人工合成丁型肝炎病毒抗原片段及其诊断应用

本文选择、设计并合成了含天然HDAg上的部分片段的27肽,用于建立检测抗HD的ELISA方法。试验结果表明,包被的合成肽抗原与已知抗HD阳性冻存血清呈抗HD阳性反应;它能与天然HDAg竞争抗HD,具天然HDAg上相应片段的抗原活性;该合成肽抗原专一性强,与正常人血清,正常小鼠血清,单纯甲型、乙型、丙型肝炎抗体阳性血清无免疫交叉反应。用建立的ELISA法,检测1992年1～5月的部分献血员及住本科各型肝炎患者血清标本的抗HD。300例献血员中1例抗HD阳性(0.33％),其ALT增高2倍以上。非乙型肝炎41例,甲型肝炎52例,抗HD均阴性。各型乙型肝炎211例,抗HD阳性21例(9.95％)。其中,携带者2/82例(2.5％);急黄肝6/43例(13.95％);慢活肝6/60例(10.00％);重肝4/18例(22.20％);肝炎肝硬化3/8例(37.50％),结果与本科已往报告基本一致。     

丁型肝炎病毒重组抗原的制备及应用

目的：获得能够应用于丁型肝炎病毒(HDV)酶免试剂的重组丁型肝炎病毒抗原。方法：采用RT-PCR自HDV感染急性期的土拨鼠肝脏扩增编码丁型肝炎病毒抗原的HDV cDNA,插入质粒pET-3α,转化大肠杆菌BL21,制备丁型肝炎病毒抗原表达菌株;对其表达蛋白的特异性、灵敏度、稳定性及活性进行鉴定;并将以该重组蛋白为主要原料制备的抗-HD酶免试剂与爱尔兰Noctech抗-HD试剂盒进行比较。结果：重组质粒PED-SHDAg和PED-LHDAg经异丙基-硫代-β-D-半乳糖苷(IPTG)诱导表达的2种蛋白经SDS-PAGE电泳后,Western Blot检测显示均与抗-HD发生强烈反应。该表达蛋白对抗-HD阳性血清抑制率为86％,37℃保存7d稳定性良好,比活性为1：8000,检测抗-HD灵敏度与国外同类试剂盒结果一致。结论：该重组丁型肝炎病毒抗原可用于抗-HD酶免试剂的制备。     

检测丙型肝炎核心抗体的合成肽酶联免疫试验

为建立一种以酶联免疫试验检测丙型肝炎病毒的核心抗体,按病毒氨基酸序列C区合成一个36-寡肽,将其作为试剂抗原包被固相,以捅获待检血清的相应抗体,以酶标抗入IgG将其检出。输血后非甲非乙肝炎32例检出抗体20例(62.5％),其中发病1个月内的13例中检出8例。特异性由抑制试验确定。17例阳性血清可用聚合酶链反应检出病毒。结果表明,完善这一系统可能发展为试剂盒。     

HepG2.9706细胞内HDV核酶对HCV RNA抑制活性的初步研究

目的探讨HDV核酶在细胞内抑制HCV RNA的可能性.方法将重组载体转染至转基因细胞HepG2.9706中,通过荧光定量PCR检测细胞和培养上清中的HCV RNA,初步探讨HDV核酶对HCV RNA的抑制活性.结果①RzCl,RzC2,RzC3 3组HDV核酶定向插入到真核表达载体.②在转基因细胞HepG2.9706中,pCl-RzCl、pC1-RzC2、pCl-RzC3对HCV-5′NCR-C RNA抑制活性分别为69.2%,38.7%、4.2%.结论①成功构建了3个HDV核酶重组表达载体.②pC1-RzC1、RC1-RzC2在转基因细胞HepG2.9706具有抑制HCV-5′NCR-C RNA的活性.     

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region

Background   Numerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed.
Methods  Based on 45 sequences, representing HCV genotypes 1-6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182-2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1-6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis.
Results  The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182-2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182-2211(R1), 2272-2301 (R7) and 2302-2331 (R9): the highly variable 2212-2241 (R3) and 2257-2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera.  Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis.   
Conclusions  The major antigenic regions in HCV NS5A region were at 2212-2241(R3), 2272-2301(R7) and 2302-2331(R9).  Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

     

