HCV核心抗原C33肽单克隆抗体的研制与初步鉴定

用ＨＣＶ核心抗原（３３肽）与鼠血清白蛋白交联后免疫ＢＡＬＢ／ｃ小鼠，用杂交瘤技术获得有实用价值的２株ＭｃＡｂ。此两株ＭｃＡｂ除与免疫抗原有较强的抗原－抗体反应外，与ＨＣＶ核心抗原ＣＰ１０也有很好的反应；但与ＨＣＶＮＳ３、ＮＳ５及核心区ＣＰ９抗原无反应性。在竞争ＥＬＩＳＡ中，对抗ＨＣＶ－ＩｇＧ阳性血清有较好的抑制作用。将此两株ＭｃＡｂ用于慢性丙型肝炎病人肝活检免疫组化研究，获得较为满意的结果。     

重组蛋白及合成肽抗原在GBV-C／HGV感染诊断中的应用

GBV-C是近几年刚发现的新病毒，对其致病性等方面的研究还不是很清楚。其诊断主要依赖于RT-PCR检测病毒RNA，但不适于临床常规检测及大面积筛查。以重组蛋白和合成肽作抗原检测血清中抗GBV-C抗体更适合于GBV-C感染的诊断和流行病学调查，有望得到广泛的应用。本文从GBV-C／HGV抗原表位研究及重组蛋白、合成肽在GBV-C感染诊断学和流行病学中的应用及其意义作一综述。     

人类白细胞抗原分子遗传中的基因重组

深入研究人类白细胞抗原（human leucocyte antigen，HLA）基因重组的分子生物学特性，对于揭示其等位基因多样性形成的演化机理以及人体所具有的抵抗多样致病微生物的防御能力具有重要意义。国外学者通过家系调查研究、统计学分析等方法估算了HLA的重组频率，证实了一些重组发生的热点，并且对重组的单倍型特异性、序列基序特异性、性别特异性等进行了深入研究。本文就HLA重组的交换频率、重组热点以及其它特性的研究现状作一综述。     

弓形虫多表位基因重组抗原在弓形虫免疫检测中的应用

目的评价弓形虫多表位基因重组抗原在弓形虫感染诊断中的效果，探索多表位抗原在弓形虫免疫检测中的应用前景。方法以Ni—NTAAgarose和割胶电渗两步纯化法。获取弓形虫多表位基因的原核表达纯化产物rMAG，以适宜浓度的rMAG包被ELISA微孔板，分别检测弓形虫急性和慢性感染血清，评价试剂盒检测的敏感性和特异性。结果经Ni—NTAAgarose和割胶纯化，得到了纯度为95．86％的弓形虫多表位重组可溶性抗原。以3μg／ml的抗原浓度包被ELISA微孔板，对兔和小鼠的急慢性弓形虫感染血清进行了检测。结果检测小鼠血清141份，其中慢性感染弓形虫小鼠血清117份、正常小鼠血清24份，检测体系的敏感性为88．88％，特异性为91．67％，一致性为89．36％。检测兔血清24份，其中急性感染弓形虫兔血清18份，正常兔血清6份，阳性血清检出率为94．4％，总一致性为91．5％。结论以弓形虫多表位重组抗原构建的ELISA试剂盒既可以检测弓形虫急性感染血清．又可以检测弓形虫慢性感染血清．具有一定的应用前景。     

TpN17重组抗原的表达及在梅毒血清学诊断中的初步应用

目的用基因工程技术表达梅毒螺旋体TpN17重组抗原,并建立操作简便、特异性好、敏感性高的梅毒血清抗体间接酶联免疫吸附试验(ELISA)检测。方法采用PCR技术扩增梅毒螺旋体TpN17基因,再进行T-A克隆及测序,然后亚克隆到原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白包被于反应板,建立检测血清中梅毒抗体的ELISA间接检测法,利用该法与梅毒螺旋体血凝试验(TPHA)、甲苯胺红不加热血清试验(TRUST)同时检测血清样品,对其结果进行比较研究。结果TpN17重组蛋白在大肠杆菌BL21中得到稳定表达,并成功建立了检测血清中梅毒抗体的ELISA间接检测法及试剂。对135例梅毒可疑患者血清进行检测,ELISA、TPHA和TRUST的检出阳性率分别为82.96%、98.52%和71.11%,而TRUST测出的8例可疑样品及31例阴性样品中,TPHA结果全部阳性、而ELISA只有2例阴性。结论TpN17重组抗原ELISA试剂在特异性和敏感性上明显优于TRUST法。     

