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。     

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

AIM：To screen human single chain Fv antibody(scFv)against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.METHODS:The phage antibody library was panned by HCVE2 antigen,Which was coated in microtiter plate.After five rounds of bipanning,56phage clones were identified specific toHCVE2antigen,The selected scFv clones were digested by SfiI/NotI and DNAwas sequenced.Then it was subcloned into the vector pCANTAB5Efor expression as E-tagged soluble scFv.The liver tissue sections from normal person and patients with choronic hepatitis B and chronic hepatitis C were immunostained with HCVE2 scFv antibody.RESULTS:The data of scFv-E2DNAdigestion and DNA sequencing showed that the scFv gene is composed of 750bp.ELISAand immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitisCE2 antigen has a specific binding character with hepatitis virus E2antigen and paraffin-embedded tissue,but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.CONCLUSION:We have successfully screened and identified HCVE2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitisC.     

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the V_H chains and V_κ chains of all three scFv antibodies were derived from V_H3 and Vκ1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.     

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the VH chains and Vkappa chains of all three scFv antibodies were derived from VH3 and Vkappa1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.     

Clinical application of hepatitis C virus core protein in early diagnosis of acute hepatitis C

A fluorescence enzyme immunoassay (FEIA) for the quantitative measurement of hepatitis C virus (HCV) core protein has recently been developed. In this study, we studied the clinical usefulness of this measurement in patients with acute hepatitis C. Eighteen patients with post-transfusion acute hepatitis C were enrolled in the study; 5 patients showed resolution of hepatitis with disappearance of HCV viremia, while the remaining 13 patients did not. A second generation HCV antibody, HCV RNA, and HCV core protein were measured in serial serum samples taken within 1 month of the onset of acute hepatitis and 3, 6, 12, 24, and 36 months after onset. Within the first month after disese onset, the positivity rates of HCV RNA (100%; P = 0.0014) and HCV core protein (89%; P = 0.0300) were both significantly higher than that of HCV antibody (56%). Six months after disease onset, the positivity rate of HCV antibody had increased, to 100%, and the pasitivity rates of HCV RNA and HCV core protein began to decrease. HCV core protein levels did not differ between patients with resolved and unresolved disease in the first month after disease onset. These findings indicate that FEIA, a simple assay, for the measurement of HCV core protein was useful for the early diagnosis of acute hepatitis C. (Received May 26, 1997; accepted Nov. 28, 1997)     

Apoptosis in hepatitis C

Summary. The apoptotic process appears to be a host defence mechanism against viral infections and tumourigenesis. However, many viral genomes encode proteins, which repress apoptosis so as to escape from immune attack by the host. Therefore, virus–host interactions may determine viral persistence, extent and severity of liver inflammation and possibly viral hepatocarcinogenesis. Apoptosis of liver cells may play a significant role in the pathogenesis of hepatitis C. Pathomorphologic features of increased apoptosis include shrinkage and fragmentation of nuclei/cytoplasm in piecemeal necrosis areas, acidophilic bodies, and focal cell dropout in the liver lobule. The hepatitis C virus (HCV) core protein exhibits both proapoptotic or antiapoptotic actions. Modulation of apoptosis may involve binding of HCV core protein to the intracellular signal transducing portion of death receptors and displacement of signalling molecules. Apoptosis may occur in the absence of significant transaminase elevation, thereby explaining the lack of correlation between biochemical activity and liver cell histological injury. Monitoring caspase activation might provide a reliable tool to estimate the efficacy of HCV therapy, and might open challenging therapeutic strategies in HCV infection. The antiviral effect of interferon may be mediated through induction of apoptosis. Lastly, administration of the antiapoptotic ursodeoxycholic acid in HCV infection is compatible with the notion that apoptosis may represent a mechanism for viral shedding rather than for viral elimination, thereby raising the concept that inhibition of apoptosis could ameliorate hepatitis C.     

Serodiagnosis of hepatitis C virus (HCV) infection with an HCV core protein molecularly expressed by a recombinant baculovirus.

An enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of hepatitis C virus (HCV) infection, using HCV core protein (p22) synthesized by a recombinant baculovirus. Among 58 clinically well-defined chronic non-A, non-B hepatitis (NANBH) patients, 49 (84.5%) were positive for p22 antibody (anti-p22), whereas 42 (72.4%) were positive for C100-3 antibody (anti-C100-3), as measured by the present assay using the HCV nonstructural protein as antigen. Thirty-nine patients (67.2%) had both antibodies. No significant level of anti-p22 was detected in sera of chronic hepatitis B patients or normal blood donors. In typical post-transfusion NANBH patients, anti-p22 could be detected at, or even before, the first alanine aminotransferase peak. Anti-p22 was also detected in blood donors who were previously shown to be involved in transmitting HCV but in whose serum anti-C100-3 was not detectable. The ELISA detecting antibody to the HCV core protein expressed and properly processed in animal cells will be useful for mass screening of donor blood as well as for early diagnosis of hepatitis C.     

