒DNA为模板,利用HA基因特异引物进行PCR,扩增出1条约1.7 kb左右的带。以间接免疫荧光法证实重组禽痘病毒能表达HA。【结论】成功构建了表达H9亚型禽流感病毒HA基因的重组禽痘病毒,且构建的重组禽痘病毒能表达具有生物学活性的HA。 |
| 关键词: H9亚型禽流感病毒 HA基因 重组禽痘病毒 基因工程疫苗 |
| DOI： |
| 分类号: |
| 基金项目:国家“十一五”科技支撑计划专项（2006BAD06A08） |
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| Construction of recombinant fowlpox virus expressing haemagglutinin gene of H9 avian influenza virus |
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| Abstract: |
| 【Objective】 Recombinant fowlpox viruses (rFPV) were developed expressing H9 avian influenza virus (AIV) haemagglutinin (HA).【Method】 HA cDNA was cloned into Bam HⅠsite of pSY538 plasmid and then subcloned into SfiⅠsite of pSY681 plasmid containing LacZ gene.The plasmid,pSY681/HA,was used to transfect on the chicken embryo fibroblasts cell (CEF) pre-infected with fowlpox virus S-FPV-017.By selection of blue plaques on the CEF overlaid with agar containing X-gal,the rFPV-HA recombinants were obtained and identified by restriction enzyme analysis and PCR.HA in the recombinalt virus-infected CEF was indentified by indirect immune of luorescence assay.【Result】 The results indicated that recombinant fowlpox viruses contained HA gene.The expression of HA was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against AIV.【Conclusion】 The recombinant fowlpox virus (rFPV-HA) was developed and had biological activity. |
| Key words: H9 avian influenza virus haemagglutinin gene recombinant fowlpox virus genetic engineering vaccine |
