利用噬菌体展示技术筛选草鱼呼肠孤病毒VP39蛋白相互作用多肽

VP39是草鱼呼肠孤Ⅲ型病毒(GCRV GenotypeⅢ, GCRV-Ⅲ)S9基因编码的蛋白,为研究VP39蛋白在GCRV-Ⅲ感染草鱼细胞过程中行使的生物学功能,将克隆VP39基因序列并构建原核表达载体pET32a-VP39,通过原核表达得到VP39-HIS融合蛋白;利用VP39蛋白溶液免疫小鼠,制备鼠抗VP39多克隆抗体,通过Western Blot对抗体进行评估;利用制备的多克隆抗体探究GCRV-Ⅲ感染细胞过程中VP39蛋白表达动力学;利用噬菌体展示技术筛选与VP39蛋白特异性结合的多肽序列并进行分析。SDS-PAGE电泳结果显示, VP39-HIS融合蛋白可良好溶于PBS中,蛋白大小约为39 kD; Western Blot检测表明实验所制备的VP39多克隆抗体在1:10000稀释比例下,既能识别原核表达的VP39-HIS融合蛋白,也能识别GCRV-Ⅲ感染CIK细胞后表达的VP39蛋白,具有良好的效价与特异性;在病毒侵染过程中, VP39前期表达量较少,在中后期大量表达;噬菌体展示技术筛选出两条多肽与VP39蛋白有高度亲和性,经过在NCBI上比对后发现草鱼基因组中有7个基因与筛...     

草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

草鱼呼肠孤病毒是引起草鱼出血病的主要病原,隶属于呼肠孤病毒科水生呼肠孤病毒属。序列分析表明,GCRVS2片段长为3877核苷酸,编码一个分子量为138kDa的蛋白VP2,具有RNA聚合酶性质。为进一步了解该病毒RNA聚合酶特性,本研究在对GCRV RNA聚合酶基因(GCRV—RdRp)保守区(约1．5kb)重组质粒pR／RRp高效表达的基础上,分别构建了编码GCRV RNA聚合酶保守区N端与C端部分基因的pR／RRpN及pR／RRpC重组表达载体,并在原核细胞中获得成功表达。筛选的重组表达菌株经IPTG诱导培养,得到分子量分别为98kDa、103kDa的目的表达融合蛋白。Western blot分析表明,该表达产物与兔抗GCRV—VP2血清呈阳性反应。通过ProBond柱亲和层析,纯化了融合有6个组氨酸的重组表达产物,并获得约90％纯的目的蛋白。上述结果为GCRV RNA聚合酶特性分析提供了依据。     

草鱼呼肠孤病毒衣壳蛋白VP7真核重组质粒的构建及免疫效果评价

为研究开发草鱼呼肠孤病毒(Grass carp reovirus, GCRV)基因疫苗, 以编码其主要衣壳蛋白VP7的序列为靶基因, 克隆构建了真核表达重组质粒 pEGFP-N1-VP7。用脂质体法将其转染真核细胞COS-1和CIK进行瞬时表达, 荧光显微镜观察及特异性RT-PCR检测结果表明, 成功转染并得到了高效表达。大量扩增重组菌, 提取并制备重组质粒pEGFP-N1-VP7, 肌肉注射免疫(205) g的健康草鱼, 重组质粒按0.5和5 g分为2组, 同时设5 g空载体组及对照组。免疫草鱼22d后, 检测草鱼肠、外周血、肾脏、脾脏的呼吸暴发活性及淋巴细胞的增殖反应; 免疫草鱼21d、28d、35d、42d、56d、70d、84d和98d后, 分别尾静脉采血并分离血清, 用双抗体夹心ELISA方法进行抗体水平的测定。结果表明, 构建的含VP7蛋白的核酸疫苗既可诱导草鱼的细胞免疫, 又可诱导特异性体液免疫, 具有明显的免疫应答作用。按照每尾0.5 g重组质粒的剂量免疫草鱼后35d进行攻毒, 免疫保护率达到67%, 此研究为GCRV基因疫苗的研制提供了实验资料。       