戊型肝炎病毒合成肽决定簇检测抗HEV的研究

利用３段分别代表ＨＥＶ３个基因区的合成多肽，研制成ＥＬＩＳＡ抗ＨＥＶ检测试剂。此试剂对ＩｇＧ及ＩｇＧ检测出阳性率在急性ＮＡＮＢＮＸ肝炎患者中分别为５４．１％（５３／９８）和３３．７％（３３／９８），在其他疾病患者正常献血员中则分别为２．７％－６．３％和０％－２．６％。与Ｇｅｎｅｌａｂｓ重组抗原生产的检测试剂比较，两者有很好的一致性，其阳性符合率为９０．０％，阴性符合率为９８．５％，总符合率为９７．     

丙型肝炎病毒核心抗原的检测及其临床意义

目的 探讨HCV 核心抗原在HCV 感染诊断和治疗中的作用.方法 丙肝核心总抗原检测采用酶联免疫法;丙型肝炎抗体检测采用第三代酶联免疫法.所有血清标本均进行HCV-RNA 和肝功能检测.丙肝核酸定量采用PCR- 荧光探针法;肝功能检测采用紫外连读检测法.结果 90 例中,HCV-cAg 检测阳性率为42.22%(38/90).HCV-cAg 阳性组HCV-RNA 阳性率100%(38/38),显著高于HCV-cAg 阴性组的71.15%(37/52).阳性组丙氨酸氨基转移酶(alanine aminotransferase,ALT)异常率63.16%(24/38),显著高于HCV-cAg 阴性组的40.38%(21/52),两组间比较(P<0.05).HCV-cAg 阴性组中59.62% (31/52)的患者ALT 在正常范围内(ALT<40U/L),显著高于HCV-cAg 阳性组的36.84%(14/38) ;而HCV-cAg 阳性组中36.84%(14/38)ALT 值均>100U/L,明显高于HCV-cAg 阴性组的9.62%(5/52).两组间比较(P<0.05).丙肝核心抗原HCV-cAg 和HCVRNA有很好的正相关性,HCV-cAg 与ALT 水平也有一定相关性,可以反映肝功损害程度.结论 丙肝核心抗原HCV-cAg是慢性丙肝感染良好的监测指标.     

新疆肝病患者HDV、HBV感染的免疫组化研究

作者以直接酶标法及过氧化物酶-抗过氧化物酶法(PAP法)对145例新疆各类肝病患者的肝组织进行HDV、HBV感染的免疫组化研究,各类标本中,HBsAg及HBcAg的合计检出率为39.3％,HDAg在HBV血清学标记阳性患者肝组织中的检出率为3.5％。胞浆型HBcAg表达多见于肝组织病变明显区域,HDAg在肝炎后肝硬化组的检出率明显高于HBV感染非肝硬化组(P<0.025),提示肝炎后肝硬化与HDV感染有关。     

Detection of anti-HEV by synthetic peptides

Ｄｅｔｅｃｔｉｏｎｏｆａｎｔｉ－ＨＥＶｂｙｓｙｎｔｈｅｔｉｃｐｅｐｔｉｄｅｓＰａｎＷｅｉ（潘卫），ＹｕＣｈａｏ（俞超），ＱｉＺｈｏｎｇｔｉａｎ（戚中田），ＣｕｉＤａｆｕ（崔大敷），ＳｏｎｇＹａｎｂｉｎｇ（宋燕斌），ＣｕｉＨｅｎｒａｎ（崔恒苒），Ｚｈｏ...     

HDV/HBV体外感染原代培养人胎肝细胞的实验研究

目的：建立ＨＤＶ／ＨＢＶ感染人胎肝细胞体外培养系统。方法：利用ＨＤＶ／ＨＢＶ阳性血清同时感染体外２的人胎肝细胞;应用ＥＬＩＳＡ、免疫组化法、原位杂交法和斑点法检测上清液和细胞中ＨＢｓＡｇ、ＨＤＡｇ、ＨＢＶＤＮＡ和ＨＤＶｃＤＮＡ。结果：上清液和细胞中ＨＢｓＡｇ、ＨＤＡｇ、ＨＢＶＤＮＡ和ＨＤＶｃＤＮＡ在感染后第２天至第１６天均可测出,其中上清液中ＨＢｓＡｇ、ＨＤＡｇ以感染后第４天至第１２天达高峰,结论     

Clinical application and analysis of hepatitis C virus NS3 antigen detection by ELISA in human serum

Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis. Methods Samples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study. Results Only 48 （1.3%） of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 （24.3%） of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 （60%） were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% （20/23） and 69.6% （16/23） respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test （r=-0.989, P〈0.05）, and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV Utres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually. Conclusions The HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.     

