用豚鼠腹腔渗出细胞代替人白细胞检测HBsAg免疫RNA活性

本文用豚鼠腹腔渗出细胞(PEC)代替人白细胞进行白细胞粘附抑制试验(LAI),检测HBsAg免疫RNA(iRNA)活性。试验结果表明,用同一豚鼠PEC对不同iRNA(HBsAg iRNA及弗氏完全佐剂(FCA))的不同批号;或用不同豚鼠PEC对不同iRNA(HBsAg iRNA,脊髓灰质炎病毒iRNA,FCA iRNA及正常羊脾RNA)进行LAI试验(以HBsAg为抗原),仅HBsAg iNRA具有过继免疫活性,其它各种iRNA则否。如事先用RNA酶处理HBsAg i RNA,则此过继免疫活性消失,证明此物质为RNA。试验结果也同时表明,这种过继免疫活性具有特异性。     

固相血球吸附试验检测iRNA致敏鼠低水平EHFV抗体

在研究抗流行性出血热病毒免疫核糖核酸(EHFV—iRNA)传递体液免疫的活性中,我们建立了检测EHFV—iRNA致敏小白鼠特异性IgM和IgG抗体的固相血球吸附试验(SPARC).该方法简便、快速、特异、敏感,在检测iRNA诱导正常小鼠产生抗体实验研究中取得了满意结果.     

快速荧光灶抑制试验的应用和改进

快速荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT)是世界卫生组织(World Health Organisation,WHO)推荐的狂犬病病毒中和抗体检测标准方法,已被应用于狂犬病疫苗免疫效果评估、狂犬病疫苗新型免疫方案评估、狂犬病诊断试剂和方法评估、狂犬病被动免疫制剂评价.对RFFIT的改进包括自动化判读结果和用表达绿色荧光蛋白(green fluorescent protein,GFP)的重组狂犬病病毒做为攻击病毒,这些改进目前尚未获得广泛应用,传统RFFIT仍然是值得推广的狂犬病病毒中和抗体检测方法.     

抗狂犬病病毒CVS-11株单克隆抗体的制备及鉴定

目的 制备抗狂犬病毒单克隆抗体,为建立快速准确的狂犬病毒抗原检测方法奠定基础.方法 将狂犬病病毒CVS-11株纯化浓缩后免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经细胞克隆和间接ELISA筛选,获得稳定分泌抗狂犬病毒单克隆抗体的杂交瘤细胞株.小鼠腹腔注射法制备大量单克隆抗体并测定腹水效价,用G蛋白亲和层析柱进行纯化,间接ELISA法和间接荧光法鉴定单克隆抗体的类型、特异性及敏感性.结果 细胞融合率达100%,经克隆筛选获4株稳定分泌抗狂犬病病毒抗体的杂交瘤细胞株,其腹水效价分别为1×104,1×105,1×104和1×105;4株单抗均为IgG类型且特异性好.结论 制备的单抗具有良好特异性和敏感性.     

狂犬病病毒糖蛋白真核表达质粒的构建及免疫原性的初步研究

目的 构建狂犬病病毒糖蛋白基因DNA真核表达质粒,并检测其免疫原性.方法 用RT-PCR法扩增和分离CTN株狂犬病病毒糖蛋白基因,测序后克隆至pcDNA5.0载体,构建重组质粒pcDNA5.0-G,提取质粒,转化293T细胞,检测糖蛋白瞬时表达,并以该重组质粒肌肉注射免疫BALB/c小鼠,狂犬病病毒CVS株攻击,观察小鼠存活情况.结果 酶切、测序结果显示重组质粒pcDNA5.0-G构建成功,瞬时表达结果显示糖蛋白获得大量表达.经肌肉注射质粒常规免疫小鼠,病毒攻击后小鼠保护率为73.3％,对照组为6.7％.结论 所构建狂犬病病毒糖蛋白真核表达质粒pcDNA5.0-G经肌肉注射免疫后可有效保护小鼠免受狂犬病病毒攻击,具有良好的免疫原性,这为后期核酸疫苗的研发奠定基础.     

