猪流行性腹泻病毒S1蛋白亲和肽的筛选与鉴定
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摘要
猪流行性腹泻(porcine epidemic diarrhea, PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)引起的一种猪的急性、高度接触性肠道传染病。自1978年报道以来在欧洲以及亚洲许多国家相继爆发,给养猪业带来巨大损失,因此对于PED的诊断以及疫苗的研究就显得尤为重要。PEDV纤突糖蛋白(S)是位于病毒粒子表面的结构蛋白,它既含有介导病毒侵入宿主细胞的受体结合域,又拥有介导机体产生病毒中和抗体的抗原表位,具有良好的免疫原性。
利用生物学软件对PEDV S基因进行了抗原位点分析,在不破坏线性抗原位点的前提下将S基因进行分割获得S基因的片段命名为S1。本试验在高效表达PEDV S1蛋白后,利用噬菌体展示技术筛选并鉴定了能与PEDV S1蛋白相互作用的多肽分子,为进行PED的快速诊断以及开发有效的新型疫苗奠定了基础。
本试验成功构建了编码PEDV S1蛋白的pET30a-S1原核重组表达质粒,并在E.Coli Rosetta中进行原核表达。将制备并纯化的重组蛋白进行复性和活性检测,结果表明这种融合蛋白具有一定的生物学活性。以重组蛋白为免疫原免疫新西兰白兔制备了多克隆抗体。免疫荧光实验以及病毒抑制实验证明此多克隆抗体具有良好的生物学活性。
利用噬菌体随机十二肽库,以PEDV S1重组蛋白为靶分子,对其进行了四轮生物淘选。对第四轮洗脱物随机淘选出的10个噬菌体单克隆的核苷酸序列进行测定,并进行多肽序列的推导,结果表明,经过对靶分子的四轮筛选后,最终筛选出三个具有高度同源性的多肽,序列分别是MPAVMSSAQVPR,NLSNRLNLSPGI,YVIHQPYAMALR。ELISA方法对合成多肽的检测结果表明这三条多肽均能与重组的PEDV S1蛋白结合。病毒体外抑制实验表明所筛选出来的其中两条多肽M和N具有良好的生物活性。体内实验通过免疫昆明鼠检测了多肽的抗病毒活性,结果表明多肽M、N在小鼠体内起到了一定的中和病毒的作用。本实验为建立新的PED诊断方法以及新型多肽疫苗的开发提供了一定的理论基础和实验依据。
Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). Since 1978, the outbreak of PED in Europe and many Asian countries have led to huge economic losses in pig industry. Thus, there is a urgent need to understand its pathological mechanism and to develop novel PED vaccine for the prevention of PED.The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a key role in the specific receptor binding, cell membrane fusion, and induction of neutralizing antibody for its possessing neutralizing epitopes and receptor binding domain. Given this, screening and identification of receptor binding domain and antigenic epitopes were carried out in this study. It will provide valuable information for development of diagnosis technology, novel vaccine, and designing antiviral immunization strategy of PED.
The experiment successfully constructed a recombinant expression vector pET-30a-S1. High level expression of S1 protein was analyzed by SDS-PAGE at different time at 37℃after IPTG induction. The S1 protein was purified and injected to a rabbit to generate polyclonal antibody. Western-blotting result showed polyclonal antibody could specifically bind to PEDV S1 protein, which indicated that recombinant fusion protein has excellent immunogenicity.
Random 12-mer Phage Display Peptide Library was used to panning the S1 protein for four rounds. Ten monoclonal phages were selected randomly and subjected to sequence comparison. The results showed that three different peptides were screened by panning on the S1 recombinat protein. Three peptides that have the highest binding activity to S1 were synthesized. Their sequences are MPAVMSSAQVPR named M, NLSNRLNLSPGI named N and YVIHQPYAMALR named Y, respectively. ELISA showed that both peptides could bind to S1 protein and inhibit cell infection by PEDV in vitro. The immunized Kunming mice were used to detect the anti-viral activity of peptides in vivo. The results showed that the peptide M and N could neutralize viral infection activity. The current study should be helpful for establishing specific diagnosis method and developing new subunit vaccine for PEDV.
利用生物学软件对PEDV S基因进行了抗原位点分析,在不破坏线性抗原位点的前提下将S基因进行分割获得S基因的片段命名为S1。本试验在高效表达PEDV S1蛋白后,利用噬菌体展示技术筛选并鉴定了能与PEDV S1蛋白相互作用的多肽分子,为进行PED的快速诊断以及开发有效的新型疫苗奠定了基础。
本试验成功构建了编码PEDV S1蛋白的pET30a-S1原核重组表达质粒,并在E.Coli Rosetta中进行原核表达。将制备并纯化的重组蛋白进行复性和活性检测,结果表明这种融合蛋白具有一定的生物学活性。以重组蛋白为免疫原免疫新西兰白兔制备了多克隆抗体。免疫荧光实验以及病毒抑制实验证明此多克隆抗体具有良好的生物学活性。
利用噬菌体随机十二肽库,以PEDV S1重组蛋白为靶分子,对其进行了四轮生物淘选。对第四轮洗脱物随机淘选出的10个噬菌体单克隆的核苷酸序列进行测定,并进行多肽序列的推导,结果表明,经过对靶分子的四轮筛选后,最终筛选出三个具有高度同源性的多肽,序列分别是MPAVMSSAQVPR,NLSNRLNLSPGI,YVIHQPYAMALR。ELISA方法对合成多肽的检测结果表明这三条多肽均能与重组的PEDV S1蛋白结合。病毒体外抑制实验表明所筛选出来的其中两条多肽M和N具有良好的生物活性。体内实验通过免疫昆明鼠检测了多肽的抗病毒活性,结果表明多肽M、N在小鼠体内起到了一定的中和病毒的作用。本实验为建立新的PED诊断方法以及新型多肽疫苗的开发提供了一定的理论基础和实验依据。
Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). Since 1978, the outbreak of PED in Europe and many Asian countries have led to huge economic losses in pig industry. Thus, there is a urgent need to understand its pathological mechanism and to develop novel PED vaccine for the prevention of PED.The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a key role in the specific receptor binding, cell membrane fusion, and induction of neutralizing antibody for its possessing neutralizing epitopes and receptor binding domain. Given this, screening and identification of receptor binding domain and antigenic epitopes were carried out in this study. It will provide valuable information for development of diagnosis technology, novel vaccine, and designing antiviral immunization strategy of PED.
The experiment successfully constructed a recombinant expression vector pET-30a-S1. High level expression of S1 protein was analyzed by SDS-PAGE at different time at 37℃after IPTG induction. The S1 protein was purified and injected to a rabbit to generate polyclonal antibody. Western-blotting result showed polyclonal antibody could specifically bind to PEDV S1 protein, which indicated that recombinant fusion protein has excellent immunogenicity.
Random 12-mer Phage Display Peptide Library was used to panning the S1 protein for four rounds. Ten monoclonal phages were selected randomly and subjected to sequence comparison. The results showed that three different peptides were screened by panning on the S1 recombinat protein. Three peptides that have the highest binding activity to S1 were synthesized. Their sequences are MPAVMSSAQVPR named M, NLSNRLNLSPGI named N and YVIHQPYAMALR named Y, respectively. ELISA showed that both peptides could bind to S1 protein and inhibit cell infection by PEDV in vitro. The immunized Kunming mice were used to detect the anti-viral activity of peptides in vivo. The results showed that the peptide M and N could neutralize viral infection activity. The current study should be helpful for establishing specific diagnosis method and developing new subunit vaccine for PEDV.
引文
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