猪流行性腹泻病毒流行株S蛋白高变区B细胞线性表位肽的鉴定

【背景】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)经典毒株与流行毒株S蛋白氨基酸序列比对显示,变异位点主要集中在S蛋白的N端。【目的】鉴定PEDV流行毒株S蛋白高变区(S10A,1-496 aa)的B细胞线性表位肽,特别是流行毒株特异性B细胞表位肽。【方法】以SDS-PAGE割胶纯化大肠杆菌表达的PEDV流行株SD2014 S蛋白高变区片段(S10ASD2014),免疫新西兰大白兔制备多克隆抗体;在E. coli中谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)融合表达覆盖S10ASD2014序列全长且彼此重叠8个氨基酸残基的系列16肽。以制备的抗S10ASD2014多抗为一抗,通过Western blot筛选系列16肽中的阳性反应性16肽,鉴定S10ASD2014上的B细胞线性表位肽;利用抗PEDV经典毒株(CV777)S蛋白高变区片段(S10ACV777)多抗血清鉴定S10ASD2014上的保守性B细胞线性表位肽。【结果】重组表达的S10ASD2014相对分子质量约为72 kD;纯化的S10ASD2014表位肽能被PEDV阳性血清识别。制备的抗S10ASD2014多抗可以识别PEDV DR13弱毒株。利用抗S10ASD2014血清和抗S10ACV777血清从61个GST融合表达的16肽中鉴定到28个阳性反应16肽,其中13个为2种血清共同识别16肽。对24株PEDV不同亚群代表毒株的对应区域进行同源性分析表明2个阳性16肽为保守性表位肽。【结论】确定了PEDV流行株SD2014 S蛋白高变区的B细胞线性表位肽,B细胞线性表位肽,特别是流行毒株特异性表位肽的确定有助于了解S蛋白的结构与功能,为建立以表位为基础的PEDV感染检测方法打下良好基础。     

鹿儿岛被毛孢分生孢子胞外黏液蛋白提取与鉴定

[背景] 分生孢子表层粘液在虫生真菌与昆虫的互作中扮演着重要角色。鹿儿岛被毛孢是寄主专一、孢子表面具粘液层的虫生真菌,但其胞外粘液成分尚不清楚。[目的] 优化鹿儿岛被毛孢分生孢子粘液蛋白的提取方法,对粘液蛋白组分进行质谱鉴定,探究分生孢子胞外粘液的蛋白成分。[方法] 用低浓度还原剂二硫苏糖醇（Dithiothreitol,DTT）对胞外粘液进行温和洗脱并优化提取条件,聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis,SDS-PAGE）检测蛋白提取浓度,用碘化丙啶（Propidium Iodide,PI）染色法检测孢子细胞膜选择通透性,通过肽段酶解、LC-MS/MS数据采集及蛋白质比对分析鉴定蛋白质。[结果] 鹿儿岛被毛孢分生孢子粘液蛋白的最佳提取条件：孢子浓度为5×10⁷ cells/mL,DTT浓度为2 mmol/L,4 ℃提取24 h;粘液蛋白提取后不影响孢子细胞膜通透性。提取的粘液蛋白共鉴定到蛋白质77个,蛋白丰度排前10的蛋白多为分泌性小分子蛋白,包括未注释的蛋白、几丁质酶、脂酶或是与昆虫体壁降解相关的酶类。[结论] 鹿儿岛被毛孢胞外粘液中富含与寄主识别、穿透相关的蛋白质,与被毛孢的粘附、识别功能密切相关。     