自身抗原PDC-E2融合蛋白在大肠杆菌中的表达及免疫学鉴定

目的：用基因工程的方法克隆表达丙酮酸脱氢酶复合体E2（PDC-E2）融合蛋白,以用于人原发性胆汁性肝硬化（PBC）的早期发现和临床诊断.方法：针对PDC-E2的cDNA序列设计引物,从正常人的淋巴细胞中提取RNA,通过反转录PCR方法扩增得到相应的基因片段,经测定序列验证后插入表达载体pET28a（＋）,构建重组表达载体pET28a（＋）-PDC-E2,转化大肠杆菌BL21（DE3）后诱导表达蛋白质.表达蛋白经SDS-PAGE、Western blot鉴定.结果：经核苷酸序列测定和酶切鉴定结果表明,成功地构建了PDC-E2重组质粒.IPTG诱导表达后,获得PDC-E2融合蛋白.经免疫学鉴定,重组抗原片段具有抗线粒体抗体二亚型（AMA-M2）的免疫原性.结论：获得PBC特异性靶抗原的多肽片段,为PBC的早期发现和临床诊断提供有力工具.     

重组蛋白及合成肽抗原在戊型肝炎病毒感染诊断中的应用

电于HEV体外培养一直未获成功，以重组蛋白或人工合成肽作为抗原来检测抗-HEV巳成为戊肝实验室诊断及流行病学调查的主要手段。本文从HEV抗原表位的研究、重组蛋白和合成肽在HEV感染诊断中的应用及其诊断学意义和存在的问题几个方面作一综述。     

HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答试验

目的：观察一种结构新颖的HCV融合抗原DNA疫苗在BALB/c小鼠的免疫效果，探讨其用于防治丙型肝炎的可行性。方法：用重叠延伸PCR拼接编码小鼠IgG kappa链信号肽和通用型辅助性T细胞表位PADRE的DNA片段，PCR分别扩增HCV核心抗原基因和包膜E2抗原基因，将3段基因插入真核表达载体pcDNA3.1，构成重组表达质粒pST-CE2t，转染COS7细胞，免疫组化检测HCV抗原的表达。将pST-CE2t和HCV核心抗DNA疫苗pcDNA3.1core分别肌肉注射接种BALB/c小鼠，检测小鼠的血清抗体、T细胞增殖和CTL反应。结果：pST-CE2t可在COS7细胞内表达HCV核心抗原和E2抗原，接种于BALB/c小鼠能有效诱导体液和细胞免疫应答，其中抗HCV核心抗原免疫应答的强度明显超过pcDNA3.1core，且更趋向于TH1型免疫应答。结论：pST-CE2t对于丙型肝炎的防治有潜在的应用价值。     

自身抗原Ro60kD重组融合蛋白的纯化和鉴定

将表达Ro60kD GST融合蛋白的重组体导入大肠杆菌JM109 中表达重组抗原, 经GST亲和层析柱纯化后用标准抗Ro血清鉴定, 经免疫印迹法证实, 纯化重组蛋白具有Ro 抗原性, 该抗原可用于临床检测及研究抗原表位与疾病的相关性     

人干燥综合征A抗原克隆表达与重组蛋白及其临床应用研究

克隆人干燥综合征A抗原基因(SSA-60kD),为SSA抗原的表达和使用重组抗原建立ELISA法用于自身抗体的临床检测.根据GenBank中检索到的人SSA-60kD cDNA序列,在5'和3'非编码区设计特异性引物,提取人源HeLa细胞总RNA作为模板,经RT-PCR扩增SSA-60kD抗原cDNA.PCR产物纯化后连接至载体PET-30a,导人大肠杆菌DH5,构建重组质粒PET-30a-sSA-60kD.对后者进行酶切鉴定、测序和分析.在大肠杆菌中表达,产生重组融合蛋白.RT-PCR扩增产物为1632bp.PET-30a-SSA-60kD经EcoR I和V双酶切证实含目的基因片段.序列分析提示与GenBank中一致.利用金属螯合亲和层析法和纯化,得到了高纯度的SSA-60kD重组蛋白.成功克隆人SSA-60kD基因,并构建PE-30a-ssA-60kD,建立了ELISA方法,检测了风湿病患者血清中抗SSA-60kD自身抗体.     