剪切HCV RNA的HDV核酶的设计及活性测定

目的：探讨丁型肝炎病毒（Hepatitis D virus,HDV）核酶用于抗丙型肝炎病毒（Hepatitis C virus,HCV）基因治疗的可能性。方法：以HDV基因组核酶的假结样结构为基础,优化其茎IV区,改建基底物结合区,获得3种针对HCV RNA的HDV核酶RzC1、RzC2和RzC3。体外转录获取含HCV RNA5‘－非编码区（5‘-noncoding region,5‘-NCR）及部分C区在内的底物RNA（HCV RNA 5‘－NCR－C）,并进行5‘端放射性标记。在pH7.5、37℃、Mg^2 20mmol/L和去离子甲酰胺2.5mol/L等条件下,将核酶和底物按摩尔比100：1混合,在不同的时间点观察剪切百分率。结论：RzC1、RzC2对底物的剪切百分率随时间延长而递增,90min分别达24.9％、20.3％;未观察到RzC3有剪切活性。结论：经过结构构优化的HDV基因组核酶在合适的位点能够剪切异源性RNA分子HCV RNA。     

乙型重症肝炎HDV重叠感染的临床意义

对８５例乙型重症肝炎患者血清进行了丁型肝炎病毒标志物（ＨＤＶＭ）及肝功能检测。结果ＨＤＶＭ阳性率为２９．４１％，表明桂林地区乙型重症肝炎患者中ＨＤＶ感染率较高；乙型重症肝炎ＨＤＶ重叠感染后肝功能损害程度明显加重，重症肝炎主要并发症发生率及死亡率明显增高，提示ＨＤＶ感染可导致乙型重症肝炎临床病情加重。同时发现乙型重症肝炎ＨＤＶ重叠感染后乙型肝炎病毒复制标志阳性率低。     

硫代反义寡脱氧核苷酸在树体内对HDV复制与表达的抑制作用

目的：在树（Ｔｕｐａｉａ）体内观察硫代反义寡脱氧核苷酸（Ｓ－ＡＳＯＤＮ）对ＨＤＶ复制、表达的抑制作用。方法：树同时接种ＨＢＶ阳性血清０．１ｍｌ、ＨＤＶ阳性血清０．３ｍｌ后，将１６只ＨＤＶ／ＨＢＶ感染成功的树随机分为两组，给药组８只树按每只每次经尾静脉注射Ｓ－ＡＳＯＤＮ３ｍｇ，隔日１次，共７次。对照组中２只注射等量生理盐水，另６只不作处理。注射后５、１０、１５、２５ｄ取血及肝组织采用免疫组化、斑点杂交及原位杂交法检测ＨＤＶＡｇ及ＨＤＶＲＮＡ。结果：于注射结束时（１５ｄ）给药组有７只树肝内ＨＤＶＡｇ及ＨＤＶＲ－ＮＡ转为阴性，对照组８只树中仅１只转为阴性。停止使用Ｓ－ＡＳＯＤＮ１０ｄ后，给药组２只阴转树肝内又可检出ＨＤＶＡｇ和ＨＤＶＲＮＡ，对照组仍有７只可检出。结论：Ｓ－ＡＳＯＤＮ在树体内能有效抑制ＨＤＶ复制及表达，但作用并不完全，可能与用药剂量不足，时间不够长及仅用硫代修饰尚不能直接导向靶基因有关     

检测乙、丙型肝炎病毒基因芯片的制备

目的：探讨研制检测乙型和丙型肝炎病毒的基因芯片。方法：以HBV、HCV高度保守的基因片段为探针,同时用没有同源性的植物基因片段为内参照基因,制备成“乙、丙型肝炎病毒双检基因芯片”。检测时抽提患者血清中的病毒核酸,进行RT－PCR扩增及荧光标记,然后与双检基因芯片杂交,结交结果用ScanArray3000扫描仪扫描。结果：双检基因芯片的内参照结果能做到绝对阳性和绝对阴性,同时对不同血清（正常人血清,乙肝E抗原阳性血清,丙肝RNA阳性血清,乙肝、丙肝阳性混合血清）能有效检测区分。结论：本研究成功地制作了乙、丙型肝炎病毒双检基因芯片,可望使用于临床。     

Distribution and significance of hepatitis C virus C33c antigen, core antigen and HBxAg in human primary intrahepato-cholangiocarcinoma tissues

