人源抗丙型肝炎病毒包膜蛋白E2单链抗体的研究

目的 研制抗丙型肝炎病毒包膜蛋白E2（HCVE2）的人源噬菌体单链抗体，并探讨其临床价值。方法 以重组的HCVE2为固相抗原，利用亲和筛选的原理，从噬菌体抗体库中经过5轮“吸附-洗脱-扩增”的筛选过程及酶联免疫吸附试验（ELISA）和DNA序列分析。获得HCVE2的人源单链抗体；用该抗体对10例石蜡包埋的丙型肝炎患者肝组织进行免疫组织化学鉴定。结果 ELISA结果表明，制备的HCVE2人源单链抗体（吸光度值A450为1.88)能与HCVE2抗原特异性结合，免疫组织化学结果表明，该抗体能够特异性识别丙型肝炎患者肝组织HCVE2抗原，与正常肝组织及乙型肝炎病毒（HBV）抗原均无交叉反应。结论 此法制备的单链抗体亲和性好，特异性强，且制备方法简便，周期短，为HCVE2病原的检测提供了新的有效的试剂。     

抗丙型肝炎病毒非结构蛋白NS5A单链抗体的制备及免疫组织化学研究

目的：研制抗丙型肝炎病毒(HCV)非结构蛋白NS5A的人源噬菌体单链可变区抗体，并探讨其在临床病理学诊断中的应用价值。方法：以大肠杆菌表达的重组HCV非结构蛋白NS5A为固相抗原，利用抗原—抗体亲和性结合的原理，从半合成的人源可变区噬菌体抗体库中经过5轮“吸附—洗脱—扩增”的筛选过程及酶联免疫吸附试验(ELISA)和DNA序列分析，获得HCV NS5A的人源单使抗体；用该抗体对772l转染全长HCV cDNA后筛选的阳性克隆细胞中的HCV NS5A抗原进行免疫组化鉴定。结果：ELISA结果表明，制备的HCV NS5A人源单链抗体能与HCV NS5A抗原特异性结合；免疫组化结果表明，该抗体能够特异性识别HCV NS5A抗原，与正常肝细胞无交叉反应。结论：此法制备的单链抗体亲和性好，特异性强，且制备方法简便，周期短，为HCV NS5A病原的检测提供了新的有效的检测手段。     

丙型肝炎病毒非结构蛋白NS5抗独特型人源单链可变区抗体的筛选与鉴定

目的：制备丙型肝炎病毒（HCV）非结构蛋白NSS（NS5）的抗独特型单链可变区抗体scFv(抗－Id scFv)，为研制HCV NS5的抗－Id scFv疫苗奠定基础。方法：采用噬菌体表面展示技术，将HCV NS5单克隆抗体固相包被于Nunc板，从噬菌体单链可变区抗体库中经过5轮“吸附－洗脱－扩增”筛选过程，随机挑选80个克隆，利用酶联免疫吸附试验（ELISA）、交叉反应和竞争抑制实验，对其进行免疫学检测，获得与HCV NS5单克隆抗体结合活性较强的抗－Id scFv阳性克隆，并对HCV NS5特异性抗－Id scFv的编码序列进行序列测定分析。结果：筛选得到的HCV NS5抗－Id scFv片段由786bp组成，具有结合HCV NS5单克隆抗体的生物学活性和特异性。结论：用噬菌体抗体库技术能够成功地获得HCV NS5的抗－Id scFv。本实验结果为开展用抗－Id scFv防治丙型肝炎的研究创造了条件。     

可溶性HCV非结构蛋白NS3人源单链可变区抗体在大肠杆菌中的表达

目的 获得可溶性的抗丙型肝炎病毒(HCV)非结构蛋白NS3的人源单链可变区抗体(ScFv),为得到纯度高、活力强的NS3 ScFv和进一步HCV的治疗奠定基础。方法 采用噬菌体表面展示技术,以重组的HCV非结构蛋白NS3为包被抗原,从噬菌体单链可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性较强的HCVNS3人单链抗体ScFv片段克隆,并对其进行DNA序列及免疫活性测定。从噬菌体抗体阳性克隆中提取质粒转化琥珀突变非抑制型大肠杆菌HB2151;IPTG诱导表达HCV NS3可溶性单链抗体;ELISA和dot blot检测其抗原结合特异性。结果 克隆了HCV NS3的单链可变区抗体基因,经DNA酶切和序列分析表明,该抗体基因由747个碱基组成。ELISA和dot blot结果表明,在大肠杆菌HB2151中经IPTG诱导表达的可溶性HCV NS3的单链可变区抗体,具有结合丙型肝炎病毒非结构蛋白NS3的特异性和免疫活性。结论 克隆、鉴定并在大肠杆菌HB2151中表达了可溶性的ScFv-NS3。     

