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1.
丙型肝炎病毒基因组C区和NS3区基因cDNA的融合及其在大肠杆菌中的表达与初步应用 总被引:3,自引:0,他引:3
通过逆转录(RT)-聚合酶链式反应(PCR),从中国人丙型肝炎病毒(HCV)携带者的血清中扩增并克隆到2段cDNA片段,即HCV基因组C区抗原基因C831cDNA片断(约530bp)和NS3区抗原基因C33ccDNA片段(约860bp)。C33ccDNA片段同C831cDNA片段经连接 肽Ser-Pro-Gly-Ser连接成为基因嵌合体C33c-C831(约1400bp)。C33c-C831基因嵌合体同温控型原核表达载体pBV220重组,构建成表达质粒pBV/C33c-C831,并在大肠杆菌细胞中获得了重组嵌合抗原C33c-CL的表达。通过酶切分析和Western免疫印迹法,对约占菌体可溶性蛋白9%的表达产物做了鉴定。采用TritonX-100和盐析处理,获得粗提表达产物。粗提的表达产物经尿素裂解和离子交换层析纯化,得到可用于检测抗HCV核壳蛋白和抗NS3区抗体的重组嵌合抗原C33c-CL。对C33c-CL做抗原性分析发现,它同时具有完整的C33c抗原和C22抗原的免疫反应活性,完全能替代单纯的C33c和C22抗原。该嵌合抗原在血清学诊断中有重要的应用价值,可望成为新一代HCVEIA诊断试剂的优选抗原。 相似文献
2.
丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究 总被引:21,自引:1,他引:21
用PCR 方法从丙型肝炎病毒(HCV) cDNA 文库中克隆了两段DNA 片段,即HCV 基因组非结构NS3区抗原基因(约0.7 kb)和核心抗原C区抗原基因(约0.6 kb)的cDNA 片段。在两段cDNA 间加入连接肽Ser- Pro- Gly- Ser 的密码子序列,构建成融合抗原基因NS3- C。将该融合基因与衣藻叶绿体基因atpA 的启动子和rbcL 基因的3′末端连接,得到丙肝病毒融合抗原基因NS3- C表达盒,再将该表达盒与选择标记基因aadA 表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体pSS6。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的PCR 和Southern 杂交分析表明,融合抗原基因NS3- C已整合到衣藻叶绿体基因组中。 相似文献
3.
丙型肝炎病毒核心蛋白基因在大肠杆菌内的表达及应用 总被引:2,自引:0,他引:2
将从中国丙肝病人血清中扩增克隆的丙型肝炎病毒核心蛋白基因(408bp)酶切处理后插入表达载体pJLA502内,获得高表达核心蛋白的重组工程菌。将重组菌经42℃热诱导5h,SDS-PAGE分析表明,表达的核心蛋白占菌体蛋白总量的20%。经分子筛和吸附层析纯化后获得的核心蛋白,ELISA检测证实有较好的抗原性和特异性。用表达的核心抗原加用表达的NS_3抗原(C_33)装配的抗-HCV试剂盒,经用标准血清验证及与国外第二代抗-HCV试剂盒比较,证实符合丙肝诊断试剂要求。 相似文献
4.
丙型肝炎病毒核酸疫苗的免疫效果观察 总被引:3,自引:1,他引:2
将丙型肝炎病毒C+E1区基因克隆到不同的真核细胞表达载体pCDNA3.1(+)和pSVL中,通过肌肉注射和皮下注射免疫BALB/C及F1小鼠后,产生了抗HCV/C区抗体。其中核酸疫苗pcDNA3.1-HCV/C+E1的免疫高峰的出现早于pSVL-HCV/C+E1,F1小鼠的抗体应答强于BALB/小鼠。肌肉注射优于皮下注射。 相似文献
5.
作者应用抗HCVNS3区C33c抗原2B6株单克隆抗体和抗HBxAg多克隆抗体,采用ABC法对102例人原发性肝细胞肝癌(PHC)组织进行了HCV及HBV抗原定位研究。HCVC3。抗原及HBxAg在PHC中的阳性检出率分别是81.4%及74.5%,C33c抗原或HBxAg阳性占所检病例94.1%,相同病例二者同时阳性为61.8%。102例PHC中50例有癌旁肝组织,其C33c抗原和HBxAg的阳性检出率分别是62%和92%。HCVC33c抗原定位于肝癌细胞的胞浆内,胞核未见阳性信号。C33c抗原阳性细胞在PHC中呈散在、局灶分布为主,在癌旁肝组织呈弥漫分布为主。本文结果提示HCV感染在PHC的发生中可能起重要作用。 相似文献
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7.
