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1.
The glycoprotein 5(GP5) of porcine reproductive and respiratory syndrome virus(PRRSV) is a multi-functional protein that plays important roles in virus assembly,entry and viral anti-host responses.In the present study,we investigated the cellular binding partners of GP5 by using lentivirus transduction coupled with immunoprecipitation and mass spectrometry.There were about 40 cellular proteins identified with high Confidence Icons by MS/MS.Ingenuity Pathway Analysis(IPA)indicated that these proteins could be assigned to different functional classes and networks.Furthermore,we validated some of the interactions by co-immunoprecipitation(Co-IP) and confocal microscopy,including those with mitofilin,a mitochondrial inner membrane protein that might be involved in PRRSV or GP5-induced apoptosis,and calnexin,a protein chaperone that might facilitate the folding and maturation of GP5.The interactome data contribute to understand the role and molecular mechanisms of GP5 in PRRSV pathogenesis.  相似文献   

2.
Ebola virus (EboV) causes rapidly fatal hemorrhagic fever in humans and there is currently no effective treatment. We found that the infection of African green monkey kidney (Vero) cells by vesicular stomatitis viruses bearing the EboV glycoprotein (GP) requires the activity of endosomal cysteine proteases. Using selective protease inhibitors and protease-deficient cell lines, we identified an essential role for cathepsin B (CatB) and an accessory role for cathepsin L (CatL) in EboV GP-dependent entry. Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection. CatB and CatB/CatL inhibitors diminish the multiplication of infectious EboV-Zaire in cultured cells and may merit investigation as anti-EboV drugs.  相似文献   

3.
根据GenBank公布的PRRSVORF3基因的核苷酸序列,设计并合成一对特异性引物,用RT-PCR方法扩增PRRSV陕西分离株ORF3基因,将其克隆入pGEM-T载体中,测序并进行序列分析。再将ORF3基因亚克隆入pET-32a中,构建原核表达载体。结果扩增到765 bp的PRRSV全长ORF3基因,序列分析结果表明,分离株与SY0608亲缘关系较近,而与CH-2、HB-2关系较远。重组原核表达质粒经酶切鉴定正确后命名为pET-GP3,为进一步研究GP3蛋白的原核表达、免疫特性、结构与功能奠定了基础。  相似文献   

4.
克隆猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因,并进行序列分析及原核表达。根据GenBank公布的PRRSV ORF5基因的核苷酸序列,设计并合成一对特异性引物,用RT-PCR方法扩增PRRSV陕西分离株糖基化囊膜蛋白基因ORF5,将其克隆入pGEM-T载体中,进行测序及序列分析。再设计另外1对引物扩增ORF5缺失编码N端31个氨基酸残基的基因片段,将截短的ORF5基因亚克隆入原核表达载体pET-32a中,在大肠杆菌BL21中进行原核表达。结果扩增到603 bp的PRRSV全长ORF5基因,序列分析表明,分离株与北美型代表株VR-2332和欧洲型代表株LV氨基酸同源性分别为87.8%和54.5%。因此推测陕西分离株属于北美型。SDS-PAGE可检测到大小约为38 ku的融合蛋白,主要以可溶性蛋白形式存在。West-ern-blot分析表明,重组蛋白可被PRRSV阳性血清所识别。  相似文献   

5.
The pathogenesis of Heymann nephritis, a rat model of human membranous glomerulonephritis, depends on the interaction of autoantibodies with a renal glycoprotein (GP330) on glomerular podocytes. Partial complementary DNAs coding for GP330 were isolated and sequenced. The deduced amino acid sequence from 4.3 kilobases of complementary DNA contains the sequences identical to two peptides derived from the isolated glycoprotein. The deduced amino acid sequence of this protein contains regions with homology to the human low density lipoprotein (LDL) receptor, an indication that GP330 and the LDL receptor may be members of the same gene family. Autoantibodies from the kidneys of rats with Heymann nephritis reacted with a nonglycosylated segment of GP330 that contains cysteine-rich 40-amino acid repeats, which are also features of the LDL receptor. GP330 is also similar in some regions to the mouse epidermal growth factor precursor.  相似文献   