ThereisacloserelationshipbetweenhepatitisCvirus(HCV)infectionandhepatocellularcarcinoma.InastudyoftherelationshipbetweenHCVandlivercirrhosisandhepatocellularcarcinomall'Zj,HCVC33cantigenwasdetectedinproliferatingepithelialcellsofthesmallbileduct.TheresultssuggestthatHCVmayinvadesmallbileductepithelialcells.Therefore,thedistributionsofHCVC33cantigenandcoreantigenwerestudiedin35casesofhumanprimaryintrahepato--cholangiocarcinoma(PIC)andtheirperlcanceroustissuesusingimmunohistochemistry.I…     

各种HBsAg阳性人群中抗D型肝炎病毒抗体的研究

采用Abbott EIA法对各种HBsAg阳性人群血清抗HDV抗体进行了调查,结果在1004份血清标本中检出有18份标本抗HDV抗体阳性,阳性率为1.8%。抗HDV抗体检出率与年龄、性别无关。但在无症状HBsAg携带者、急性乙型肝炎、慢性乙型肝炎(包括慢性活动性肝炎、慢性迁延性肝炎、肝硬化)、肝癌患者血清中,抗HDV抗体检出率有显著性差别,分别为0.6%、0%、1.7%、和12.1%。这些阳性血清中抗HDV抗体滴度<1:10,间隔2～6个月后对部份患者重新采血复查时,抗HDV抗体转为阴性。研究结果提示了低感染率的D型肝炎在我国的存在。     

HIV infection among new synthetic drug abusers: a pilot study in Beijing

Background  Following the increased human immunodeficiency virus (HIV) notification (infection) rates via sexual transmission and increased abuse of new synthetic drugs instead of heroin in China, a study on HIV infections and risk factors among synthetic drugs and heroin users in Beijing was conducted to provide a cue for further development of targets and strategies of HIV/AIDS control and prevention for different drug populations.

Methods  A total of 428 subjects including 224 heroin users and 204 new synthetic drug users was recruited from communities in Beijing by convenience sampling. A c² test was used to compare HIV and syphilis infections between the two subpopulations. Logistic regression models were used to evaluate factors related to HIV, syphilis infections and abuse of synthetic drugs.

Results  Unbalanced distributions of demographics were detected between the two subpopulations. Compared with heroin users, most users of synthetic drugs were migrants, single and of younger age (age £30 years). Ten HIV infections and twenty-four syphilis infections were confirmed among 428 subjects. No significant difference in HIV infection was found between the two subpopulations (crude odds ratio (OR) =0.46, 95% confidence interval (CI): 0.07–15.80 and adjusted odds ratio (AOR) =1.26, 95% CI: 0.09–18.28) while four times as many syphilis infections were identified among users of synthetics drugs (AOR=3.92, 95% CI: 1.06–14.56). HIV and syphilis infections appeared to work synergistically. The migrants who were of Han ethnicity, single and who had stayed in Beijing for more than six months were more likely to abuse synthetics drugs.

Conclusions  The new synthetic drug users and heroin users are different subpopulations. We optimistically speculate that HIV has not yet been introduced into the former in Beijing, but considering higher syphilis infections in synthetic drug users and the role of syphilis in HIV infection, synthetic drug users, as a possible and important driver of the HIV/AIDS epidemic in Beijing, should be paid more attention in HIV prevention and control strategies.

     

HDV感染麻鸭的实验研究

为建立丁型肝炎的实验动物模型，分别筛选４只携带ＤＨＢＶ的麻鸭（实验组）和４只正常麻鸭（对照组）进行了ＨＤＶ实验感染。结果：实验组１只麻鸭在接种后第二周血清中出现ＨＤＶ抗原，另１只麻鸭血清中检出抗－ＨＤ。对照组麻鸭血清中未检出ＨＤＶ标记物。揭示在麻鸭中ＤＨＢＶ可辅助ＨＤＶ进行复制和表达。     

丁型肝炎抗体诊断试剂的研制

取乙型肝炎患者Delta抗体(抗—HD)阳性血清,经硫酸铵盐析和DE—52柱层析,得到纯化的抗—HD抗体,用辣根过氧化物酶标记。用丁型肝炎病毒(HDV)感染慢性携带土拨鼠肝炎病毒的东方土拨鼠(Woodchuck)后,取其肝组织制成匀浆,离心后经Sepharose 4B柱,免疫亲和层析柱,获得高丰度的HDAg。双抗体夹心法测定其滴度高达1∶10~6。制备成ELISA竞争抑制法检测抗HD试剂盒,其特异性、敏感性、重复性与爱尔兰同种试剂比较结果一致。