丙型肝炎病毒核心蛋白人源单链可变区抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（HCV）核心蛋白的人源单链可变区抗体（ScFv)。方法 采用噬菌体表面展示技术，以重组的HCV核心蛋白为包被抗原，从噬菌体单链可变区抗体库中经过3轮“吸附－洗脱－扩增”筛选过程，获得抗原结合活性较强的HCV核心蛋白特异性人源单链可变区抗体片段阳性克隆，并对其进行免疫学及核苷酸序列测定。结果 筛选得到的ScFv片段具有抗HCV核心蛋白的特异性，基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征。结论 利用噬菌体抗体库技术，成功获得HCV核心蛋白的特异性人源单可变区抗体的编码基因。     

丙型肝炎病毒NS3蛋白人源基因工程单链抗体的表达

目的在大肠杆菌XL1-Blue中表达可溶性的抗HCV非结构蛋白NS3的人源单链可变区抗体（single-chainvariablefragmentantibody,ScFv）。方法以重组的HCVNS3为抗原,利用噬菌体抗体库技术筛选含有抗-HCVNS3ScFv基因的噬菌体克隆。从噬菌体抗体阳性克隆中提取质粒,经SfiI/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行DNA序列测定;异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside,IPTG）诱导表达HCVNS3可溶性单链可变区抗体。ELISA和斑点吸印杂交检测其与不同来源的抗原的结合活性。结果筛选到的HCVNS3的单链抗体基因,经限制性内切酶酶切和序列分析表明,该抗体基因由750bp组成,ELISA和斑点吸印杂交结果表明,在大肠杆菌XL1-Blue中表达的HCVNS3的单链抗体,可与不同来源的NS3抗原结合。结论大肠杆菌XL1-Blue表达的NS3-ScFv具有结合不同来源的HCVNS3的活性和特异性。     

丙型肝炎病毒非结构蛋白2反式激活蛋白的原核表达及多克隆抗体制备

目的 对HCV非结构蛋白2反式激活蛋白(NS2TP)进行原核表达,制备多克隆抗体,并探讨其在不同肝组织中的表达情况.方法 PCR法获得HCV NS2TP基因,将其克隆至原核表达载体pET-32a(+)上,并转化入E.coli BL21.在大肠埃希菌中诱导表达,表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,经免疫印迹分析验证后,大量表达并纯化该重组蛋白.将重组蛋白免疫家兔后获取多克降抗体,应用免疫组织化学技术检测健康人及慢性HCV患者的肝组织.结果 成功获得丁重组目的 蛋白(相对分子质量为33×103)及高滴度、高特异度的多克隆抗体.慢性HCV患者肝组织内NS2TP的表达高于健康肝组织,且以细胞核内分布为主.结论 明确了慢性HCV患者肝组织内NS2TP表达量及细胞内定位的变化,为进一步研究NS2TP的生物学功能和HCV的致病机制奠定了基础.     