丙型肝炎病毒核心蛋白DNA结合区的测定

目的 阐明丙型肝炎病毒（HCV）核心蛋白的DNA结合特性及其意义。方法 在大肠杆菌中表达谷胱甘肽转移酶（GST）融合HCV核心蛋白不同长度片段，并进行纯化。经聚丙烯酰胺凝胶电泳后，通过更换缓冲液的方法将这些蛋白片段相对固定在凝胶中，用γ－^32P[4667-0091]ATP标记人工合成的DNA进行2次电泳，通过放射自影检测核心蛋白的DNA结合区。结果 HCV核心蛋白N端第10－16位氨基酸残基和第46－70位氨基酸残基可独立地结合DNA，核心蛋白既能和单链DNA结合又能和双链DNA结合，对单双链DNA的结合域相同。对所结合的DNA序列无选择性。结论 HCV核心蛋白的N端含有两个DNA结合区，结合区序列与核内转移信号的部分重迭以及对结合靶DNA序列的非选择性可能是HCV核心蛋白具有多功能的基础。     

Serum antibodies against the hepatitis C virus E2 protein mediate antibody-dependent cellular cytotoxicity (ADCC)

BACKGROUND/AIMS: The role of antibody dependent cellular cytotoxicity (ADCC) in HCV infection is unclear at present. Antibodies mediating ADCC are usually directed against viral envelope proteins. As cell surface expression of the HCV envelope E2 protein has been shown, the HCV E2 protein is an especially promising candidate target for ADCC. METHODS: Sera from patients with acute (n=6), self-limited (n=11) and chronic (n=19) HCV infection were analyzed in this study. Sera reacting with cell-bound HCV antigens were examined in a flowcytometric cytotoxicity assay using antigen-coated JOK-1 cells as targets. RESULTS: We found that sera from all stages of HCV infection reacted with cells loaded with HCV E2. E2-specific ADCC was observed in patients with acute (n=3/6), self-limited (n=5/11) and chronic (n=13/19) hepatitis C and was closely related to fluorescence intensity in the E2-binding assay (r=0.67, P<0.001). CONCLUSIONS: We conclude that E2-antibodies from all stages of HCV infection can mediate ADCC. Thus, the role of this process in the pathogenesis of chronic hepatitis C should be further elucidated.     

一种用于HCV核心蛋白体外表达研究的CHO细胞模型的建立

目的:建立丙型肝炎病毒(hepatitis C virus,HCV)核心(core,C)蛋白体外表达的非肝细胞模型.方法:核酸酶切法鉴定含有HCV1b基因型C蛋白编码基因的重组质粒pCMH6K的稳定性,将pCMH6K瞬时及稳定转染于中华仓鼠卵巢(China hamster ovary,CHO)细胞并连续传代110 d,...     

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。     

Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting

Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9). In vitro, scFv9B9 retains the ability of the parental antibody to recognize human and rat ACE when expressed both on the surface of phage and as a soluble protein in prokaryotic and eukaryotic expression systems. The ability of scFv 9B9 presented by phage or the soluble protein labeled with I(125) to recognize ACE in the pulmonary circulation was also confirmed in an in vivo rat model. Sequence analysis revealed a putative glycosylation site in close proximity to the complementarity determining region 2 (CDR2) of the scFv 9B9 heavy chain. Mutation of Asn68 to Gln in the heavy chain of scFv 9B9 eliminated the glycosylation site and significantly improved the binding affinity of scFv 9B9 to human ACE as determined by cell ELISA and Western Blot. Moreover, Asn68Gln scFv 9B9 showed a greater rate of secretion at 30°C than wild type scFv 9B9, but had a decreased thermal stability at 37°C. The development of a stable and functional single-chain format of mAb 9B9 which specifically recognizes human and rat ACE represents a novel antibody-based reagent suitable for targeted delivery of drugs/genes to the pulmonary circulation.     

Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor α and carnitine palmitoyl acyl-CoA transferase 1A

AIM: To elucidate the role of the peroxisome proliferator-activated receptor α (PPARα) and its target gene carnitine palmitoyl acyl-CoA transferase 1A (CPT1A)in the pathogenesis of hepatitis C virus (HCV) infection.METHODS: Liver samples were collected from the patients with chronic HCV infection and controls. HepG2cells were transfected with vector pEF352neo carrying.Two independent clones (clone N3 and N4) stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction (PCR) using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS: Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls (1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4+1, ,P = 0.004). Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls (2.3±0.3 vs 3.6±0.2,P = 0.009). The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION: The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.     

Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor α and carnitine palmitoyl acyl-CoA transferase 1A

AIM: To elucidate the role of the peroxisome proliferator-activated receptor alpha (PPARalpha) and its target gene carnitine palmitoyl acyl-CoA transferase 1A (CPT1A) in the pathogenesis of hepatitis C virus (HCV) infection. METHODS: Liver samples were collected from the patients with chronic HCV infection and controls. HepG2 cells were transfected with vector pEF352neo carrying. Two independent clones (clone N3 and N4) stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARalpha and CPT1A mRNAs were quantified by real-time polymerase chain reaction (PCR) using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARalpha antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARalpha. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARalpha. For immunohistochemical staining of PPARalpha, sections were incubated with the primary goat polyclonal antibody directed against PPARalpha at room temperature. RESULTS: Real-time PCR indicated that the PPARalpha level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls (1.8+/-2.8 vs 13+/-3.4, P = 0.0002; 1.1+/-1.5 vs 7.4+/-1, P = 0.004). Western blot results showed that the level of PPARalpha protein in the livers of hepatitis C patients was lower than that in controls (2.3+/-0.3 vs 3.6+/-0.2, P = 0.009). The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARalpha staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARalpha mRNA levels were significantly lower than that in the controls. CONCLUSION: The impaired intrahepatic PPARalpha expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARalpha and CPT1A at the level of not only mRNAs but also proteins. PPARalpha plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.     

丙型肝炎病毒核心蛋白结合蛋白激酶R的功能区域研究

目的 构建并表达丙型肝炎病毒(HCV)不同病毒株：癌中心株(BT)、癌旁珠(BNT)、HCV-J及BT不同截短片段谷胱甘肽(GST)-核心融合蛋白；寻找核心蛋白(Core)与蛋白激酶R(PKR)相互作用区域，探讨它们在HCV持续感染及肝细胞癌(HCC)发病机制中的作用。方法 用聚合酶链反应扩增不同片段HCV核心蛋白基因，并将7个不同的基因片段分别克隆到原核表达载体pGEX-4T-1，诱导表达并纯化表达蛋白，与两株细胞(HepG2和Huh-7)的PKR进行相互作用试验。结果7个不同片段Core在体外都得到相应表达，不同片段结合PKR的能力存在一定差异。BT、BNT、C191的Core N端1～172氨基酸(aa)3个片段均能与PKR发生直接结合，BT与PKR结合的区域在Core N端的1～58aa。结论 不同片段Core在原核细胞中获得较好的表达；Core／PKR相互作用，在HCV持续感染和HCC的发病机制中可能起重要作用。     

Inhibition of hepatitis C virus NS3-mediated cell transformation by recombinant intracellular antibodies

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be of importance. With the aim of inhibiting NS3-mediated cell transformation, we used antibody-phage display to isolate NS3-specific single-chain antibodies (scFv). METHODS: We have isolated and characterized eight anti-NS3 scFvs. We investigated the phenotypic changes that NS3-expressing cells undergo upon intracellular expression of these antibodies in different subcellular compartments (intracellular immunization), assayed by their proliferation rate and their ability to grow anchorage independently. The intracellular location of NS3 and the scFvs were analyzed by immunofluorescent staining using confocal microscopy. RESULTS: Nuclear targeted anti-NS3 intrabodies shuttle NS3 from the cytosol to the nucleus with concomitant inhibition of cell proliferation and loss of the transformed phenotype. CONCLUSIONS: Intracellular immunization-based gene therapy strategies may emerge as a promising antiviral approach to interfere with the life cycle and tumorigenicity of HCV.     

丙型肝炎病毒核心基因免疫研究

目的研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫用于预防和治疗ＨＣＶ感染的可行性和有效性．方法将ＨＣＶＣ基因片段插入真核表达载体ｐｃＤＮＡ３质粒ＣＭＶ启动子的下游，在证实其可以在小鼠骨髓瘤细胞ＳＰ２／０（Ｈ２ｄ）中表达之后，将重组质粒注射ＢＡＬＢ／ｃ（Ｈ２ｄ）小鼠股四头肌，ＥＬＩＳＡ检测血清中抗体产生水平；３ＨＴｄＲ掺入法测定免疫小鼠淋巴细胞ＨＣＶＣ抗原特异性增殖能力，４ｈ５１Ｃｒ释放法检测免疫小鼠细胞毒Ｔ细胞（ＣＴＬｓ）体外杀伤功能．结果免疫小鼠２０只，初次免疫２ｗｋ后，血清中均出现了ＨＣＶＣ抗体，且增加免疫剂量可提高抗体滴度；淋巴细胞增殖指数为６１０，明显高于对照组（Ｐ＜００１），ＣＴＬｓ体外特异性杀伤率为６３１％，也高于对照组（Ｐ＜００１）．结论ＨＣＶＣ基因免疫不仅可以诱导机体产生特异性的体液免疫，而且产生特异性的细胞免疫，它可能是防治ＨＣＶ的有效方法．