传染性牛鼻气管炎病毒单克隆抗体的制备及其对病毒结构蛋白的分析

采用改进的融合技术得到318个杂交瘤细胞系。经ELISA筛选,这些细胞系所寸泌的抗体均和病毒抗原发生反应。克隆出26个杂交瘤细胞系,制出特异的单克隆抗体,并采用ELISA、放射免疫沉淀试验(RIPA)、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Immunoblet、病毒中和试验等对单克隆抗体的特性作了鉴定。利用单克隆抗体对牛疱疹病毒I型[Bovine Herpcs Virus I(BHV—1)]主要结构糖蛋白作了分析。结果表明,这些杂交瘤细胞系分泌IgG和IgM两类抗体,其中IgG有192个(60.4％,其余126个39.6％)为IgM。免疫球蛋白IgG具有强的病毒中和作用。利用[~(35)S]—标记BHV-1病毒感染细胞溶解物,经SDS-PAGE分析、病毒至少有25个结构多肽成分,其中15个是主要的,被抗BHV-1免疫血清和单克隆抗体所识别的结构多肽分子量有180Kd、150Kd、130Kd、97Kd、71Kd、5bKd、37/33Kd和25Kd,除低分子量37/33Kd和25Kd外、其余均为糖蛋白。71Kd和97Kd与病毒的致病性有关,其相对应的单克隆抗体＃132、＃186、＃191和＃181、＃128、＃101、＃213等有强的或较强的病毒中和力;在还原条件下,单克隆抗体＃33、＃74、＃76可识别3个多肽,其分子量分别为130Kd、71Kd、56Kd,而在非还原条件下仅能识别130Kd,并证明130Kd/71Kd/56Kd为一三聚体。     

杂交瘤细胞系筛选方法Dot-ELISA的建立和应用

自杂交瘤技术问世以来,已在生物学、医学、兽医学等领域内推广应用,并利用杂交瘤技术成功地研究和揭示了许多疑难问题。在杂交瘤的筛选中,常用的有ELISA、间接荧光抗体及放射免疫等法,本文采用Dot-ELISA法进行猪伪狂犬病病毒单克隆抗体杂交瘤细胞系的筛选试验,经反复试验证明,它是一种杂交瘤细胞系筛选试验有用的方法。     

用基因工程表达的HBcAg制备抗-HBcAg单克隆抗体及其初步应用

用基因工程表达的NBcAg免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞Sp2/0融合,以ELISA抑制法检测抗体,获得28株分泌抗-HBc McAb的杂交瘤细胞系。对其中7株进行了克隆化,培养上清抗体滴度1:320～1:2560,免疫小鼠腹水和血清滴度1:10万～1:80万。Ig类型测定2株IgG1、2株IgG2a、1株IgG3、2株IgM。杂交瘤细胞系在体外连续传代培养6个月及液氮冻存的细胞系经复苏后仍能稳定地分泌McAb。得到的McAb只与HBcAg反应且能被HBcAg特异阻断,证明其特异性高。用ELISA间接法测定了McAb的相对亲和力,从50％最大结合的浓度分析结果,7株McAb的相对亲和力为2E6>2F5>2B11>2C10>1A7>1B7>1H7,浓度范围为0.6～10μg/ml。2E6和2C10 2株McAb与HRP结合,用ELISA双抗休夹心法筛选出2F5、2B111和H7 3株McAb适合作为包被抗体。2F5与2E6-HRP配对,建立了快速McAb-ELIS抑制法测定患者血清中抗-HBc。用本法测定了222份临床患者血清标本,结果与“华美”试剂盒测定结果基本一致。     

抗成骨肉瘤mAb的制备、鉴定及其细胞毒效应

目的 建立抗人成骨肉瘤mAb杂交瘤细胞系并对其抗体特性进行研究。方法 利用我室自建的骨肉瘤细胞系（OS——9607）免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞Sp2/0进行融合,经筛选和克隆化后,建立杂交瘤细胞系,以免疫组化和Western Blot等方法研究抗体特性,以MTT法研究细胞毒作用,结果 得到1株能够稳定分泌抗体,效价高的杂交瘤细胞系3D9,免疫组化结果显示着色区主要分布在细胞核上,相应抗原在成骨肉瘤标本和成骨肉瘤细胞系高度表达（83%）,正常组织未见表达,WesternBlot显示3D9相应分子量为Mr54000。MTT法示该抗体对成骨肉瘤细胞的杀伤率明显高于对照组。结论 得到的杂交瘤细胞系分泌的mAb体具有成骨肉瘤细胞特异亲和性,并有一定的细胞毒效应,可能成为新的成骨肉瘤生化标志,在肿瘤的免疫治疗方面有广阔的应用前景。     