重叠乙型和丙型肝炎病毒感染的临床与病理分析

目的 观察HBV和HCV重叠感染的临床与病理,探讨HBV和HCV相互作用的特点.方法 收集226例慢性肝病患者的血清学指标,实时荧光定量PCR法测定HBV DNA和HCVRNA,ELISA检测HBV血清标志物、抗-HCV抗体.行肝穿刺活组织病理检查、免疫组织化学HBsAg、HBcAg和原位杂交HBV DNA、HCV RNA检测.计数资料比较采用X2检验或Fisher确切率检验.结果 HBV和HCV重叠感染的重度慢性肝炎患者比例为62.50%,高于HBV或HCV单独感染者的27.05%和30.56%(X2=14.70,P＜0.01).HBV感染组的血ALT、AST、TBil、DBil和Alb高于HBV和HCV重叠感染组和HCV感染组,差异均有统计学意义(X2=8.52,P＜0.05).重叠感染组和HBV感染组的血HBsAg与肝内HBsAg一致率比较,差异均有统计学意义(X2=15.60,P＜0.01).HBV和HCV重叠感染组血清HBV DNA阳性率为12.5%,低于HBV单独感染组的87.7%(X2=17.66,P＜0.01);而HBV和HCV重叠感染组HCV RNA阳性率为75.0%,低于HCV单独感染组的80.6%,差异无统计学意义(P＞0.05).结论 HBV和HCV重叠感染导致的肝损伤更明显.     

Infection with an unenveloped DNA virus (TTV) in patients with acute or chronic liver disease of unknown etiology and in those positive for hepatitis C virus RNA

BACKGROUND/AIMS: An unenveloped single-stranded DNA virus (TTV) has been reported in association with elevated transaminase levels in patients with posttransfusion hepatitis and in those with acute or chronic liver disease of unknown etiology. To further evaluate the association of TTV with liver disease, TTV DNA was searched for in patients with acute or chronic liver disease of various etiologies. METHODS: TTV DNA was determined by polymerase chain reaction with hemi-nested primers in 64 patients with acute or chronic liver disease of unknown etiology and in 100 with acute or chronic liver disease positive for antibody to hepatitis C virus (HCV) as well as HCV RNA. RESULTS: TTV DNA was detected in two of the seven (29%) patients with acute hepatitis of unknown etiology, but in none of the four patients with acute HCV-associated hepatitis. It was detected in 27 of the 57 (47%) patients with chronic liver disease of unknown etiology at a frequency significantly higher (p<0.001) than that in 17 of the 96 (18%) patients with chronic HCV-associated liver disease. By contrast, RNA of hepatitis G virus was detected in none of the patients with acute hepatitis, and only in one of the 57 (2%) patients with chronic liver disease of unknown etiology as well as in six of the 96 (6%) patients with chronic HCV-associated liver disease. CONCLUSIONS: Based on the obtained results, TTV has a role in the development of acute and chronic liver disease of unknown etiology.     

Hepatitis B Virus Gene in Liver Tissue Promotes Hepatocellular Carcinoma Development in Chronic Hepatitis C Patients

The hepatitis B virus (HBV) gene has been detected in hepatocellular carcinoma (HCC) tissue negative for the hepatitis B surface antigen and positive for the hepatitis C virus (HCV) antibody, but the precise role of the HBV gene in hepatocarcinogenesis has yet to be clarified. We studied the HBV gene in liver tissue several years before the emergence of HCC. Eleven patients diagnosed with HCV-positive chronic liver disease and who developed HCC were assigned to group A. HBV DNA was detected in 8 of the 11 patients (73%). Twenty-five patients, who did not develop HCC, were selected as group B. Six of the group B patients were classified as DNA-positive (24%). The HBV DNA in liver tissue was found to be significantly related to HCC development (P < 0.01). Thus, the presence of the HBV gene in patients with chronic HCV associated-liver injury appears to promote hepatocarcinogenesis, although prospective studies are needed to confirm this result.     

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.     

Low-level hepatitis C viremia and humoral immune response to NS4 in chronic hepatitis B virus-hepatitis C virus coinfection

