摘要
【目的】为新型马立克氏病(MD)疫苗的开发奠定基础。【方法】以SPF鸡肝脏中提取RNA为模板,利用RT-PCR反应扩增鸡HSP108基因;利用PCR方法扩增MDV gB CTL表位基因,并将MDV gB CTL表位基因与鸡HSP108融合在一起,构建融合蛋白基因rgBHSP108,将该融合蛋白基因克隆入原核表达载体pET-28a中,构建重组表达载体pET-gBHSP108,将该重组载体转化E.coliBL21(DE3),并用IPTG诱导融合蛋白rgBHSP108的表达,对rgBHSP108进行SDS-PAGE电泳、可溶性分析、蛋白纯化及Western-blot分析。【结果】扩增到鸡2.4kb的HSP108基因和330bp的MDV gB CTL表位基因;克隆的鸡HSP108基因序列与GenBank上发表的鸡输卵管HSP108(NM204289)和来源于肝脏HSP108(AF387865)核苷酸的同源性分别为99.7%和99.0%。rgBHSP108融合蛋白基因构建成功;融合蛋白rgBHSP108以包涵体的形式获得高效表达并纯化。Western-blot分析表明,针对MDV gB CTL表位的多抗可以与融合蛋白发生特异性反应。【结论】成功构建了鸡HSP108融合蛋白基因,利用原核表达系统成功表达rgBHSP108。
[Objective] The study was to supply the basis on developing a new type of MD vaccine. [Method] HSP108 gene was amplified by RT-PCR from SPF chicken liver,and MDV gB CTL epitope amplified by PCR. The fusion gene for expressing gB CTL epitope and chicken HSP108 was obtained by fusing the gene fragment encoding a MDV gB CTL epitope to 5＇end of chicken HSP108 gene. And the fusion gene was inserted into pET-28a to construct the recombinant plasmid,named pET-gBHSP108. The recombinant protein named rgBHSP108 was analyzed by SDS-PAGE, solubilized in urea （8 mol/L）, purified by His. Bind affinity chromatography and identified by western blotting. [Result] The chicken HSP108,a fragment of about 2.4 kb in length and MDV gB CTL epitope,a fragment of about 330bp were successfully obtained. The cloned HSP108 gene showed 99. 7% homology with the HSP108 gene of chicken oviduct （GenBank Accession No. NM 178423） ,and 99.0% homology with the HSP108 gene of chicken liver （GenBank Accession No. AF 387865） at nucleotide levels. The fusion gene rgBHSP108 was successfully constructed,and rgBHSP108 was expressed as inclusion body and successfully purified. Immunoblotting analysis showed that the expressed fusion protein was recognized by antibody against MDV gB CTL epitope. [Conclusion] This study successfully constructed a fusion gene encoding chicken HSP108 gene and gB CTL epitope gene of MDV. And the fusion gene was successfully expressed by E. coli.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第5期1-6,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
2007年人事部高层次留学人才回国资助项目“马立克氏病毒突破宿主细胞p53防御机制的研究”
关键词
CTL表位
原核表达
融合蛋白基因
热休克蛋白
CTL epitope
prokaryotic expression
fusiong protein gene
heat shock protein