猪流行性腹泻病毒S2截短肽的原核表达及其线性B细胞表位区鉴定

【背景】由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的猪流行性腹泻给养猪业造成了巨大的经济损失。PEDV S蛋白可以诱导宿主产生中和抗体。【目的】原核表达PEDV CV777疫苗株S2截短肽(aa:961-1 382)并制备其多克隆抗体;鉴定表达的S2截短肽上的线性B细胞表位区。【方法】将经密码子优化的PEDV S2截短肽编码DNA (s2t)克隆至载体p ET-28a中并转化Escherichia coli BL21(DE3),利用IPTG诱导S2截短肽表达。以经SDS-PAGE切胶纯化的重组S2截短肽免疫新西兰大白兔制备多克隆抗体。在E. coli BL21中GST融合表达覆盖S2截短肽序列全长、彼此重叠8个氨基酸残基的系列16肽。以制备的抗S2截短肽兔血清为一抗,通过Westernblot(WB)筛选系列16肽中的阳性反应性16肽,鉴定S2截短肽上的线性B细胞表位区。【结果】重组PEDV S2截短肽的相对分子质量约为50 kD;诱导4 h表达量最高,且主要形成包涵体。WB结果显示,纯化的S2截短肽能被猪抗PEDV血清识别;以纯化的S2截短肽免疫新西兰大白兔制备多抗血清,ELISA法检测抗体效价位于1:25 600-1:102 400之间。免疫组化和间接免疫荧光分析均表明,制备的多抗血清可以识别Vero细胞培养的PEDV DR13弱毒株。以制备的多抗血清通过WB从52个GST融合表达的16肽中鉴定到11个阳性反应性16肽。WB分析显示,得到的阳性反应性16肽都可以被猪抗PEDV血清识别。鉴定到的阳性16肽在S2截短肽上形成4个线性B细胞表位区(aa:969-984;1 065-1 096;1 225-1 280;1 361-1 382)。【结论】高效价抗PEDV S2截短肽多克隆抗体的制备和S2截短肽上线性B细胞抗原表位区的确定有助于了解S蛋白的结构与功能,为建立有效的PEDV检测方法奠定了基础。     

B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

稻田除草剂二氯喹啉酸降解菌15#的分离、鉴定及降解特性

[背景] 二氯喹啉酸（Quinclorac,QNC）是一种高选择性、激素类、低毒性除草剂,主要用于防治稻田稗草,持效期长,易于在土壤中积累而影响后茬作物的生长发育,而且环境中残留的QNC可对动物生长发育产生不良影响,并影响微生物的群落结构和丰度。[目的] 从稻田土壤中分离筛选出一株可降解除草剂QNC的菌株,鉴定并明确其降解特性。[方法] 通过形态学、生理生化试验、磷脂脂肪酸（Phospholipid Fatty Acid,PLFA）微生物鉴定、16S rRNA基因测序及分析鉴定菌株。通过单因素实验探究菌株的降解特性。[结果] 筛选得到一株编号为15^#的QNC降解菌,被鉴定为无色杆菌属菌株（Achromobacter sp.）。降解特性研究结果表明,菌株15^#的最佳培养条件为：30℃、pH为6.0、初始QNC浓度为100 mg/L、接种量为7%、添加质量分数为0.1%的酵母浸粉、氮源为蛋白胨,在此条件下培养21 d后QNC的降解率可达43.0%。[结论] 筛选到降解QNC新菌株并为该菌株的进一步研究提供理论基础。     

重组人骨形成蛋白-3的克隆及其在大肠杆菌中的表达

骨形成蛋白(Bone Morphogenetic Protein,BMP)是一类能诱导异位骨及软骨形成,并在动物的发育和分化中起作用的蛋白质^[1,2,3]。自Urist及其同事发现骨形成蛋白以来⁴。已对8种人的BMP进行了克隆,除BMP-1外^[5],BMP-2至BMP-8均与TGF-β家族相关,它们能诱导细胞分化,促进骨、软骨及牙本质的形成^[1,6]。并在发育、分化和形成过程中起重要作用。最新的研究认为BMP-1是一种胶原蛋白酶^[7],进一步揭示了BMP家族成员的生物学作用。人的BMP-3基因定位于第4染色体上,BMP-3蛋白由472个氨基酸组成,包括N端的信号肽、中间的前肽及C端的成熟肽三部分。BMP-3的C末端与MBP-2A及BMP-2B有49％的序列相同^[5]。本实验室曾检测了BMP-3和BMP-5在不同 组织和细胞中的表达情况,发现它们在一些与骨形成无关的组织和细胞中均有表达,说明了BMP在动物和人中有着其他重要的作用^[8]。在此基础上,我们对BMP-3进行了克隆及在大肠杆菌中高效表达BMP-3-GST融合蛋白,并用Western印迹证明了其活性。     