BACKGROUND: There is a limited amount of published data on the interference of hepatitis B virus (HBV) on hepatitis C virus (HCV). The aim of this study was to investigate the effect of concurrent HBV infection on serum titers of HCV RNA and HCV antibody profiles in chronic HCV infection. METHODS: The clinical and virological profiles (serum titers of HCV RNA, HCV genotypes and antibody profiles) of 25 patients with chronic HBV-HCV coinfection were compared with those of 25 age- and sex-matched patients with HCV infection alone. RESULTS: Among the 25 patients with HBV-HCV coinfection, only 3 were found hepatitis Be antigen (HBeAg) and HBV DNA positive by hybridization assays, and the other 11 were found HBV DNA positive by polymerase chain reaction. Genotype 1b was dominant in both HBV-HCV coinfection and HCV infection alone (64% versus 84%, P > 0.1). Patients with HBV-HCV coinfection had significantly lower alanine aminotransferase (ALAT) levels and inflammatory scores but higher fibrosis scores than those with HCV infection alone. Serum titers of HCV RNA were significantly lower in HBV-HCV coinfection than in HCV infection alone. The frequency and relative intensity of antibody response to core, E2/NS1, NS3, and NS5 showed no significant difference between the two groups, but antibody response to NS4 was diminished significantly in HBV-HCV coinfection. CONCLUSIONS: In HBV-HCV coinfection, serum levels of HBV DNA are usually low or undetectable. Concurrent HBV infection, however, could interfere with HCV replication and suppress antibody response to NS4. The biological significance of selective inhibition of humoral immune response to NS4 in HBV-HCV coinfection should be further studied.     

Specific targeting of hepatitis C virus core protein by an intracellular single-chain antibody of human origin

The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84-87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein-mediated pathogenesis.     

Hepatitis delta virus RNA in serum of patients with chronic delta hepatitis

We studied 28 patients with chronic delta hepatitis for the presence of hepatitis delta virus (HDV) RNA in serum. The hot start polymerase chain reaction (PCR) method, in which the reaction begins at 60–80° C, showed a higher sensitivity than conventional PCR reaction. Additionally, the presence of hepatitis B (HBV) and C virus (HCV) infections were determined by PCR. HDV RNA was detected in 26 patients (93%), HBV DNA in 22 (79%), and HCV RNA in only one. Detection of HDV RNA correlated very well with detection of hepatitis delta antigen by immunostaining in the liver. In six patients HDV RNA was detectable despite the absence of HBV DNA in serum, suggesting that high levels of HBV are not required for HDV replication. Of 29 control patients with chronic hepatitis B without antibody to HDV, none had detectable HDV RNA, while all had HBV DNA in serum. Detection of HDV RNA with PCR proved highly sensitive and specific, demonstrating that virtually all patients with chronic HDV infection had ongoing viral replication.     

GB virus C/hepatitis G virus in serum and liver of patients with chronic C and non B non C hepatitis: molecular and immunological aspects

GB virus C/hepatitis G Virus (HGV) is a single-stranded RNA virus that is transmitted parenteraly. This study investigates GB virus C in 62 patients with chronic hepatitis C (CHC) and non B non C hepatitis (CNBNC). The viral E2 protein was examined in the sera of the patients (using western blott assay) while viral replication in the liver was examined by detecting the negative strand of HGV-RNA and its E2 protein in liver tissue using in situ hybridization and immunohistochemical staining respectively. E2 protein was detected in 28% of patients with chronic hepatitis C and in 13.3% of patients with non B non C chronic hepatitis, while not detected in healthy blood donors (0%). HGV- E2 protein and the negative strand of HGV -RNA were detected in hepatocytes of only 3 out of the 13 examined liver biopsies from HGV infected patients (23%). The mean level of ALT in chronic HCV hepatitis patients who were +ve for HGV was significantly lower than those who were -ve for HGV. There was a significant difference between the mean value of HCV -RNA level by real time PCR in sera of hepatitis C positive patients with + ve HGV-E2 when compared with HCV patients with - ve HGV-E2 (p < 0.001). It is concluded that HGV co-infection may occur in some cases with CHC and CNBNC. Sites of replication, other than liver, are suggested as the virus was detected in liver tissue of only 23 % of cases inspite of its presence in their sera.     

人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。     

Low‐level hepatitis C viremia and humoral immune response to NS4 in chronic hepatitis B virus‐hepatitis C virus coinfection