草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

草鱼呼肠孤病毒是引起草鱼出血病的主要病原,隶属于呼肠孤病毒科水生呼肠孤病毒属.序列分析表明,GCRV S2 片段长为3 877核苷酸,编码一个分子量为138kDa 的蛋白VP2,具有RNA聚合酶性质.为进一步了解该病毒 RNA聚合酶特性,本研究在对GCRV RNA聚合酶基因(GCRV-RdRp)保守区(约1.5kb)重组质粒pR/RRp高效表达的基础上,分别构建了编码GCRV RNA聚合酶保守区N端与C端部分基因的 pR/RRpN及pR/RRpC重组表达载体,并在原核细胞中获得成功表达.筛选的重组表达菌株经IPTG诱导培养,得到分子量分别为98kDa、103kDa的目的表达融合蛋白.Western blot分析表明,该表达产物与兔抗GCRV-VP2血清呈阳性反应.通过ProBond柱亲和层析,纯化了融合有6个组氨酸的重组表达产物,并获得约90%纯的目的蛋白.上述结果为GCRV RNA聚合酶特性分析提供了依据.     

草鱼细胞系CIK酵母双杂交cDNA文库的构建及初步应用

旨在探索草鱼呼肠孤病毒与宿主细胞蛋白质间的相互作用,运用SMART技术构建了草鱼肾组织细胞系CIK(Ctenopharyngodon idellus kidney)的酵母双杂交cDNA文库。提取CIK细胞总RNA后,分离纯化mRNA,然后以mRNA为模板,反转录合成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR,扩增双链cDNA。利用SMART技术,通过同源重组的方法,在酵母株Y187中构建了草鱼CIK细胞全长cDNA文库。经检测,未扩增文库的转化率为1.6×105,文库容量为2.4×106,插入的双链cDNA片段的长度为250-2 000 bp,文库滴度为7×107 CFU/mL,重组率为98%,此文库具有良好的cDNA片段多态性和完整性。利用构建的CIK酵母双杂交文库,以草鱼呼肠孤病毒的VP7和VP5蛋白作为诱饵进行筛选试验,得到VP7相互作用蛋白的阳性菌落,未得到VP5相互作用蛋白的阳性菌落。草鱼CIK细胞酵母双杂交cDNA文库的构建为研究草鱼呼肠孤病毒与宿主细胞间的互作机制提供了重要研究工具。     

草鱼呼肠孤病毒HZ08株S4基因序列分析

草鱼呼肠孤病毒HZ08株是本实验室从患出血病草鱼体内分离到的一个新毒株,已完成部分基因序列的分析,其氨基酸序列的同源性和873株相比,仅为20%~30%之间.因序列差异较大,无法通过设计特异性引物来扩增和分析其基因序列,采用单引物扩增技术,对HZ08株S4基因进行序列分析表明:S4全长为2263 bp,最大的ORF编码717个氨基酸,推导出其表达的蛋白约为79 kDa.正如其他基因节段,基因末端也含有保守碱基序列5′(GUAAUUU…UUCAUC),3′.S4基因推导的氨基酸序列与同宿主的其他呼肠孤病毒的非结构蛋白NS1同源性最大,其次是和哺乳动物正呼肠孤病毒的非结构蛋白mu-NS以及禽呼肠孤病毒非结构蛋白NS1同源性较大,表明S4可能表达细胞骨架相关蛋白.基于S4推导出的氨基酸序列构建的系统进化树HZ08株单独作为一个分支,与同宿主的其他呼肠孤病毒亲缘关系比较近,而与其他呼肠孤病毒则相对较远.这提示HZ08株可能是多个毒株的遗传信息经长期的遗传进化而得,综合其它已知序列信息,推测HZ08株可能为呼肠孤病毒的一个新成员.     