应用原位分子杂交及免疫组分方法,使用地高辛标记HCV5’非编码区探针及抗HCV NS3区C33c单克隆抗体,对35例人原发性肝内胆管细胞癌(PIC)和癌旁肝组织的HCV RNA及其NS3抗原进行检测结果发现HCV RNA在PIC中的阳性率为83%,HCV RNA定位于癌细胞浆中,个别病例并在淋巴细胞、肝窦内皮细胞和枯否氏细胞中发现阳性信号。HCV NS3区C33c抗原在PIC的阳性率为89%,阳性 相似文献
8.
PCR与ELISA检测丙型肝炎病毒的比较李京培王明丽史百芬陆应玉(安徽医科大学230032)丙型肝炎病毒(HCV)感染后,患者血清中可以检测其特异的核酸序列HCV-RNA和抗-HCV等。目前国内临床大多数HCV的实验室诊断主要依靠抗HCV-IgG检查,但急性期抗体检出率仅能达到60%(37.6~82.8%),部分病例可呈阴性反应[1]。说明用ELISA检测HCV有相当部分漏检,不能发现早期患... 相似文献
9.
用人工合成的丁型肝炎病毒抗原(HDV-Ag)肽建立了检测抗HDV-IgM抗体的ELISA方法。本法操作简便、快速、重复性好、特异性强,与抗HAV-IgM'抗HBc-IgM、抗HBs-IgM、抗HCV-IgM、抗CMV-IgM、抗RV-IgM、类风湿因子(RF)及抗核抗体(ANA)阳性血清均不起反应,且可被2-巯基乙醇阻断而不起反应。经初步临床应用,31例正常人血清抗HDV-IgM全部阴性,28例慢 相似文献
10.
本研究通过反转录从丙肝病人血清中克隆了丙型肝炎病毒(HCV)C33c基因,通过DNA合成法又分别合成了编码HCV核心抗原基因,NS4抗原及NS5抗原部分抗原决定簇的基因,利用基因工程手段,在大肠杆菌中表达了含有上述四种抗原的融合蛋白。该融合蛋白具有良好的抗原性和特异性,以该融合蛋白为抗原制备的抗-HCVELIS试剂检测了中国药品生物制品检定所198份标准血清时,其阳性,阴性符合分别达97%和98% 相似文献
11.
丙型肝炎病毒核壳蛋白抗原的化学合成及其在血清学诊断中的应用 总被引:1,自引:0,他引:1
选取丙型肝炎病毒(HCV)核壳蛋白区两个可能含有优势抗原表位的19和16氨基酸的肽殴(C-19肽和C-16肽),利用固相多肽合成技术进行了化学合成。经用反相高压液相层析(HPLC)及脉冲液相多肽测序技术检定合成肽产品的纯度及序列后,以C-19和C-16肽作为包被抗原组装成检测HCV血清抗体的ELISA诊断试剂。用所组装的合成肽试剂检测中国药品生物制品检定所提供的HCV标准参比血清,并比较利用该试剂和美国Abbott实验室生产的HCV第二代EIA诊断试剂平行检测109份献血员血清的结果,表明C-19肽能够特异、重复、灵敏地检出HCV血清抗体,可以作为我国第一代HCV诊断试剂的合成肽抗原。 相似文献
12.
A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity. 相似文献
13.
系统研究了通过霍乱毒素B亚基与HCV的4个抗原决定簇进行基因融合所表达的12种融合蛋白中HCV抗原决定簇的反应原性,探索了以融合蛋白为抗原,进行抗-HCV检测的途径。结果表明,多数融合蛋白中HCV抗原决定簇均能与对应的HCV抗体结合。以融合蛋白95082为抗原研制的抗-HCV ELISA试剂检测122名献血员血清,结果与美国雅培公司抗-HCV ELISA试剂检测的结果完全一致,经药品生物制品检定所检定,其特异性、灵敏度、精密性及稳定性均达到国家卫生部抗-HCV ELISA试剂的暂行检定标准。 相似文献
14.
用含谷胱甘肽转硫酶(GST)基因的pGex-2T为表达载体在大肠杆菌中高效表达了含HCV核心区部分基因片段(约122个氢基酸)的融合蛋白,表达产物C27经测定占菌体总蛋白的30%,C27纯化后,用我国HCV诊断试剂第二代血清参考品为标准与G11核心抗原进行比较,结果显示二者具有相同的总体检出率和近似的抗原活性。因此,推测表达的部分区段基因可能是核心区重要功能区域。另外融合蛋白对提高重组蛋白的抗原活性和分离纯化等均有一定作用。 相似文献
15.