RNAi技术抑制HCMV-IE1表达的在体实验研究

目的:研究RNA干扰(RNA interference,RNAi) 对体内人类巨细胞病毒-立即早期基因1(human cytomegalovirus immediate-early gene 1,HCMV-IE1)表达的沉默作用,为靶向HCMV-IE1的基因治疗提供科学的实验依据.方法:选取60只雄性昆明种小鼠,并分为3组,分别为实验对照组(注射生理盐水)、病毒感染组(注射HCMVAD169株病毒液)和RNA干扰组(染毒后第2天注射转染了表达RNAi载体的人胚肺成纤维细胞液);采用PCR法检测小鼠体内HCMV基因的表达水平;采用ELISA检测动物血清中HCMV-IgM的含量;采用免疫组织化学和Western免疫印迹技术分别测定小鼠体内HCMV-IE1的蛋白水平.结果:PCR结果表明干扰组比病毒感染组的HCMV的基因表达下调20%;ELISA结果表明RNAi能使动物血清中HCMV的含量降低为19%;免疫组织化学和Western免疫印迹结果证实,干扰组和病毒感染组的HCMV-IE1蛋白水平分别降低为17.6%和18.5%.结论:RNAi技术能有效地抑制目的基因在体内的表达,有可能用于靶向HCMV-IE的基因治疗.     

抗孕酮单克隆抗体的制备与鉴定及其初步应用

目的:获得一株特异性强,灵敏度高的抗孕酮单克隆抗体,用辣根过氧化物酶标记孕酮-11α-羟基孕酮半琥珀酸酯制成酶标记物,建立酶联免疫分析(ELISA)方法,用于检测人血清中孕酮含量。方法:用免疫原11α-OH-孕酮-BSA免疫BALB/c小鼠,采用杂交瘤技术,得到14株抗孕酮单克隆抗体;用反射免疫分析(RIA)方法测定抗孕酮单克隆抗体的亲和常数和交叉反应率。结果:获得的14株抗孕酮单克隆抗体具有较高的亲和性和较小的价差反应率;建立ELISA方法学的灵敏度为0.12 ng/ml;批内变异系数为7.5%～11.4%;批间变异系数为:8.2%～12.1%;回收率93.8%～124.7%;高值临床样品系列倍比稀释后,测定值与稀释度呈线性相关,相关系数为r=0.995 7;与全自动化学发光测定的临床样品值比对结果较好,线性方程为y=0.789 4x+0.760 0,r=0.912 6。结论:方法学鉴定结果符合免疫分析的基本要求。     

抗内毒素Fab'的制备

目的 研究抗内毒素卵黄免疫球蛋白(IgY)的活性片断Fab',探讨防治内毒素血症的新途径.方法 用内毒素(LPS)作为抗原免疫25周龄德国罗曼鸡,改良水溶法提取抗内毒素IgY,胃蛋白酶切后提取Fab'片断,光密度法测抗内毒素Fab'的浓度和含量、ELISA检测抗内毒素Fab'效价、SDS-聚丙烯酰胺凝胶电泳检测其分子量及纯度. 结果 抗内毒素Fab'含量为4.2 mg/mL蛋黄液,效价为1∶51 200,纯度为92%,相对分子质量为44 000. 结论 抗内毒素Fab'产量大、效价高、特异性强.     

运用流式细胞术快速检测汉滩病毒滴度

目的:建立用流式细胞仪快速测定汉滩病毒滴度的方法.方法:汉滩病毒76-118株感染Vero-E6细胞,以汉滩病毒核蛋白单克隆抗体(mAb) 3G1为一抗,FIX标记的羊抗小鼠抗体为二抗,用流式细胞术(FCM)检测阳性细胞率,评价感染后不同时间点,以及不同病毒接种量的阳性细胞率,并与间接免疫荧光法对比.结果:感染后36 h阳性细胞百分率为(10.06±0.42)%,最低检测的病毒滴度为100 TCID50/ML.结论:相对于传统的空斑实验及间接免疫荧光滴定法,FCM是一种简单、快速、有效检测汉滩病毒滴度的方法.     

