猪繁殖与呼吸综合征病毒nsp4抗体制备与鉴定

为了获得猪繁殖与呼吸综合征病毒(PRRSV)nsp4的抗体,根据HP-PRRSV TA-12株(Gen Bank Accession No.HQ416720)的nsp4基因序列,设计并合成一对引物。用RT-PCR扩增后克隆到原核表达载体p ET-28a(+)中,构建重组质粒p ET28a-nsp4,转化至Trasseta(DE3),经IPTG诱导重组蛋白获得了高效可溶性表达,大小约为26 k Da。经镍离子亲和柱(Ni+-NTA)纯化获得了高纯度重组蛋白,将纯化的nsp4蛋白免疫新西兰大白兔制备了多克隆抗体。ELISA检测抗体效价可达106,Western blotting和IFA检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性,能够识别PRRSV感染宿主细胞中的nsp4蛋白。本研究成功制备了针对nsp4的多克隆抗体,为进一步研究nsp4的功能及PRRSV致病机制奠定了基础。     

猪流行性腹泻病毒非结构蛋白nsp9互作宿主蛋白对病毒复制的影响

猪流行性腹泻病毒(porcineepidemicdiarrheavirus,PEDV)导致仔猪和育肥猪发生急性肠道传染病，是危害养猪业最重要的病原体之一。目前发现PEDV能够编码至少16个非结构蛋白，其中nsp9能够结合至单链RNA中，但是其功能机制还不清楚。本研究通过免疫沉淀联合蛋白质谱分析，筛选出潜在的与PEDV nsp9宿主互作蛋白。通过进一步免疫共沉淀(co-immunoprecipitation, Co-IP)和激光共聚焦技术确认了nsp9与热休克蛋白HSPA8、Toll相互作用蛋白Tollip、热休克蛋白HSPA9、线粒体外膜蛋白TOMM70互作。其中，过表达HSPA8使nsp9的表达量先上调而后下调，并促进PEDV的增殖；过表达Tollip使nsp9的表达量显著上调，并抑制PEDV的增殖；过表达TOMM70使nsp9的表达量显著下调，但对PEDV的增殖无明显影响；过表达HSPA9对nsp9的表达以及PEDV的增殖均无明显影响。该研究为探索nsp9互作蛋白在PEDV感染过程中的功能提供了重要信息。     

马西尼罗热病毒E蛋白部分基因在大肠杆菌中的表达

参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a( )。重组质粒pET32a-E转化BL21(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为32kD的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的33．1%。表达产物纯化后,Wester-blotting分析证明表达产物能被WNV的阳性血清所识别,为下一步建立以表达产物为包被抗原建立检测马的WNV的ELISA方法打下了基础。     

鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备

目的用RT-PCR技术扩增鸭坦布苏病毒(DTMUV)AH-F10株E基因,并克隆至pET32a(+)载体,构建重组表达质粒pET32a-E,表达E蛋白。方法重组表达质粒转化感受态细胞BL21(DE3),经IPTG诱导后,获得含6个His标签的融合蛋白,大小约54kDa。表达的蛋白以包涵体形式存在。对目的蛋白进行纯化,用纯化的E蛋白免疫Balb/c小鼠,制备多克隆抗体血清。结果SDS-PAGE和Western blot试验结果表明E基因在大肠埃希菌中成功表达,并能与抗DTMUV多克隆抗体产生特异性反应,具有良好的反应原性。间接免疫荧光试验表明免疫小鼠后获得的多克隆抗体能与DTMUV反应。结论本研究为DTMUV新型疫苗和诊断试剂盒的进一步研究奠定了基础。     

东部马脑脊髓炎病毒的分子生物学进展

东部马脑脊髓炎病毒属虫媒病毒,能引起人和马发生急性脑炎。东马病毒为单股正链RNA病毒,可分为南美型和北美型,包括两个开放读码框架,分别编码结构蛋白（E1,E2,E2,C,6K）和非结构蛋白（nsp1,nsp2,nsp3,nsp4)。其中E1/E2包膜糖蛋白以异二聚体的形式病毒颗粒外刺突。非结构蛋白主要能与负链RNA的合成,近来,随着研究深入,病毒受体越来越受到广泛关注。本介绍东部马脑脊髓炎病毒结构、进化、复制、组装等方面的分子生物学进展。     

