戊型肝炎病毒Ⅳ型的ORF3及ORF2蛋白的分片段表达、纯化及抗原性分析

目的：对戊型肝炎病毒（HEV）Ⅳ型ORF3的全长基因片段和4个覆盖全长ORF2的互相重叠的基因片段进行了表达，对表达产物进行了纯化及抗原性分析。方法：将O3、FB5、E4、F2－2和E5基因片段分别连接到融合性表达质粒pThioHis，用IPTG进行诱导表达，并用反向、分子筛、离子交换和亲和层析方法进行纯化，用纯化的蛋白分别制备检测抗HEV IgG的诊断试剂，用该试剂检测急性散发性的戊肝病人和非戊肝病人血清。结果：位于ORF2－N端的FB5蛋白在急性期的戊肝病人血清中具有最强的反应性；而且仍有部分被HEV I型或／和Ⅱ型的诊断试剂排除的急性非戊肝病人血清对HEV Ⅳ型的多肽吴阳性反应。结论：为早期诊断戊肝病毒的感染，其诊断试剂应含有HEVⅣ型的ORF2N－端的蛋白片段。     

酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定

目的 对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定。方法 以原核细胞表达的重组HEV ORF2抗原作为对照，应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性；并测定不同保存时间对抗原稳定性的影响。同时，比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性。结果 应用酵母抗原可检测到HEV抗体的最低包被量为12．5ng／ml，可检测到HEV IgM、IgG抗体的血清最大稀释度皆为l：51200重组蛋白与其他类型肝炎患者血清无交叉反应，37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性。各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性，其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍。结论 应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位，具有良好抗原性、特异性和稳定性。提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原。     

一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性

目的以非包涵体形式表达含戊型肝炎病毒(HEV)中和抗原表位的新型HEV重组蛋白,并对其进行鉴定和分析。方法将HEV开放阅读框架2(ORF2)编码452~617位氨基酸的基因片段连接到载体pET28a( ),转化大肠杆菌,获取表达克隆。以Ni-NTA层析柱纯化表达的蛋白,并用SDS-PAGE、Westernblot和直接电镜负染等方法分析鉴定,最后免疫小鼠检测特异性抗体的产生水平。结果表达的重组蛋白天然可溶,相对分子质量(Mr)约为22000,并可形成直径为20nm左右的病毒样颗粒,能与戊型肝炎患者血清发生Westernblot阳性反应,免疫小鼠后可诱导产生高滴度的特异性抗体。体外中和试验显示产生的抗体具有中和HEV的活性。结论原核表达的、含HEV中和抗原表位的、长度仅166个氨基酸的HEVORF2近3′端编码蛋白能够形成病毒样颗粒,而且该新型病毒样颗粒具有良好的抗原性和免疫原性。     

戊型肝炎病毒ORF2 C端抗原片段的表达及其免疫学特性研究

目的　表达戊型肝炎病毒 (hepatitisEvirus ,HEV)ORF2C端 12 8个氨基酸残基的抗原片段ORF2 3 ,并进行其免疫学特性的研究。方法　用pBV2 2 0载体进行抗原的非融合表达 ,包涵体经变性凝胶过滤层析及离子交换层析纯化后透析复性 ,以Westernblot、ELISA、动物免疫与抗原捕获RT nPCR等方法研究其免疫学特性。结果　获得了ORF2 3的高效表达 ,表达量占菌体总蛋白 5 0 %左右 ,纯化后纯度达 98%以上 ,Westernblot表明表达抗原可与戊肝患者阳性血清特异性结合 ;在HEV感染恒河猴实验中用于IgG抗体的检测 ,具有较好灵敏度与特异性 ;用其免疫豚鼠 ,抗体经ELISA法测定效价为 1∶80 0 0 ;用制备的豚鼠抗血清捕获戊型肝炎病毒颗粒 ,经RT nPCR扩增出特异性片段。结论　ORF2 3抗原片段具有HEV抗体识别的抗原表位 ,能够刺激机体产生抗体 ,抗血清具有一定的识别与结合戊型肝炎病毒颗粒的能力     