抗人巨细胞病毒单克隆抗体的建立及初步临床应用

应用人巨细胞病毒AD169株(HcMV-AD169)免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,获得两株(1F9 2H10)分泌抗HCMV单克隆抗体(McAb)的杂交瘤细胞系。经鉴定,两株McAb的Ig亚类为IgG1,腹水效价间接ELISA法为10~(-5)和10~(-6)。两株McAb仅与HCMV反应,而与其他疱疹病毒无反应,2H10有中和病毒作用而1F9则无。用HCMV-McAb建立抗体捕获ELISA法测定150例孕妇血清中HCMV-IgM抗体,阴阳性总符合率与间接ELISA法相比较,为99.3％(149/150)。文中尚对HCMV-McAb用途作了讨论。     

基因工程乳腺癌特异性人单链抗体的表达与特性

研究解决分泌人单抗的杂交瘤细胞系难于稳定持久分泌抗体的难题，制备单链抗体，使单抗分子小型化，为进一步研究其在肿瘤诊断和治疗中的应用作准备。从分泌抗乳腺癌人单抗的杂交瘤细胞CM－1总RNA中，利用RT－PCR技术分别扩增出人单抗重链可变区VH基因和轻链可变区VL基因，将扩增产物纯化后克隆于pGEM-T载体中，进行DNA测序和序列比较分析后，将两者共克隆于表达载体中诱导表达，利用斑点免疫印迹及竞争抑制法检测表达产物的抗原性。所克隆的CM－1人单抗重链可变区和轻链可变区基因片段，分别属于人免疫球蛋白IgM Ⅲ亚群，和鼠κ轻链V亚群，ⅩⅦ家族，用斑点免疫印迹法检测可见表达产物能与人乳腺癌细胞特异结合；人CM－1单克隆抗体对此单链抗体与人乳腺癌细胞的结合有竞争性抑制作用，抑制率为75．7％。结论：成功地制备了可特异结合乳腺癌细胞的CM－1单链抗体。     

丁酸钠对杂交瘤细胞增殖及抗体分泌的影响

用含不同浓度丁酸钠完全培养液和无血清培养液培养分泌抗组织型纤溶酶原激活剂(t-PA)单克隆抗体的杂交瘤细胞。当丁酸钠浓度为0.25～0.5 mmol/L时,对杂交瘤细胞分泌抗体能力具有刺激作用,提高抗体效价2～4倍,对细胞生长无抑制作用。表明丁酸钠可作为一种添加剂用以提高杂交瘤细胞分泌抗体的能力。     

抗人IgD单克隆抗体的制备及其特性鉴定

本文采用Ultrogel AcA34凝胶过滤,DEAE离子交换及亲和层析法,从骨髓瘤病人血清中分离高纯度的IgG,以此为抗原免疫小鼠,利用常规杂交瘤技术制备了3株分泌抗人IgD单克隆抗体的杂交瘤细胞株,分别命名为ZD2、ZD61和ZD94,此3株杂交瘤细胞分泌的抗体与IgD有特异性反应,而与人其它类别的免疫球蛋白重、轻链无交叉反应,均属IgG1亚类。免疫转印结果显示,均能识别分子量为67kD的IgD重链条带。用ELISA法测定3株抗体分别识别两种不同的抗原决定簇。     

抗人重组SCF单克隆抗体杂交瘤细胞系的建立及初步鉴定

本研究利用PEX31B作为载体,在大肠杆菌表达出重组人SCF融合蛋白。用该蛋白作为抗原,免疫BALB/c小鼠。通过鼠-鼠杂交瘤技术,成功地获得4株分泌抗人重组SCF单克隆抗体(下称单抗)的杂交瘤细胞系。经检测,它们所分泌的抗体亚类均为IgG1,免疫印迹试验和抗体特异性鉴定证实,4株单抗均能特异性地识别可溶性SCF。抗SCF单抗杂交瘤细胞系的建立,为深入研究SCF的生物学特性、功能以及SCF产品的纯化,提供了有力的工具。     

条件培养液中杂交瘤生长因子对人—鼠杂交瘤生长、克隆化及抗体分泌的影响

本文报道了含有杂交瘤生长因子的人纤维母细胞系CRL1506和EB病毒转化经克隆化的人B淋巴母细胞系N23两种条件培养液,对分泌肾综合征出血热病毒人单克隆抗体的人-鼠杂交瘤和人—(人—鼠)杂交瘤细胞系的生长、克隆化及抗体分泌有明显促进作用,并排除了白细胞介素2和白细胞介素4作用的可能性。结合文献报道,初步认为来自这两种条件培养液的杂交瘤生长因子很可能含有白细胞介素6。正确选择促进人—鼠杂交瘤生长、克隆化和抗体分泌的条件培养液,对于克服生产人单克隆抗体杂交瘤技术所存在的融合率低、克隆化难、抗体产量少等困难具有较大的应用价值。     