寨卡病毒囊膜E蛋白的原核表达及其抗体制备

目的:在大肠杆菌BL21(DE3)中重组表达寨卡病毒(ZIKV)囊膜(E)蛋白的200个氨基酸(138～338)截短片段ZIKV-E~(200),制备其多克隆抗体,为寨卡病毒亚单位疫苗后续研究提供检测抗体。方法:用全基因序列合成方法合成寨卡病毒E蛋白全长基因,以该基因为模板,用PCR方法克隆ZIKV-E~(200)基因片段,经EcoRⅠ/XhoⅠ双酶切后连接到pET22b载体并转化大肠杆菌DH5α感受态细胞,获得重组质粒pET22b-ZIKV-E~(200),将其转化大肠杆菌BL21(DE3)感受态细胞得到重组表达菌株pET22b-ZIKV-E~(200)。将37℃诱导表达后的菌液超声波破碎处理后制备ZIKV-E~(200)蛋白,将制备的抗原ZIKV-E~(200)免疫BALB/c小鼠,经3次免疫后,采集血清制备其相应的多克隆抗体,采用Western印迹检测多克隆抗体的特异性。结果:获得ZIKV-E~(200)基因片段,并构建了其相应的原核表达载体pET22b-ZIKV-E~(200),在大肠杆菌中表达了重组ZIKV-E~(200)蛋白,其相对分子质量约为25 000,与理论值一致;用分离的蛋白ZIKV-E~(200)免疫小鼠后获得了抗ZIKV-E蛋白的多克隆抗体,该多克隆抗体检测到了酵母表达的寨卡病毒E蛋白。结论:ZIKV-E~(200)蛋白的多克隆抗体可用于酵母表达的寨卡病毒E蛋白的检测等研究。     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a( )中,从而构建了原核表达质粒pET30-VP1。将pET30-VP1转化至感受态细胞E.coliBL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达。该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清。以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上。以Westernblot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性。VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础。     

猪流行性腹泻病毒nsp1蛋白对Ⅰ型干扰素应答的影响

猪流行性腹泻病毒 (PEDV) 能抑制宿主Ⅰ型干扰素及其诱导的细胞抗病毒免疫应答,但是PEDV抑制Ⅰ型干扰素应答的分子机制尚不明了,尤其是PEDV非结构蛋白 (Nonstructural proteins,nsps) 在Ⅰ型干扰素应答中的调控作用研究不多。为研究PEDV非结构蛋白1 (nsp1) 对细胞Ⅰ型干扰素应答的影响,构建了真核表达载体pCAGGS-nsp1,采用Western blotting和间接免疫荧光试验确定nsp1在细胞中的表达。通过报告基因法、ELISA以及病毒复制抑制试验评估nsp1对Ⅰ型IFN的影响。结果显示,nsp1在转染细胞和病毒感染细胞中均高效表达;双荧光报告基因试验结果表明,nsp1能显著抑制IFN-β启动子活性,且具有剂量依赖性。ELISA结果显示,nsp1能显著抑制IFN-β蛋白的表达。水泡性口炎病毒 (VSV) 复制抑制试验结果显示,nsp1明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用。结果提示,nsp1作为PEDV的保守蛋白,具有拮抗Ⅰ型干扰素启动子活性和应答的功能,为揭示PEDV逃逸宿主天然免疫应答的机制和研发新型高效抗PEDV疫苗奠定基础。     