Immunodetection of a Hepatitis C Virus (HCV) Antigen and Th1/Th2 Cytokines in Cerebrospinal Fluid of Meningitis Patients

Abstract

Infection with hepatitis C virus (HCV) has become the most important public health problem in Egypt. HCV infection has been implicated in diseases of the central nervous system. Cerebrospinal fluid (CSF) and serum samples from 91 patients with meningitis (62 males and 29 females, mean age of 37 years) were investigated. Anti‐HCV antibodies and HCV antigen were evaluated in patients CSF and serum using enzyme linked immunosorbent assay. The levels (mean?±?SD?pg/ml) of Th1 cytokines (IFN‐γ and TNF‐α) and Th2 interleukines (IL‐10 and IL‐4) were also determined. The anti‐HCV antibodies were detected in high percentages both in CSF samples (71%) and in sera (90%). Also, the HCV antigen was detected in about 60% of tested CSF and serum samples. The levels of IFN‐γ and IL‐10 cytokines were significantly higher (P?<?0.05) in both serum and CSF of patients positive for HCV antigen than those negative. HCV antigen was detected in the CSF of meningitis patients with a significant upregulation of Th1 and Th2 responses. The high incidence of HCV infection may draw light on the etiological role of HCV in the pathogensis of meningitis diseases in our study group.     

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users

BackgroundHepatitis C virus (HCV) core antigen is a serological marker of current HCV infection.ObjectivesThe aim of this study was mainly to evaluate the performance characteristics of the ARCITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users.Study designA total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test.ResultsHCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9–94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959.ConclusionsHCV core antigen testing may be reliably used to identify current HCV infection.     

丙型肝炎病毒抗体与核心抗原联合检测的诊断效能研究

目的 探讨丙型肝炎病毒抗体(HCV-Ab)与核心抗原(HCV-cAg)联合检测对丙型肝炎的诊断效能.方法 经重组免疫印迹试验确证的92例阳性标本,81例阴性对照标本,同时进行HCV-Ab和HCV-cAg检测,分析HCV-Ab检测法、HCV-cAg检测法及两者联合检测法的敏感性、特异性和ROC曲线下面积.结果 HCV-Ab检测法敏感性为79.3％,特异性为93.8％;HCV-cAg检测法敏感性为87.0％,特异性为90.1％;HCV-cAg与HCV-Ab联合检测法敏感性为92.4％,特异性为88.9％.三种试验比较,敏感性差异有统计学意义(P＜0.05),特异性差异无统计学意义(P＞0.05).三种方法ROC曲线下面积分别为0.866、0.885和0.906.HCV-cAg与HCV-Ab联合检测法,诊断准确性较高.结论 HCV-cAg与HCV-Ab检测方法的联合应用对于丙型肝炎的诊断效能较高.     

Protective effect of HLA-B57 on HCV genotype 2 infection in a West African population

Recovery from Hepatitis C virus (HCV) infection is considered infrequent (<20%) in western populations but reaches 50% in West Africa where genotype 2 infection is predominant. To investigate the role of cellular immune responses and host genetics in this phenomenon, samples from 104 Ghanaian blood donors reactive with anti-HCV assays were collected between 2000 and 2005. HCV antibody was confirmed by Western blot using genotype 2 recombinant core, E2 and NS3 proteins. Viral load and genotype were determined. Samples were stratified into 37 chronic, 35 recovered infections and 32 false positive. Eighty-one percentage of subjects with chronic infection (RNA positive) carried genotype 2 HCV. Cellular immune response was investigated in 35 frozen peripheral blood mononuclear cell (PBMC) samples suitable for interferon-gamma ELISPOT assay. Twelve out of 24 confirmed recovered, 1 out of 5 chronically infected and none of the 6 false-positive controls reacted to recombinant proteins. HLA-A, -B and -DR types were determined by DNA methodology. HLA-B*57 was significantly more frequent in the group which had recovered from HCV infection compared with chronically infected subjects (P = 0.0053, OR = 8.02). In conclusion, it is hypothesized that the dominance of genotype 2 HCV strains may be an important factor explaining the high rate of recovery from HCV infections in Ghana via an efficient contribution of HLA-B*57 which is relatively frequent in the population.     