戊型肝炎病毒Ⅰ型ORF3蛋白的表达、纯化及抗原性分析

目的　表达戊型肝炎病毒 (HEV)Ⅰ型ORF3全长基因片段 ,纯化表达产物并进行抗原性分析。方法　将Ⅰ型HEVORF3基因连接到融合表达载体pThioHisB ,IPTG诱导表达 ;Westernblot分析表达产物的抗原活性 ;用经高效液相色谱纯化的表达产物作为抗原 ,制备血清抗 HEVIgG及抗 HEVIgM诊断试剂 ,用国家参考品进行考核 ;用所得抗原检测临床血清中抗 HEV抗体 ,比较其与Genelabs试剂盒检测结果的差异。结果　含有Ⅰ型pThioHisB/ORF3质粒的菌株表达了相对分子质量(Mr)为 2 6× 10 3 的融合蛋白 ,免疫印迹表明其能与戊型肝炎患者恢复期血清发生特异性反应。国家参考品考核结果显示 ,用表达产物制备的抗HEVIgG诊断试剂阳性符合率为 10 0 % (10 / 10 ) ,阴性符合率为 96 .7% (2 9/ 30 ) ,总符合率为 97.5 % (39/ 4 0 ) ;制备的抗 HEVIgM诊断试剂阳性符合率为 83.3%(10 / 12 ) ,阴性符合率为 10 0 % (2 0 / 2 0 ) ,总符合率为 93.8% (30 / 32 )。与Genelabs公司试剂盒检测结果比较 ,抗 HEVIgG和抗 HEVIgMELISA总符合率分别为 94 .5 %和 87.0 %。结论　本实验所得全长ORF3基因工程表达产物具有良好的抗原性 ,可用于制备抗 HEV诊断试剂。     

4型戊型肝炎病毒ORF2多肽的二聚体特性和抗原性特征

目的 研究4型戊型肝炎病毒(hepatitis E virus,HEV)开放阅读框架2(open readingframe 2,ORF2)编码的多肽二聚体特性和抗原性特征.方法 从患者血清克隆HEV ORF2基因,在大肠杆菌中表达不同长度的ORF2多肽,并表达点突变多肽,纯化后在SDS-PAGE不同条件下分析二聚体特性,同时用Westem blot分析其抗原性特征.结果 由459～607位氨基酸构成的ORF2多肽能形成二聚体,而在氨基端截短13个氨基酸或在羧基端截短13个氨基酸均不形成二聚体,562位天冬酰胺、595位异亮氨酸分别突变后在SDS作用下仅以单体存在.Westem blot显示,抗-HEV仅与二聚体的ORF2多肽反应,而不与单体ORF2多肽或截短肽反应.结论 4型HEV ORF2多肽形成二聚体必须含有459～607位氨基酸,其中562位和595位氨基酸突变能影响二聚体的稳定性.该多肽的抗原性取决于二聚体结构.     

一个能诱生戊型肝炎病毒中和抗体的ORF2编码蛋白短片段

目的：比较戊型肝炎病毒（HEV）第4基因型ORF2编码蛋白片段pN472-C617（aa472～617）和pN477-C613（aa477～613）的免疫原性，找到能诱生HEV中和抗体的ORF2编码蛋白的更短片段。方法：表达和纯化pN472-C617和pN477-C613，分别免疫BALB/c小鼠，以间接ELISA检测免疫血清的抗体效价，并以基于PCR的体外中和试验检测免疫血清的中和活性。结果：pN472-C617和pN477-C613可在大肠杆菌高效可溶性表达，纯化后的重组蛋白能在小鼠体内诱导出高效价的抗体。基于PCR的体外中和试验显示pN472-C617免疫血清可有效中和HEV，阻断其在敏感细胞表面的吸附和细胞内复制；而两端仅比pN472-C617各短5个氨基酸的pN477-C613的免疫血清不具有中和HEV的活性。结论：重组蛋白pN472-C617具有良好的抗原性和诱生中和抗体的能力，是目前文献报道中含有HEV中和抗原表位的最短ORF2编码蛋白片段，可作为重组亚单位候选疫苗用于HEV疫苗的开发。     