化学发光法和酶联免疫法作为筛选试验测定丙肝抗体的评价

目的比较化学发光法(CLIA)和酶联免疫法(EIA)测抗-HCV对诊断HCV感染的检出率,分析CLIA测定抗-HCV作为HCV感染初筛试验的重要临床意义。方法分别用CLIA、EIA方法检测所有临床标本中的抗-HCV,选取抗-HCV初筛阳性样本进行HCV RNA确认试验。结果 6461例样本中,EIA和CLIA测抗-HCV阳性率分别为2.86%和3.17%;确认试验中,CLIA法抗-HCV阳性与HCV RNA的符合率为97.1%,显著高于EIA组(91.9%)。EIA法测抗-HCV,S:CO>5.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为69.7%;CLIA法测抗-HCV,S:CO>2.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为92.9%。有31例样本CLIA法抗-HCV和HCV RNA阳性,而EIA法抗-HCV阴性;有4例样本EIA法检测为阳性,而CLIA法和PCR确认试验均为阴性。结论CLIA法测抗-HCV作为HCV感染初筛实验,与传统EIA相比,特异性和阳性预测值更高,有效降低了假阳性率,有利于临床医生对HCV感染患者的早期诊断和治疗。     

Cell preparation in immune response by immune ribonucleic acid. II. Independence of T lymphocytes in immune response against T-independent antigens.

The immune response of congenitally athymic (nude) mice induced by immune ribonucleic acid (iRNA) to lipopolysaccharides of Escherichia coli 0-55 (LPS) was studied. The thymus-independent nature of the immune response of mice to LPS was confirmed and nude mice responded to LPS in a manner similar to normal mice. An iRNA preparation extracted from the spleen of nude mice immunized with LPS could induce the proliferation of rosette-forming cells (RFC) in nude mice. iRNA preparations were insensitive to treatment with deoxyribonuclease and pronase, but were inactivated by ribonuclease treatment. The active fraction of the iRNA preparation had sedimentation values in a sucrose density gradient between 7 and 16 S and comprised only a small fraction of the total RNA present in the spleen cells, thereby indicating that the active moiety was one or more species of RNA. The anamnestic response was induced by treatment with iRNA made from the spleen of nude mice immunized with LPS. An increase in the number of rosette-forming cells (RFC), plaque forming cells (PFC) and formation of humoral antibody to LPS was seen after injection with a small amount of LPS 4 weeks after iRNA treatment.     

Cell participation in immune response by immune ribonucleic acid. I. The role of T lymphocytes in immune response by immune RNA against T-dependent antigens

T- and B-cell participation in the immune response induced by immune ribonucleic acid (iRNA) preparations against T-dependent antigens was studied using athymic nude, neonatally thymectomized (NT) and cyclophosphamide-treated (CY) mice. The iRNA(T + B) preparations were made from the spleen of BALB/c mice immunized with these antigens. Injection of the iRNA into nude or NT mice caused an increase in the number of specific rosette-forming cells (RFC) and of memory cells capable of responding to secondary stimulus with a small dose of the corresponding antigen. Injection with T-dependent antigens or with iRNA(T + B) did not cause any immune response in CY mice, suggesting depletion of the B-cell function. The iRNA(T) and iRNA(B) were prepared, respectively, from the thymuses of BALB/c mice and from the spleens of nude mice which had been immunized with T-dependent antigens. Injection of nude mice with both iRNA(T) and iRNA(B) caused an increase in the number of specific RFC and the secondary antibody formation response after boosting with a small dose of the corresponding antigen. Injection of iRNA(T) preparation into nude mice could induce the anamnestic response after boosting. However, neither of the iRNA(T) or iRNA(B) preparation could induce in nude mice the proliferation of the number of specific RFC. These results indicate the presence of at least two kinds of iRNA preparations against T-dependent antigens and that the cooperation of iRNA(T) and iRNA(B) was required for the induction of immune response against T-dependent antigens.     

iRNA对家兔急性实验性血清病的免疫调节作用

本实验以一次性静脉注射BSA诱发的家兔急性实验性血清病(AESS)为模型。于免疫后连续4天用iRNA处理模型动物,观察了iRNA对AESS家兔的血清学和免疫病理学影响,并建立了家兔血清补体旁路活性的检测方法。结果表明:iRNA能降低AESS家兔循环特异性抗体和特异性免疫复合物水平,减轻血清补体消耗,减少肾小球内免疫复合物的沉积和炎性细胞的浸润,使肾炎病变得以缓解。提示:iRNA可能通过抑制特异性体液免疫应答,发挥对AESS的免疫调节作用,从而减轻其免疫病理损伤的程度。     