Background: There is a limited amount of published data on the interference of hepatitis B virus (HBV) on hepatitis C virus (HCV). The aim of this study was to investigate the effect of concurrent HBV infection on serum titers of HCV RNA and HCV antibody profiles in chronic HCV infection. Methods: The clinical and virological profiles (serum titers of HCV RNA, HCV genotypes and antibody profiles) of 25 patients with chronic HBV‐HCV coinfection were compared with those of 25 age‐ and sex‐matched patients with HCV infection alone. Results: Among the 25 patients with HBV‐HCV coinfection, only 3 were found hepatitis Be antigen (HBeAg) and HBV DNA positive by hybridization assays, and the other 11 were found HBV DNA positive by polymerase chain reaction. Genotype 1b was dominant in both HBV‐HCV coinfection and HCV infection alone (64% versus 84%, P?>?0.1). Patients with HBV‐HCV coinfection had significantly lower alanine aminotransferase (ALAT) levels and inflammatory scores but higher fibrosis scores than those with HCV infection alone. Serum titers of HCV RNA were significantly lower in HBV‐HCV coinfection than in HCV infection alone. The frequency and relative intensity of antibody response to core, E2/NS1, NS3, and NS5 showed no significant difference between the two groups, but antibody response to NS4 was diminished significantly in HBV‐HCV coinfection. Conclusions: In HBV‐HCV coinfection, serum levels of HBV DNA are usually low or undetectable. Concurrent HBV infection, however, could interfere with HCV replication and suppress antibody response to NS4. The biological significance of selective inhibition of humoral immune response to NS4 in HBV‐HCV coinfection should be further studied.     

High prevalence of anti-hepatitis B virus serological markers in patients with hepatitis C virus related chronic liver disease in Japan

BACKGROUND/AIMS: Evidence is accumulating that hepatitis B virus (HBV) is present in patients who are hepatitis B surface antigen negative but have antibody to hepatitis B core antigen (anti-HBc). Furthermore, recent studies have shown that patients with hepatocellular carcinoma who have antibody to hepatitis C virus (HCV) often possess HBV related serological markers. Data on the seroprevalence of HBV infection in patients with HCV related chronic liver disease were collected to evaluate the significance of the presence of antibodies to HBV. METHODS: The prevalence of HBV related serological markers was analysed in a total of 2014 Japanese patients with HCV infection. The control group comprised 352 subjects without liver disorder. RESULTS: A large number of patients (49.9%) with HCV related chronic liver disease including hepatocellular carcinoma were positive for anti-HBc. In addition, the prevalence of anti-HBc closely correlated with the clinical stage of the liver disease. There was no relation between a past history of blood transfusion and the prevalence of anti-HBc. Notably, anti-HBc was the only serological marker for HBV infection in a significant number of patients with HCV related chronic liver disease (24.1%). CONCLUSIONS: Our data provide further evidence for the high prevalence of anti-HBc in patients with HCV related chronic liver disease, particularly those with hepatocellular carcinoma, suggesting that HBV infection, probably including latent infection, may play an important role in carcinogenesis in these patients.     

Hepatitis B virus DNA in sera and liver tissue of HBsAg negative patients with chronic hepatitis C

BACKGROUND/AIMS: Hepatitis B virus (HBV) DNA has been detected in HBsAg-negative patients with hepatitis C. We determined the rate and explored the clinical significance of HBsAg negative HBV coinfections in Austrian patients with chronic hepatitis C. METHODS: Sera (n=82, group I) or liver tissue (n=16, group II) from 98 HBsAg negative Austrian patients with chronic hepatitis C were examined for HBV DNA by nested polymerase chain reaction (PCR). For control purposes, sera from 15 patients with chronic HBV infection (8 HBsAg positive, 7 HBsAg negative, all HBV PCR positive) were examined. RESULTS: HBV DNA was detected in 22% of sera and 19% of liver tissue specimens of patients with chronic hepatitis C. No significant difference in mean aminotransferase values, markers of HBV infection, inflammatory disease activity, or degree of hepatic fibrosis was observed in patients with or without HBV DNA. Anti-HBc alone as a marker of past HBV infection was more frequent in chronic hepatitis C patients compared to control individuals. Negative HCV PCR was more common (p=0.009) among patients with positive HBV PCR in serum. When examining repeat sera for HBV DNA, positive results were obtained in previously negative, but also negative results in previously positive patients. CONCLUSIONS: Coinfection with HBV can be demonstrated by PCR in a considerable number of HBsAg negative Austrian patients with chronic hepatitis C. HBV infection seems to suppress HCV replication even in HBsAg negative patients with dual infection. HBV coinfection in HCV infected patients cannot be excluded by negative HBsAg status alone. Repeat PCR examinations are needed to exclude dual infections.