非核糖体肽Skyllamycin B生物合成中I型硫酯酶底物杂泛性的初步研究

[背景] Skyllamycins是一类从链霉菌中发现的具有血小板生长因子抑制和生物膜抑制作用的非核糖体肽类,其环肽环合反应是由非核糖体肽合成酶中的硫酯酶功能域催化完成。[目的] 克隆和表达Skyllamycin非核糖体肽合成酶最后一个模块中的硫酯酶（Skyxy-TE）基因,合成Skyxy-TE底物类似物,通过体外催化实验表征Skyxy-TE的底物杂泛性。[方法] 采用Ligation Independent Cloning（LIC）方法,从一株含有Skyllamycin B生物合成基因簇的链霉菌Streptomyces sp.PKU-MA01239中克隆和表达skyxy-TE,通过镍离子柱亲和层析纯化Skyxy-TE。运用固相多肽合成法合成2个底物类似物1和2,进行Skyxy-TE的体外催化实验。[结果] 通过对Skyxy-TE的表达纯化,获得了纯度较好的可溶性蛋白;通过固相多肽合成,得到了能够模拟Skyllamycin B底物类似物的化合物1和2,硫酯酶蛋白体外催化化合物1和2得到了化合物3和4,化合物3和4通过核磁共振和高分辨质谱确认为环肽。[结论] Skyllamycin B生物合成中Skyxy-TE表现出一定的底物杂泛性,可以识别底物类似物催化环化反应,该研究为将来利用化学-酶联法制备更多环肽类似物提供了依据。     

腺相关病毒载体工程毒株精细化纯化

[背景] 大量制备高纯度的重组腺相关病毒（Recombinant Adeno-Associated Virus,rAAV）,是以腺相关病毒（AAV）为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。[目的] 利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。[方法] 对组装rAAV的3种质粒用HiPrep^TM10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoad^TM10Q和HiLoad^TM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。[结果] HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到（2.46±0.37）×10¹² TCID₅₀/mL （MOI=1.0×10⁴）。[结论] 通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。     

基于铁氧化/还原菌改善黄铁矿-水草酸解液体系下低品位辉铜矿生物浸出过程

[背景] 高效的生物浸出与微生物介导活跃的铁硫代谢紧密关联,低品位辉铜矿（Cu₂S）铁代谢匮乏严重制约其效能。[目的] 强化铁硫代谢及“接触”机制改善低品位辉铜矿生物浸出。[方法] 基于自主筛选的嗜酸杆菌属（Acidiphilium sp.）及双层平板筛选的嗜铁钩端螺旋菌（Leptospirillum ferriphilum）,与硫氧化菌喜温嗜酸硫杆菌（Acidithiobacillus caldus）协作,加以Fe²⁺/Fe³⁺-黄铁矿-纤维质废弃物酸解液（废-废资源利用）干预,系统分析浸出生化参数差异性。[结果] 扫描电子显微镜（Scanning Electron Microscope,SEM）结果表明矿渣表面大量微孔及坑壑,表明活跃的菌体作用;傅立叶变换红外光谱仪（Fourier Transform Infrared Spectroscopy,FTIR）揭示N-H、C=O、O-H等键与胞外聚合物（Extracelluler Polymer Substance,EPS）紧密相关,S=O、C-O-S等吸收峰波动表明更剧烈的硫代谢;激光共聚焦扫描显微镜（Confocal Laser Scanning Microscope,CLSM）结果表明优化体系呈现更多附着细胞及EPS,为“接触”机制奠定基础,浸出40 d游离/附着细胞量分别提高2.51倍及5.73倍,最大比生长速率（μ_max）出现时间提前1.5-5.3 d,最高浸出率达67.6%。[结论] 铁氧化/还原菌及外源含铁物质干预强化浸出体系铁硫代谢加速矿物溶解,酸解液促进铁元素循环及菌体生长,附着细胞及EPS分泌增多强化“接触”机制从而有效改善浸出微环境和效能。     

