H5亚型AIV HA1基因与鸡IL-18成熟蛋白基因重组杆状病毒表达载体的构建及其表达
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摘要
白细胞介素-18(interleukin-18,IL-18)是Okamura等(1995)从小鼠肝脏中克隆获得的。Nakamori等(2003)研究发现,IL-18在抗肿瘤、抗感染及免疫调节等方面有着重要的作用,具有免疫治疗及免疫佐剂的作用,有潜在的应用前景。同时,随着畜牧业的发展,禽病特别是禽流感呈上升趋势,迫切需要新型疫苗与疫苗佐剂的研发,而禽流感病毒的主要抗原决定簇位于HA1基因,故HA1蛋白是研制基因工程亚单位苗的理想候选抗原。鉴于此,本实验室对鸡白介素18成熟蛋白基因(mature Chicken interleukin-18,mChIL-18)与HA1基因进行了真核双表达,旨在探索利用mChIL-18能促进机体免疫功能的特性,将其应用于禽流感的预防及临床治疗。本试验针对如何获得高效表达且具有生物学活性的mChIL-18蛋白这些问题进行了探讨,主要包括以下两部分:
试验一H5亚型AIV HA1基因与鸡IL-18成熟蛋白基因的杆状病毒共表达载体的构建
参考GenBank上发表的H5亚型禽流感(AIV)血凝素(HA)基因序列及鸡白细胞介素18成熟蛋白(mChIL-18)的cDNA基因序列,分别设计了HA1和mChIL-18的cDNA的两对引物。然后分别以pMD18-T-HA质粒和pGEX-mChIL-18质粒为模板,扩增出了H5亚型AIV的HA1基因和mChIL-18的cDNA片段。再以杆状病毒pFastBacTM dual为载体,将H5亚型AIV HA中的HA1基因和mChIL-18基因分别插入到双表达载体pFastBacTM dual的p10启动子(P_(P10))和多角体蛋白启动子(P_(PH))的下游,构建携带HA1基因和mChIL-18基因的重组pFastBac~(TM) dual-HA1-mChIL-18真核表达质粒。最后将构建的真核表达质粒转座入大肠杆菌DH10Bac,为下一步进行昆虫细胞的转染奠定了基础。这个重组载体的构建为考察mChIL-18作为免疫增强剂的作用及下一步AIV疫苗的研究奠定了坚实的基础。
试验二H5亚型AIVHA1基因与鸡IL-18成熟蛋白基因在昆虫杆状病毒表达载体中的表达
将试验一构建好的pFastBac~(TM) dual-IL-18-HA1质粒转化DH10Bac感受态细胞,将转化混合物涂到LB琼脂平板上,用小量法从培养物中提取质粒DNA,在此基础上通过一对通用引物M13(该引物扩增重组质粒Bacmid LacZα互补区域内的mini-attTn7位点两端的片段,任何外源性基因片段插入pFastBac~(TM) dual质粒内都会使PCR产物增大)进行PCR扩增,鉴定pFastBac~(TM) dual-IL-18-HA1质粒克隆到杆状病毒表达载体,通过转染昆虫细胞sf9,收获重组杆状病毒,得到了共表达IL-18和HA1的高效表达产物。
The gene encoding interleukin-18 (IL-18) was firstly cloned by Okamura in 1995. IL-18 plays important roles in antitumor, anti-infection and immunoloregulation. And it has potential prospect in immunologic therapy and adjuvant. With the development of prologue industry in China, the poultry’s disease especially Avian Influenza (AI) is becoming severity. So a newly vaccine and its adjuvant are urgently demand to research. And major antigen determinants of AIV lie in HA1 gene, so HA1 protein is the ideal electing antigen of studying gene engineering subunit vaccine. The gene encoding mature Chicken IL-18(mChIL-18) was cloned and expressed in our laboratory in order to prevent and therapy the poultry’s diseases. In the present study, the efficiently expressed mChIL-18 protein with biological activity was obtained. It was included two parts as following:
Experiment 1: Construction of Recombinant Baculovirus Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Mature Chicken Interleukin-18 Gene
The hemagglutinin(HA) gene of Avian Influenza Virus(AIV) subtype H5 and mature chicken interleukin-18(mChIL-18) gene were amplified by designing two pairs of primers according to the sequence published on GenBank.Recombinant baculovirus coexpressing HA1 gene from AIV subtype H5 plus chicken interleukin-18 gene were constructed by inserting cDNA copy of HA gene and cDNA copy of mChIL-18 gene into the baculovirus expression plasmid pFast Bac~(TM)Dual,furthermore cDNAs respectively inserted into the downstreams of P10 promoter and PH promoter.Finally,the constructing eukaryotic expression plasmid pFastBac~(TM) dual-HA1-mChIL-18 was transposited into Bacterium Coli DH10Bac,which is based of next step insect cell’s transfection.The construction of this recombinant plasmid is benefic to investigate the effect of mChIL-18’s immunologic enhancement and study a new AIV recombinant vaccine.