Atsushi Sato Nobuo Ida Mari Ishikawa Kazuhiro Tanahashi Haruji Nakamura Yukihiko Sho Terukatsu Arima Tetsunosuke Kunitomo 《Microbiology and immunology》1993,37(4):295-304
An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non-fused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR. 相似文献
16.
丙型肝炎病毒基因组C区、NS3区、NS4区基因的融合、表达及初步应用 总被引:1,自引:0,他引:1
通过PCR技术从HCV全长基因组中扩增并克隆到Core区、NS4区的部分优势表面抗原肽的基因和C33a基因。再将它们以Core\,CC3c\,NS4的顺序和C33c、NS4的顺序分别拼接融合成融合抗原CCN、CN。片段之间以Gly-Gly-Gly-Gly柔性连接肽连接。经核苷酸序列分析表明,拼接点序列正确。将融合抗原CN、CCN基因分别克隆至T7启动子控制下的表达质粒PET24(a)+,PET22(b)+中,转化至大肠杆菌BL21(DE3)后,经IPTG诱导可高表达分子量约为43kD、58kD的融合抗原,表达产物以包涵体形式存在。表达产物经Ni2+IDA亲和柱纯化后,并对融合抗原CN、CCN的抗原性做了初步的鉴定。 相似文献
17.
He J Xiu B Wang G Chen K Feng X Song X Zhu C Yang X Bai G Ling S Zhang H 《Protein and peptide letters》2011,18(8):839-847
Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection. 相似文献
18.
提高对丙型肝炎患者实验室检测的灵敏度和特异性,对相关人群进行筛查和早期诊断,是控制丙型肝炎病毒(HCV)流行与传播的有效措施。为了建立更为可靠的HCV诊断方法,通过采用PCR方法从J6/JFH12a型病毒中克隆出HCV ns3基因片段,将其连接到p ET-28a载体上,重组载体p ET-28a-ns3转化大肠杆菌BL21(DE3)后诱导表达,以10%SDS-PAGE进行鉴定,获得表达的NS3重组蛋白分子量为72 k Da。将纯化的NS3蛋白免疫BALB/c小鼠,第4次免疫后采集血液并分离血清进行抗体活性鉴定,小鼠抗体效价为1∶256 000。进一步的Western blotting和间接免疫荧光结果显示,以重组NS3蛋白免疫小鼠制备的多克隆抗体可以很好地识别HCV感染Huh7.5.1细胞中的NS3蛋白,为下一步开展单克隆抗体制备和检测试剂盒研制工作奠定了基础。 相似文献
19.
To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven
by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3)
gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed
several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous
mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of
HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with
lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded
within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular
levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star
topology suggesting that a wild-type HCV sequence is maintained, unlike HIV.
Received: 2 November 2000 / Accepted: 1 October 2001 相似文献
20.
Li W Krishnadas DK Li J Tyrrell DL Agrawal B 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(10):6065-6075
Hepatitis C virus (HCV)-specific T cell responses have been suggested to play significant role in viral clearance. Dendritic cells (DCs) are professional APCs that play a major role in priming, initiating, and sustaining strong T cell responses against pathogen-derived Ags. DCs also have inherent capabilities of priming naive T cells against given Ags. Recombinant adenoviral vectors containing HCV-derived Core and NS3 genes were used to endogenously express HCV Core and NS3 proteins in human DCs. These HCV Ags expressing DCs were used to prime and stimulate autologous T cells obtained from uninfected healthy donors. The DCs expressing HCV Core or NS3 Ags were able to stimulate T cells to produce various cytokines and proliferate in HCV Ag-dependent manner. Evidence of both CD4(+) and CD8(+) T cell responses against HCV Core and NS3 generated in vitro were obtained by flow cytometry and Ab blocking experiments. Further, in secondary assays, the T cells primed in vitro exhibited HCV Ag-specific proliferative responses against recombinant protein Ags and also against immunodominant permissive peptide epitopes from HCV Ags. In summary, we demonstrate that the dendritic cells expressing HCV Ags are able to prime the Ag-specific T cells from uninfected healthy individuals in vitro. These studies have implications in designing cellular vaccines, T cell adoptive transfer therapy or vaccine candidates for HCV infection in both prophylactic and therapeutic settings. 相似文献