6.
【目的】bZIP类转录因子参与植物的生长发育、激素信号、抗病性及抗逆性等多种生物胁迫过程。前期研究表明黄瓜花叶病毒(Cucumber mosaic virus,CMV)或烟草花叶病毒(Tobacco mosaic virus,TMV)侵染本氏烟(Nicotiana benthamiana)上调内质网应激（endoplasmic reticulum stress,ERs）因子NbbZIP28;NbbZIP28沉默导致病毒积累量上升,本研究旨在验证NbbZIP28对病毒侵染胁迫的响应机制。【方法】通过CRISPR/Cas9基因编辑技术和烟草遗传转染创建NbbZIP28基因突变植株,以野生型植株为对照,分别浸润接种侵染性克隆TMV-GFP、摩擦接种TMV-GFP或CMV接种液,检测突变体植株对病毒侵染胁迫的敏感性变化,接种CMV后0-48 h,采用qRT-PCR检测内质网应激相关的未折叠蛋白反应（unfolded protein response,UPR)基因的表达。本氏烟接种TMV 24 h、接种CMV 48 h后,在接种叶上浸润融合蛋白NbbZIP28-GFP,瞬时表达48 h后,采用Western blot检测病毒诱导NbbZIP28蛋白的水解激活;采用在线搜索工具PlantCARE分析NbbZIP28启动子区域中参与防卫和应激反应的顺式作用元件。【结果】CRISPR/Cas9定点敲除NbbZIP28后,目的基因靶位点缺失了10个碱基,导致翻译错误、蛋白功能变化。在正常生长条件下,转基因阳性植株与野生型无显著的表型差异。植株接种TMV-GFP后4-8 d,突变体中的病毒浸润斑亮度或初侵染点数目均显著高于野生型,扩展至新叶的速度较快。接种CMV后12-48 h,突变体中UPR相关基因BiP、PDI、CAM及NbbZIP28下游基因NF-YC2的表达量显著低于野生型;5-7 d突变体中的病毒外壳蛋白（coat protein,CP）基因表达量显著高于野生型,花叶及皱缩症状更显著。蛋白质序列比对分析显示NbbZIP28具有与拟南芥AtbZIP28相同的S1P和S2P蛋白酶水解位点。Western blot检测发现,与接种清水对照相比,TMV或CMV侵染显著促进了全长的融合蛋白NbbZIP28-GFP在S1P和S2P位点发生裂解。NbbZIP28启动子序列中包含5个参与热应激反应的顺式作用元件（heat stress response element,HSE）、3个参与低温反应的顺式作用元件（low temperature response element,LTR）、1个参与防卫和应激反应的顺式作用元件（TC-rich repeats）。【结论】病毒侵染促进NbbZIP28的水解激活并上调相关的UPR基因;NbbZIP28敲除导致植株对病毒的敏感性上升,病毒诱导的UPR基因表达被抑制。NbbZIP28为病毒侵染胁迫下的UPR调控因子,在病毒侵染早期通过上调UPR信号和提高寄主基础防卫反应而延缓病毒的侵染和增殖。  相似文献   

7.
利用构建的猪瘟病毒C株感染性克隆作为骨架,利用猪繁殖与呼吸综合征病毒GP5基因作为标记基因,将GP5基因引入猪瘟病毒感染性cDNA中,体外转录得到RNA,转染SK6细胞后,检测了传代细胞中重组猪瘟病毒包含GP5区段的一段重组序列。结果表明,GP5基因稳定地插入在重组病毒基因组中,改造后的重组病毒可望作为C株活病毒标记疫苗。  相似文献   

8.
Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.  相似文献   

9.
The structure of kistrin, which is a member of a homologous family of glycoprotein IIb-IIIa (GP IIb-IIIa) antagonists and potent protein inhibitors of platelet aggregation, has been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The 68-residue protein consists of a series of tightly packed loops held together by six disulfide bonds and has almost no regular secondary structure. Kistrin has an Arg-Gly-Asp (RGD) adhesion site recognition sequence important for binding to GP IIb-IIIa that is located at the apex of a long loop across the surface of the protein.  相似文献   

10.
将克隆到的猪繁殖与呼吸综合征病毒全长GP5基因插入到pAdTrack-CMV穿梭质粒中,再将重组质粒pAdTrack-CMV/GP5用PmeⅠ线性化后电转化携带有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183感受态细胞,经细菌内同源重组产生重组腺病毒质粒pAdEasy-GP5,经酶切充分暴露反向末端重复序列,再与脂质体混合转染AD-293细胞,成功获得了AD-GP5复制缺陷型腺病毒,经检测证实AD-GP5稳定性好,安全性高.转录水平检测证实,GP5蛋白基因得到了转录;荧光检测发现,外源蛋白已得到表达.  相似文献   

11.
以猪繁殖与呼吸综合症病毒(PRRSV)主要中和表位所在的糖蛋白GP5为模式抗原,采用肌肉注射、皮下注射和滴鼻免疫方式免疫6～8周龄ICR小鼠,利用间接ELISA检测血清抗体,MTT法检测淋巴细胞增殖.结果表明:三免后GP5弗氏佐剂乳化组小鼠血清抗体的水平最高,LTB+GP5免疫组的抗体水平显著高于单独GP5免疫组,肌肉注射免疫组的抗体水平明显高于皮下注射组,滴鼻免疫组没有检测到特异性血清抗体;各GP5免疫组均能不同程度地诱导脾脏B淋巴细胞增殖,其中GP5弗氏佐剂乳化组、LTB+GP5肌肉注射和LTB+GP5皮下注射组B淋巴细胞增殖显著,除了GP5弗氏佐剂乳化免疫组外,其他各免疫组均无明显的脾脏T淋巴细胞增殖反应.  相似文献   