Experiment 2: Expression of Recombinant Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Chicken Interleukin-18 Gene
After constructing pFastBac~(TM) dual-IL-18-HA1 plasmid ,we make it transformate into DH10Bac competent cells.Then transformations are spreaded on fresh LB plates and the recombinant bacmid DNA is extracted.On the basis of M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis),we analyze the recombinant bacmid DNA by PCR.Through transfecting insect cells, trcombinant baculovirus is harvested.Then we gain the high performance expressing product of IL-18 and HA1,which is benefic to investigate the effect of mChIL-18’s immunologic enhancement and study a new AIV recombinant vaccine.
试验一H5亚型AIV HA1基因与鸡IL-18成熟蛋白基因的杆状病毒共表达载体的构建
参考GenBank上发表的H5亚型禽流感(AIV)血凝素(HA)基因序列及鸡白细胞介素18成熟蛋白(mChIL-18)的cDNA基因序列,分别设计了HA1和mChIL-18的cDNA的两对引物。然后分别以pMD18-T-HA质粒和pGEX-mChIL-18质粒为模板,扩增出了H5亚型AIV的HA1基因和mChIL-18的cDNA片段。再以杆状病毒pFastBacTM dual为载体,将H5亚型AIV HA中的HA1基因和mChIL-18基因分别插入到双表达载体pFastBacTM dual的p10启动子(P_(P10))和多角体蛋白启动子(P_(PH))的下游,构建携带HA1基因和mChIL-18基因的重组pFastBac~(TM) dual-HA1-mChIL-18真核表达质粒。最后将构建的真核表达质粒转座入大肠杆菌DH10Bac,为下一步进行昆虫细胞的转染奠定了基础。这个重组载体的构建为考察mChIL-18作为免疫增强剂的作用及下一步AIV疫苗的研究奠定了坚实的基础。
试验二H5亚型AIVHA1基因与鸡IL-18成熟蛋白基因在昆虫杆状病毒表达载体中的表达
将试验一构建好的pFastBac~(TM) dual-IL-18-HA1质粒转化DH10Bac感受态细胞,将转化混合物涂到LB琼脂平板上,用小量法从培养物中提取质粒DNA,在此基础上通过一对通用引物M13(该引物扩增重组质粒Bacmid LacZα互补区域内的mini-attTn7位点两端的片段,任何外源性基因片段插入pFastBac~(TM) dual质粒内都会使PCR产物增大)进行PCR扩增,鉴定pFastBac~(TM) dual-IL-18-HA1质粒克隆到杆状病毒表达载体,通过转染昆虫细胞sf9,收获重组杆状病毒,得到了共表达IL-18和HA1的高效表达产物。
The gene encoding interleukin-18 (IL-18) was firstly cloned by Okamura in 1995. IL-18 plays important roles in antitumor, anti-infection and immunoloregulation. And it has potential prospect in immunologic therapy and adjuvant. With the development of prologue industry in China, the poultry’s disease especially Avian Influenza (AI) is becoming severity. So a newly vaccine and its adjuvant are urgently demand to research. And major antigen determinants of AIV lie in HA1 gene, so HA1 protein is the ideal electing antigen of studying gene engineering subunit vaccine. The gene encoding mature Chicken IL-18(mChIL-18) was cloned and expressed in our laboratory in order to prevent and therapy the poultry’s diseases. In the present study, the efficiently expressed mChIL-18 protein with biological activity was obtained. It was included two parts as following:
Experiment 1: Construction of Recombinant Baculovirus Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Mature Chicken Interleukin-18 Gene
The hemagglutinin(HA) gene of Avian Influenza Virus(AIV) subtype H5 and mature chicken interleukin-18(mChIL-18) gene were amplified by designing two pairs of primers according to the sequence published on GenBank.Recombinant baculovirus coexpressing HA1 gene from AIV subtype H5 plus chicken interleukin-18 gene were constructed by inserting cDNA copy of HA gene and cDNA copy of mChIL-18 gene into the baculovirus expression plasmid pFast Bac~(TM)Dual,furthermore cDNAs respectively inserted into the downstreams of P10 promoter and PH promoter.Finally,the constructing eukaryotic expression plasmid pFastBac~(TM) dual-HA1-mChIL-18 was transposited into Bacterium Coli DH10Bac,which is based of next step insect cell’s transfection.The construction of this recombinant plasmid is benefic to investigate the effect of mChIL-18’s immunologic enhancement and study a new AIV recombinant vaccine.
Experiment 2: Expression of Recombinant Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Chicken Interleukin-18 Gene
After constructing pFastBac~(TM) dual-IL-18-HA1 plasmid ,we make it transformate into DH10Bac competent cells.Then transformations are spreaded on fresh LB plates and the recombinant bacmid DNA is extracted.On the basis of M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis),we analyze the recombinant bacmid DNA by PCR.Through transfecting insect cells, trcombinant baculovirus is harvested.Then we gain the high performance expressing product of IL-18 and HA1,which is benefic to investigate the effect of mChIL-18’s immunologic enhancement and study a new AIV recombinant vaccine.
引文
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