HCV核心蛋白在HepG2细胞中对COX-2表达的影响

目的研究丙型肝炎病毒(HCV)核心蛋白是否影响环氧化酶-2(COX-2)的表达。方法用PCR从含HCVH77株全长基因组序列质粒中扩增HCV核心蛋白基因,并克隆至pcDNA3.1载体中,构建HCV核心蛋白基因的真核表达载体HCV-C/pcDNA3.1。用HCV-C/pcDNA3.1和含COX-2启动子的荧光素酶报告载体COX2pro1.5kb/luc瞬时共转染HepG2细胞,测定萤火虫荧光素酶的活性,并用Westernblot检测COX-2蛋白的表达水平。结果成功地构建了HCV-C/pcD-NA3.1重组质粒。瞬时转染后的HepG2细胞中,COX2启动子荧光素酶的活性显著增强。Westernblot检测,发现COX-2的表达明显升高。结论HCV核心蛋白在HepG2细胞中激活COX-2启动子,并且明显诱导COX-2的表达,为进一步研究COX-2与HCV致病性的关系提供了新的实验依据。     

酶联法抗-HCV和HCV-cAg联检对丙型肝炎实验室诊断的应用价值

目的 探讨联合丙型肝炎病毒核心抗原(HCV-cAg)及抗-HCV抗体酶联免疫检测对丙型肝炎实验室诊断的应用价值.方法 289份血清标本按抗-HCV阳性、弱阳性、阴性及HCV-cAg阳性、弱阳性及阴性分组,用PCR扩增法检测其HCV RNA含量.结果 单项抗-HCV阳性及弱阳性标本其HCV RNA检测阳性符合率分别为87...     

鼠抗丙型肝炎病毒ns－5区单克隆抗体的研究

用ＨＣＶ非结构区ｎｓ－５合成多肽抗原交联后免疫小鼠，成功地建立了３株抗ｎｓ－５单克隆抗体（ＭｃＡｂ），经检测这３株ＭｃＡｂ属于同一位点，与其它区域无明显交叉反应，具有高度特异性。ＭｃＡｂ的研制为检测ｎｓ－５抗原奠定了一定基础。     

HCV core antigen testing in HIV- and HBV-coinfected patients, and in HCV-infected patients on hemodialysis

Background

A quantitative HCV core antigen (HCVcoreAg) immunoassay has been developed for the confirmation of viremia in patients with hepatitis C.

Objectives

We evaluated the correlation of HCV RNA and HCVcoreAg in different patient populations without HCV-specific treatment: HIV/HCV-coinfection, HBV/HCV-coinfection, and patients with end-stage renal disease.

Study design

HCVcoreAg was quantified by a fully-automated immunoassay. Correlation of HCVcoreAg with HCV RNA was studied cross-sectionally in HIV/HCV- and HBV/HCV-coinfected patients, as well as before and after hemodialysis in patients with end-stage renal disease.

Results

A concordant positive or negative test result for both HCV RNA and HCVcoreAg was observed in 68 of 71 (96%), 55 of 57 (96%), and in 109 of 109 (100%) samples of patients with HIV- or HBV/HCV-coinfection, and patients undergoing hemodialysis, respectively. HCVcoreAg showed high correlation with HCV RNA in samples from HIV/HCV-coinfected patients and HCV-infected patients undergoing hemodialysis (r = 0.97 and r = 0.94, p < 0.001). There was no overall correlation between HCVcoreAg and HCV RNA in HBV/HCV-coinfected individuals (r = 0.04, p = 0.822). Excluding patients with HCV RNA to HCVcoreAg ratios below 100 and above 10,000 kIU/fmol led to improved correlation (r = 0.53; p = 0.02), but remained worse than for the other cohorts. Overall, HCV RNA to HCVcoreAg ratios did not differ significantly between the different patient populations, though variation tended to be higher in HBV/HCV-coinfected patients. Patients with lower HCV RNA levels tend to have lower HCV RNA/HCVcoreAg ratios.

Conclusions

HCVcoreAg represents a reliable marker of viral replication showing a good correlation with HCV RNA in various patient populations, with some limitations in HBV/HCV-coinfection.