戊型肝炎病毒ORF3蛋白研究进展

戊型肝炎病毒(hepatitis E virus,HEV)是戊型肝炎的病原体,是肝炎病毒科戊型肝炎病毒属的惟一成员,无包膜、正义、单链RNA病毒,病毒基因组全长约7.2 kb,由3个开放阅读框ORF1、ORF2和ORF3组成[1],其中ORF1编码与病毒复制、转录相关的酶等非结构蛋白[2-3],ORF2编码660 aa的病毒衣壳蛋白[4],ORF3编码小分子的磷蛋白.ORF2蛋白是公认的病毒抗原,是病毒的主要结构蛋白,对其研究的比较透彻[5].近年来,科学家们对于ORF3蛋白的研究逐渐增多,发现ORF3是一个多功能蛋白,是病毒感染、释放所必需,并参与细胞内的信号转导调控等生物过程[6].     

戊型肝炎病毒嵌合重组蛋白的表达及免疫原性研究

我国是戊型肝炎主要流行区之一，人群感染率达17．2％，且呈上升趋势。由于缺乏有效的病毒体外长期培养系统，阻碍了戊型肝炎病毒(HEV)疫苗的研制和诊断试剂的开发。用基因工程手段表达HEV主要结构蛋白已成为研究的热点。本文报道用原核表达系统表达含HEV主要抗原表位的ORF2和ORF3的嵌合蛋白，并对其免疫原性进行研究。     

重组猪戊型肝炎病毒ORF2区主要抗原域的原核表达及鉴定

目的：在大肠杆菌中表达猪戊型肝炎病毒ORF2区主要结构蛋白,并对其进行血清学鉴定。方法：通过RT-PCR技术从一份猪粪中扩增并克隆戊型肝炎病毒主要的结构基因片段,将该片段插入pET-32a表达载体,在原核系统中融合表达蛋白,Western blot和间接ELISA方法分析该蛋白的抗原性。结果：SDS-PAGE分析结果表明,获得45kD的目的蛋白,该重组蛋白可以与HEV阳性血清反应,而不与阴性血清反应,表明该蛋白具有良好的抗原性。结论：本研究获得了猪戊型肝炎病毒重组抗原,可与HEV阳性血清产生特异性的结合反应,可以作为诊断用的重组蛋白,为研制敏感、特异的HEV诊断试剂盒,行之有效的HEV基因工程疫苗及为HEV感染的预防和临床治疗提供资料。     

Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti‐HEV immunoglobulin M

Following infection with hepatitis E virus (HEV), anti‐HEV immunoglobulin (Ig) M is thought to develop before anti‐HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti‐HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti‐HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N‐terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti‐HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti‐HEV IgM against the N‐ or C‐terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C‐terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N‐ and C‐terminal polypeptides from ORF2 complemented one another in detecting anti‐HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N‐ and C‐terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti‐HEV IgM. J. Med. Virol. 81:1052–1061, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis E virus ORF3 antigens derived from genotype 1 and 4 viruses are detected with varying efficiencies by an anti-HEV enzyme immunoassay