利用噬菌体展示技术淘选草鱼呼肠孤病毒的单链抗体

草鱼呼肠孤病毒（GCRV）是引起我国大面积草鱼幼鱼出血病暴发的主要病原,其外衣壳蛋白VP5和VP7在病毒入侵宿主细胞过程中起着至关重要的作用。研究以原核表达的VP7、全长VP5、VP5的N端片段及C端片段为靶蛋白,利用已构建的噬菌体展示单链抗体文库进行淘选。经过3轮淘选后,共获得7个针对VP7、VP5、VP5N和VP5C的单链抗体。经过验证,识别原核表达的VP7的两个单链抗体能够成功识别天然GCRV病毒。此结果对于进一步研究GCRV与宿主细胞的相互作用机理奠定了基础。       

鸡髓样分化因子88的原核表达及单克隆抗体制备

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。     

质粒DNA单次脾内注射制备单克隆抗体

探索一种新的快捷有效DNA免疫制备单克隆抗体的方法,辅助实现构建高通量无蛋白纯化体系单克隆抗体制备和筛选。分别通过“重叠PCR”和“无模板PCR”在pVAX1真核载体中分别引入IL-2信号肽、IgG kappa链信号肽构建分泌型真核表达载体,将代表抗原基因的profilin 1基因克隆到经改造带有信号肽基因的表达载体上,构建重组质粒pVAX-IL2-prof1和pVAX-Igκ-prof1,单次脾内注射重组质粒DNA免疫BALB/c小鼠。经过细胞融合、ELISA筛选,获得两株抗profilin 1的单克隆抗体。单抗亚型分别为IgM和IgG3。单次脾内质粒DNA免疫便捷有效,是制备单克隆抗体的有效方法。     

质粒DNA单次脾内注射制备单克隆抗体

探索一种新的快捷有效DNA免疫制备单克隆抗体的方法,辅助实现构建高通量无蛋白纯化体系单克隆抗体制备和筛选。分别通过“重叠PCR”和“无模板PCR”在pVAX1真核载体中分别引入IL-2信号肽、IgG kappa链信号肽构建分泌型真核表达载体,将代表抗原基因的profilin1基因克隆到经改造带有信号肽基因的表达载体上,构建重组质粒pVAX-IL2-prof1和pVAX-Igκ-prof1,单次脾内注射重组质粒DNA免疫BALB/c小鼠。经过细胞融合、ELISA筛选,获得两株抗profilin1的单克隆抗体。单抗亚型分别为IgM和IgG3。单次脾内质粒DNA免疫便捷有效,是制备单克隆抗体的有效方法。     

High level expression of grass carp reovirus VP7 protein in prokaryotic cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.     

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus （GCRV） s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein （OCP）. To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid, which was named as pR/GCRV-VP7, was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover, the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody （mouse） and anti-GCRV serum （rabbit）. This work provides a research basis for further structure and function studies of GCRV during entry into cells     

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella

Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus’s structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse naïve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5–7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.     

Expression of outer capsid protein VP5 of grass carp reovirus in <Emphasis Type="Italic">E.coli</Emphasis> and analysis of its immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.     

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus （GCRV） is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses （MRV） compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.     

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.     

一种新型草鱼呼肠孤病毒人工感染方法

研究从患病草鱼中新分离到一株草鱼呼肠孤病毒（Grass carp reovirus,GCRV）,对其进行了病毒纯化与电镜观察、基因组RT-PCR分型以及病毒量定量分析等,并在此基础上探索了一种新的病毒人工感染方法。取病鱼肌肉组织进行病毒纯化与电镜观察,观察到大量病毒粒子,直径在70-80 nm。病毒基因组RT-PCR扩增结果表明,该草鱼呼肠孤病毒新分离株属基因型Ⅱ型GCRV;通过绝对定量的方法,对病毒悬液的浓度进行了测定,为2.97×103 copy/μL。通过灌胃法,对3个组别的实验鱼分别感染不同浓度的病毒液,同时设置灌胃PBS的组别作为对照组。结果显示,3个实验组死亡率均在80%左右,而对照组仅出现一例死亡个体。实验组死亡个体体表发黑,腹部、鳍条基部以及鳃盖处均有明显的出血症状,为草鱼出血病的典型症状。随机选取死亡的个体进行RT-PCR检测,均能检测出Ⅱ型GCRV的条带。以上结果说明,灌胃法可以作为一种新的方法,用于草鱼的GCRV人工感染实验。     

Construction and co-expression of grass carp reovirus VP6 protein and enhanced green fluorescence protein in the insect cells

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein) genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5 days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro. Undergraduate training student from College of Life Sciences, Wuhan University.