猪流行性腹泻病毒nsp1对Ⅰ型干扰素应答的影响

猪流行性腹泻病毒(PEDV)能抑制宿主Ⅰ型干扰素及其诱导的细胞抗病毒免疫应答,但是PEDV抑制Ⅰ型干扰素应答的分子机制尚不明了,尤其是PEDV非结构蛋白(Nonstructural proteins,nsps)在Ⅰ型干扰素应答中的调控作用研究不多。为研究PEDV非结构蛋白1(nsp1)对细胞Ⅰ型干扰素应答的影响,构建了真核表达载体p CAGGS-nsp1,采用Western blotting和间接免疫荧光试验确定nsp1在细胞中的表达。通过报告基因法、ELISA以及病毒复制抑制试验评估nsp1对Ⅰ型IFN的影响。结果显示,nsp1在转染细胞和病毒感染细胞中均高效表达。双荧光报告基因试验结果表明,nsp1能显著抑制IFN-β启动子活性,且具有剂量依赖性。ELISA结果显示,nsp1能显著抑制IFN-β蛋白的表达。水泡性口炎病毒(VSV)复制抑制试验结果显示,nsp1明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用。结果提示,nsp1作为PEDV的保守蛋白,具有拮抗Ⅰ型干扰素启动子活性和应答的功能,为揭示PEDV逃逸宿主天然免疫应答的机制和研发新型高效抗PEDV疫苗奠定基础。     

含CSFV E2基因A/D区原核表达载体的构建、表达及其抗原性研究

应用RT PCR方法扩增了编码猪瘟病毒石门株 (CSFVshimenstrain)囊膜糖蛋白E2全基因 ,然后将其克隆到pMD 1 8T质粒中 ,获得重组质粒pMD E2。再以pMD E2为模板 ,另行设计两对引物 ,同时扩增其中一段适于在E .coli中表达且抗原反应性较好的基因片段 (E2蛋白A D抗原区基因序列 ) ,将扩增的两片段串联插入原核表达载体pET 32a中构建成重组质粒pET 2e。用酶切和序列分析鉴定插入目的基因的正确性。SDS PAGE和Western blot分析表明 ,经pET 2e转化、IPTG诱导的受体菌可表达目的蛋白 ,克隆在硫氧还蛋白 (thioredoxinprotein ,TrxA)基因下游的E2蛋白基因与TrxA基因获得了高效融合表达 ,并且具有免疫学反应活性 ,这为猪瘟的血清学诊断方法的建立打下了基础 。     

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.     

Sequence analysis and structural prediction of the severe acute respiratory syndrome coronavirus nsp5

The non-structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3-D models of the SARS-CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide-nucleotide basic local alignment search tool (BLASTN) and a protein-protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS-7 cells and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study provided the foundation for further exploration of the protein‘s biological function, and contributed to the search for anti-SARS-CoV drugs.     

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.     

SARS冠状病毒非结构蛋白Nsp的原核表达

严重急性呼吸综合征病毒,即SARS冠状病毒((Severe acute respiratory syndrome coronavirus,SARS-CoV),为具有囊膜的单股正链RNA病毒,基因组约长29~31kb。基因组从5'到3'端依次编码复制酶蛋白(Rep)、刺突蛋白(S)、囊膜蛋白(E)、膜蛋白(M)和核蛋白(N)以及其他一些辅助性蛋白[1]。编码复制酶蛋白的基因,从基因组5'端起约占全长的2/3区域(≈21.2kb),在该区域的nt13392-13398存在保守的UUUAAAC位点,此位点含有-1位的核糖体翻译移框(frameshift),可引发自单一起始位点的蛋白翻译扩展,即由ORF1a编码的Pp1a(约486kDa)扩展为由ORF1b编…     

Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain

The severe acute respiratory syndrome (SARS) epidemic was caused by the spread of a previously unrecognized infectious agent, the SARS-associated coronavirus (SARS-CoV). Here we show that SARS-CoV could inhibit both virus- and interferon (IFN)-dependent signaling, two key steps of the antiviral response. We mapped a strong inhibitory activity to SARS-CoV nonstructural protein 1 (nsp1) and show that expression of nsp1 significantly inhibited the activation of all three virus-dependent signaling pathways. We show that expression of nsp1 significantly inhibited IFN-dependent signaling by decreasing the phosphorylation levels of STAT1 while having little effect on those of STAT2, JAK1, and TYK2. We engineered an attenuated mutant of nsp1 in SARS-CoV through reverse genetics, and the resulting mutant virus was viable and replicated as efficiently as wild-type virus in cells with a defective IFN response. However, mutant virus replication was strongly attenuated in cells with an intact IFN response. Thus, nsp1 is likely a virulence factor that contributes to pathogenicity by favoring SARS-CoV replication.