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein product remains unclear but it is able to induce a strong antibody response following HEV infection. Therefore, it has been used in some enzyme immunoassays (EIAs) for detecting anti-HEV antibody. In order to evaluate the difference in antigenicity of HEV ORF3 polypeptides derived from genotypes 1 and 4, two EIAs were developed, based on ORF3 polypeptides from genotypes 1 and 4 HEV. Serial weekly serum samples from two rhesus monkeys vaccinated with ORF3 antigens derived from the genotype 4 ORF3 protein and nine rhesus monkeys experimentally infected with genotypes 1 and 4 HEV were tested for anti-HEV using the assays. HEV ORF3 antigens derived from viruses of genotypes 1 and 4 showed different patterns of reactivity with sera obtained from monkeys immunized with ORF3 antigens or infected experimentally with HEV. The genotype 1 ORF3 polypeptide exhibited stronger reactivity with the sera from monkeys infected with genotype 1 than the genotype 4 ORF3 polypeptide. The genotype 4 ORF3 polypeptide demonstrated stronger reactivity with the sera from monkeys infected with genotype 4 than did the genotype 1 ORF3 polypeptide. The HEV ORF3 polypeptide contains genotype-specific antigens and the antigen-antibody reactions between the same genotypes were stronger than those between different genotypes.     

多型别HCV-E1复合表位抗原研究

目的 设计多型别HCV-E1表位复合免疫原,通过免疫小鼠,探讨其在丙型肝炎治疗性疫苗与诊断试剂研究中的应用.方法 分析比较HCV-E1包膜糖蛋白B细胞表位序列,选取几个代表性基因型的中和性优势抗原表位,再结合泛DR辅助性T细胞表位(PADRE),构建含有不同HCV型别E1表位抗原基因和通用T辅助细胞表位基因的原核表达质粒.结果 成功构建了含有HCV 1a、1b、2a、3a、4a和6a等基因型E1中和性表位以及通用T辅助细胞表位的质粒pBVIL1/E1s-PADRE,该质粒转化大肠杆菌后获得的工程菌,通过诱导培养可以高效表达重组多型别HCV-E1表位复合抗原,所获得的多型别HCV-E1表位复合免疫原可在BALB/c小鼠体内诱发强烈的体液免疫反应,ELISA检测抗体水平可达到1:12 800.结论 新构建的HCV-E1表位复合免疫原具有很好的免疫原性,为进一步研究HCV疫苗奠定了基础.     

Genotyping of varicella zoster virus strains isolated in Mongolia

This study analyzed 50 varicella zoster virus (VZV) samples collected during 2004 to 2007 from patients with VZV infection, who were treated at the National Center of Communicable Diseases, Ulan-Bator, Mongolia. The method based on amplification of specific DNA fragments of the ORF21, ORF22, and ORF50 genes was used, followed by the sequencing and detection of the status of characteristic point mutations in these fragments. The results indicated that the collected samples belonged to genotypes J (62%), M1 (18%), E1 (12%), E2 (4%), and M2 (4%). Moreover, restriction endonuclease polymorphism in ORF 62 for the cleavage site Smal and Mspl, in ORF 38 and ORF 54 for the cleavage site Pstl and Bgll were analyzed. All the samples were Sma- Msp-. All samples with genotype E were Pst+ Bgl-; all samples with genotype M1 and M2 were Pst+ Bgl+. Out of 31 samples with genotype J, 29 and 2 were Pst+ Bgl+ and Pst+ Bgl+, respectively. The study could identify the genotypes of VZV circulating in Mongolia and confirmed the absence of mutations characteristic for the vaccine strain.     

The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA

Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3' end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3' non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3' end of the 504-nt 3' untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101-283, did not interact with these 3' IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.     

Preparation of recombinant polypeptides containing antigenic determinants of hepatitis E virus]

DNA fragments complementary to hepatitis E Burma strain ORF2 and ORF3 obtained by oligonucleotide synthesis were cloned in expressing bacterial system. Recombinant polypeptides isolated from E. coli producer strains, immobilized on solid phase (polystyrene plates and nitrocellulose membranes), are studied in enzyme immunoassay to detect their ability to react with sera of patients with acute viral hepatitis from an Uzbekistan region endemic for hepatitis E. Two polypeptides reacting with the greatest number of sera, containing hepatitis E virus ORF2 and ORF3 gene fragments, were selected for further study of antigenic specificity.     

Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins]

Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E. coli cells, is analyzed. Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control. 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide. No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings. Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen. Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993.