谷物蛋白对白酒发酵过程中微生物群落及其代谢多样性的调控

[背景] 在白酒发酵过程中,原料中的谷物蛋白可为微生物的生长提供氮源等营养物质,进而形成多种代谢产物。谷物蛋白可分为清蛋白、球蛋白、醇溶蛋白和谷蛋白。然而,谷物蛋白对微生物多样性及其代谢产物多样性的调控尚不明确。[目的] 揭示白酒发酵过程中与微生物多样性及其代谢产物多样性显著相关的关键谷物蛋白种类及其调控作用。[方法] 通过Osborne法测定不同品种高粱中谷物蛋白的组成;采用多组学联用技术解析4种高粱在发酵过程中的微生物菌群多样性及代谢产物多样性;通过模拟发酵揭示原料中影响微生物群落及其代谢多样性的关键蛋白。[结果] 4种高粱中的谷物蛋白组成存在显著差异（ANOSIM：R=0.85,P=0.001）;4种高粱在发酵第5天时,S4高粱的细菌多样性显著（P<0.05）高于其他3种高粱,S3高粱中微生物的代谢产物多样性显著（P<0.05）高于其他3种高粱;清蛋白和球蛋白含量与发酵第5天的优势细菌多样性（R²=0.34,P<0.05;R²=0.58,P<0.05）和代谢产物多样性呈显著正相关（R²=0.58,P<0.05;R²=0.36,P<0.05）,被定义为关键蛋白;模拟发酵实验验证了优势细菌多样性和代谢产物多样性可随着2种关键蛋白即清蛋白和球蛋白含量的升高而升高。当清蛋白含量在3.0 g/L时,优势细菌多样性及代谢产物多样性可分别达到0.72和0.65;当球蛋白含量在3.0 g/L时,优势细菌多样性及代谢产物多样性可分别达到0.66和0.81。[结论] 研究揭示了酿造原料中的清蛋白和球蛋白对发酵过程中细菌多样性及代谢产物多样性的调控作用,为提高白酒发酵的可控性及质量提供了依据。     

HMCES蛋白促进猪流行性腹泻病毒在Vero细胞中的复制

【目的】鉴定能够调控猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)复制的关键宿主蛋白。【方法】利用LC-MS/MS技术结合串联质谱标签(tandem mass tag,TMT)，分析PEDV感染Vero细胞36 h后和未感染组的蛋白组学差异。鉴定筛选了114个显著差异表达蛋白，其中宿主胚胎干细胞特异性5-羟甲基胞嘧啶结合蛋白(5-hydroxymethylcytosine binding,ES cell-specific protein,HMCES)显著上调。进一步构建HMCES真核表达质粒，通过蛋白免疫印迹和实时荧光定量PCR检测过表达HMCES对PEDV复制的影响；合成针对HMCES基因的特异性si RNA，利用Western blotting和RT-q PCR检测si RNA对HMCES表达的干扰效果及HMCES被干扰后对PEDV复制的影响。【结果】过表达HMCES能显著促进PEDV在Vero细胞中复制，并且复制水平随着HMCES的剂量递增呈现剂量依赖式增加；si RNA-341下调内源性HMCES表达进而抑制PEDV复制。【结论】H...     

Identification of a novel linear B-cell epitope in the M protein of avian infectious bronchitis coronaviruses

This report describes the identification of a novel linear B-cell epitope at the C-terminus of the membrane (M) protein of avian infectious bronchitis virus (IBV). A monoclonal antibody (MAb) (designated as 15E2) against the IBV M protein was prepared and a series of 14 partially-overlapping fragments of the IBV M gene were expressed with a GST tag. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using MAb 15E2 to identify the epitope. A linear motif, ¹⁹⁹FATFVYAK²⁰⁶, which was located at the C-terminus of the M protein, was identified by MAb 15E2. ELISA and western blotting also showed that this epitope could be recognized by IBV-positive serum from chicken. Given that 15E2 showed reactivity with the ¹⁹⁹FATFVYAK²⁰⁶ motif, expressed as a GST fusion protein, in both western blotting and in an ELISA, we proposed that this motif represented a linear B-cell epitope of the M protein. The ¹⁹⁹FATFVYAK²⁰⁶ motif was the minimal requirement for reactivity as demonstrated by analysis of the reactivity of 15E2 with several truncated peptides that were derived from the motif. Alignment and comparison of the 15E2-defined epitope sequence with the sequences of other corona-viruses indicated that the epitope is well conserved among chicken and turkey coronaviruses. The identified epitope should be useful in clinical applications and as a tool for the further study of the structure and function of the M protein of IBV.     

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.

Methods and Results

To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope ¹⁷³LPAPTS¹⁷⁸ is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.

Conclusions and Significance

We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.     

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

Background

The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.

Methods and Results

This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched ²¹³SVQYHPL²¹⁹ of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that ²¹³SVQYHPL²¹⁹ was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.

Conclusions and Significance

We identified ²¹³SVQYHPL²¹⁹ as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101) or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.     

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the ²²²⁶DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The ²²²⁶DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.