12.
A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH   总被引：28，自引：0，他引：28  
A stable cell line expressing a complementary DNA clone encoding the vesicular stomatitis virus glycoprotein fused and formed polykaryons at pH 5.5. The formation of polykaryons was dependent on the presence of glycoprotein anchored at the cell surface and could be prevented by incubation of cells with a monoclonal antibody to the glycoprotein. Fusion occurred at a pH 0.5 unit lower than that observed for cells infected with vesicular stomatitis virus.  相似文献   

13.
参照已公布的猪繁殖与呼吸综合征病毒(PRRSV)美洲株GP5蛋白基因序列,设计并合成1对引物,以RT-PCR方法扩增出PRRSV的GP5基因片段(603 bp)。回收目的基因片段,克隆到pMD18-T载体内构建成重组质粒pMD18T-G。经酶切鉴定、测序正确后,再回收目的基因片段,克隆到真核表达载体pIRES-neo内,经单酶切和双酶切鉴定,成功构建基因疫苗表达载体pIRES-G。免疫小鼠试验表明,2次免疫后抗体水平显著提高,但总的抗体水平体低于灭活苗。  相似文献   

14.
Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.  相似文献   

15.
Somatostatin （SS） is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus （PRRSV）. A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.  相似文献   

16.
The envelope of the human immunodeficiency virus type 1 (HIV-1) plays a central role in the process of virus entry into the host cell and in the cytopathicity of the virus for lymphocytes bearing the CD4 molecule. Mutations that affect the ability of the envelope glycoprotein to form syncytia in CD4+ cells can be divided into five groups: those that decrease the binding of the envelope protein to the CD4 molecule, those that prevent a post-binding fusion reaction, those that disrupt the anchorage of the envelope glycoprotein in the membrane, those that affect the association of the two subunits of the envelope glycoprotein, and those that affect post-translational proteolytic processing of the envelope precursor protein. These findings provide a functional model of the HIV envelope glycoprotein.  相似文献   

17.
 【目的】对豆天蛾核型多角体病毒（CbNPV）的gp41同源基因进行克隆和序列分析，为进一步研究该病毒基因组的结构打下基础。【方法】从豆天蛾幼虫尸体中分离纯化了豆天蛾核型多角体病毒，提取其基因组DNA，用shotgun法构建了DNA片段的文库，并进行了全基因组测序。对CbNPV的gp41同源基因进行序列分析。【结果】CbNPV的gp41同源基因长度为933 bp，编码310个氨基酸。氨基酸序列同源性分析结果表明，CbNPV GP41与I类NPV及II类NPV GP41的同源性分别为53%～61%和56%～73%。【结论】初步推测，CbNPV与II类NPV的亲缘关系可能更近。  相似文献   

18.
The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects. In the construction of CRISPR/Cas9-mediated dsLmRNase2~(–/–) mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newlylaid eggs that were less tanned. However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G_1 progeny was similar between adults derived from the tanned or less tanned engineered eggs. Further, the similar effective mutant ratios in the normally developed eggs and G_0 adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9. Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs. We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection. Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure. In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.  相似文献   

19.
为了制备猪繁殖与呼吸综合征病毒(PPRSV)的单克隆抗体,利用猪繁殖与呼吸综合征病毒(PPRSV)GP5重组蛋白免疫BALB/c小鼠,取脾细胞和NS0浆细胞瘤细胞进行融合,经间接ELISA和阻断ELISA筛选、有限稀释法克隆化获得3株能稳定分泌抗PRRSV GP5蛋白单克隆抗体的(mAb)杂交瘤细胞株,命名为4B7、2C5和2E4。间接免疫荧光(IFA)和免疫过氧化物酶单层试验(IPMA)结果显示,3株mAb均与感染PRRSV BJ 4株的Marc 145细胞特异性反应,表明其可识别天然病毒蛋白。mAb的Ig亚类均为IgG1,细胞培养上清和腹水的ELISA效价分别为1∶256和1∶51 200,Western blot分析表明,mAb均特异性识别PRRSV GP5蛋白。  相似文献   

20.
基因编辑技术及其在作物育种中的应用与安全管理   总被引：3，自引：0，他引：3  
基因组编辑技术是研究基因功能和对生物体基因进行定向改造的有力工具。随着近几年CRISPR/Cas9技术的快速发展,基因组编辑技术在作物育种领域起着越来越重要的作用。介绍了ZNFs、TALENs和CRISPR/Cas9系统的原理及在作物育种领域的研究进展,重点论述了CRISPR系统相关的变体和该系统在植物基因功能研究和作物育种中的进展。同时,也论述了基因编辑作物的检测方法及不同国家和地区对基因编辑作物的监管态度,重点介绍了美国、欧盟以及我国目前的监管态度,并分析了基因编辑作物存在的问题和发展趋势。为我国基因编辑作物的研究、安全管理和商业化批准提供了参考。  相似文献   

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