严重烧伤病人PMNCD11b/CD18受体表达及特异性iRNA对其调节作用

本文探讨了严重烧伤对病人外周血中性粒细胞 (PMN)CD11b/CD18受体表达的影响及特异性免疫核糖核酸 (iRNA)对其调节作用。结果发现 :(1)严重烧伤病人PMNCD11b/CD18受体表达率明显下降 ,至伤后第 10天时 ,分别只有正常的6 7 1%和 6 8 9% ,且其下降程度与烧伤面积成正比 ;(2 )伤后早期应用特异性iRNA可明显提高烧伤病人PMNCD11b/CD18受体表达率 ;(3)临床观察发现 ,治疗组伤后创面细菌培养阳性率 ,创面脓毒症及菌血症的发生率明显低于对照组 (P均 <0 0 5 ) ,创面愈合时间明显短于对照组 (P <0 0 1)。该结果为临床应用特异性iRNA防治烧伤后感染提供了实验依据。     

Properties of maize chlorotic dwarf virus and its ribonucleic acid

Maize chlorotic dwarf virus (MCDV) was purified by a procedure in which virus was precipitated at low ionic strength. MCDV contained 36.2% single-stranded ribonucleic acid (RNA). An RNA molecular weight of 3.2 × 10⁶ was found by sedimentation of formaldehyde-treated RNA in linear-log sucrose gradients containing formaldehyde and by electrophoresis of formaldehyde-treated RNA in formaldehyde-containing polyacrylamide gels. The relationship between distance sedimented and RNA molecular weight for the sedimentation analysis is described. The nucleotide composition of the RNA was 29.8% Ap, 28.6% Up, 24.4% Gp, and 17.2% Cp.MCDV appears to be unique among small, single-stranded RNA plant viruses. A large genome and high virion molecular weight (8.8 × 10⁶) probably account for its fast sedimentation rate and high buoyant density.     

Transfer of specific immunity with RNA.

The term "immune RNA" (iRNA), as presently used, refers not to a characterized species of RNA but to phenol extracts of lymphocytes from immunized animals, which have the capacity to transfer a variety of immune products or activities. These include synthesis of specific antibody, development of specific cellular (delayed) hypersensitivity, and production of immunoglobulin allotypic or idiotypic specificities. Recent evidence that iRNA might be used as a specific therapeutic agent to induce immunity to tumors has stimulated an increased interest in this area of research. The purpose of this article is to review the current status of studies on iRNA.     

Translation of potyvirus RNA in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) RNA in the mRNA-dependent rabbit reticulocyte lysate produced products which comigrated with potyviral inclusion proteins on polyacrylamide gels. The TEV RNA translation products comigrating with the 49,000 and 54,000 molecular weight TEV nuclear inclusion proteins were also immunoprecipitated by antisera specific to the nuclear inclusion proteins. The proteolytic peptide map of the comigrating in vitro translation products for TEV RNA was similar to that generated by the nuclear inclusion proteins. The PeMV RNA translation product comigrating with the cylindrical (pinwheel) inclusion protein was immunoprecipitated by antiserum to PeMV cylindrical inclusion protein. These results provided direct evidence that the inclusions associated with potyviruses consist of virus-coded, nonstructural proteins.     

Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

转移因子和免疫核糖核酸对LAK细胞抗肿瘤活性的影响

本研究用重组白细胞介素２（ｒＩＬ－２）联合转移因子（ＴＦ）或抗肿瘤免疫核糖核酸（ｉＲＮＡ）作用于外周血单个核细胞（ＰＢＭＣ），测定其对Ｋ５６２，Ｒａｊｉ及Ｈ７４０４细胞的杀伤活性。结果发现，高浓度ＴＦ（０．２５ｕ／ｍｌ）可抑制ＬＡＫ活性，ＴＦ进一步稀释（０．１２５ｕ／ｍｌ）可使其抑制作用消失。ＴＦ和抗肿瘤ｉＲＮＡ不能进一步提高最适剂量ｒＩＬ－２（５００ｕ／ｍｌ）诱导的ＬＡＫ活性，但能显著增强亚适剂量ｒＩＬ－２（２００ｕ／ｍｌ）诱导的ＬＡＫ活性，诱导ＬＡＫ活性增高的ＴＦ最适剂量为０．０３１ｕ／ｍｌ。ＴＦ或抗肿瘤ｉＲＮＡ单独不能诱导出ＬＡＫ活性，而当两者联合应用可诱导ＰＢＭＣ产生ＬＡＫ活性。本文为ＴＦ及抗肿瘤ｉＲＮＡ协同ＬＡＫ细胞疗法在临床应用提供实验基础。