抗HSPC238单克隆抗体的制备和初步鉴定

目的制备抗HSPC238的单克隆抗体，为HSPC238的功能研究奠定基础。方法以纯化的重组蛋白HSPC238为抗原，免疫BALB／c小鼠，运用杂交瘤技术制备HSPC238单克隆抗体，并用间接ELISA法和Western—blot法对单克隆抗体的特性进行鉴定。结果成功建立两株稳定分泌抗HSPC238的单克隆抗体杂交瘤细胞株，分别命名为E001和E002。ELISA检测抗HSPC238单克隆抗体的腹水效价为1：12800和1：25600。两株单克隆抗体的免疫球蛋白亚类均为IgG1。通过Western—blot实验证实，两株单克隆抗体均能特异性结合真核细胞内源性HSPC238蛋白。结论成功制备了两株效价高、特异性好的抗HSPC238单克隆抗体，制备的抗HSPC238单克隆抗体可用于HSPC238蛋白的鉴定，为HSPC238蛋白的生物学功能研究奠定了基础。     

The neutralization of pseudorabies virus by anti-alpha-galactocyl natural antibody in normal serum

Pseudorabies virus (PrV), a member of herpesviridae alphaherpes subfamily, can infect human cells in vitro. However, the transmission to Old World primates including humans is strongly restricted. In this study, we report the neutralizing activity of normal human serum against PrV grown in CPK cells derived from pig. PrV grown in all Old World primates-derived cells, which was tested in this study, were not neutralized by normal human serum. The virion of PrV grown in CPK cells harbored Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal epitope) on its surface, while PrV grown in Vero cells did not. Depletion of antibodies reacting to surface antigens of CPK cells negated the neutralization activity of human serum. Blockade of anti-alpha-gal antibodies by adding soluble Galalpha1-3Gal to normal human serum also prevent the inactivation of PrV grown in CPK cells. Although normal swine serum did not neutralize PrV grown in CPK cells, swine serum supplemented with exogenous anti-alpha-gal antibodies did. These results indicate that anti-alpha-gal antibodies in normal human serum contribute to the neutralization of PrV. Anti-alpha-gal antibodies in normal human serum may prevent transmission of PrV into humans.     

Acetylcholine activates latent pseudorabies virus in pigs

Summary Pseudorabies virus (PrV) was isolated from the nasal swabs and the cultured trigeminal ganglia of latently infected pigs after they were treated with acetylcholine (ACH). These results indicated that ACH activates latent infections of PrV.     

Production, isolation and characterization of monoclonal antibodies to cytochromes c of beef heart and Paracoccus denitrificans

Hybridoma cell lines secreting monoclonal antibodies which bind beef heart cytochrome c or Paracoccus denitrificans cytochrome c have been produced using spleen cells from BALB/c mice immunized with cytochrome c. Immunization was performed with either the native cytochrome c, succinylated hemocyanin-conjugated cytochrome c, or beef heart cytochrome c polymerized with glutaraldehyde. Of 10 such fusions, the hybridization frequency ranged from 0 to 42%. The cell fusion efficiency, the possible factors involved in the cell fusion efficiency and the frequency of antibody producing hybridomas are described. The percentage of hybridomas positive for anti-cytochrome c antibody production as screened for by radioimmunoassay or ELISA was 2%. Of the antibodies from 12 hybridoma cell lines which resulted from 10 fusions, three were specific to beef heart cytochrome c, another three were specific to P. denitrificans cytochrome c, and the remainder reacted with both cytochromes c. These groups of monoclonal antibodies react to different sets of sites on these two cytochromes c. The monoclonal antibodies from ten representative clones have been isolated and characterized by different methods.     

Pseudorabies virus infectivity for swine skin characterized in vitro

Summary The infectivity of pseudorabies virus (PrV) was demonstrated in a cell substrate derived from swine skin explant cultures designated primary porcine skin cells (^c/cSLA PPSC).^c/cSLA PPSC infected with either wild type or TK^– PrV strain Kaplan (Ka) developed typical cytopathologic changes (CPE) as early as 4 h post inoculation (p.i.). The CPE caused by PrV on^c/cSLA PPSC was specifically neutralized by covalescent swine sera. Synthesis of late viral proteins was demonstrated in PrV-infected^c/cSLA PPSC by indirect fluorescent antibody staining using monoclonal antibodies (mAbs) specific for PrV gIII. PrV induced protein synthesis was further confirmed by specific immunoprecipitation of³⁵S-methionine labeled viral polypeptides from PrV-infected^c/cSLA PPSC with PrV convalescent swine serum, PrV immune mouse serum or mAb to PrV gIII. Moreover, the virus progeny derived from^c/cSLA PPSC was shown to be infectious for MDBK cells and this infection was specifically neutralized by PrV convalescent swine serum. The capacity^c/cSLA PPSC to support a complete growth cycle of PrV and the relative ease of deriving these cells from pigs can be applied in an autologous fashion in studies of cellular immunity where the MHC needs to be matched.     

乙型脑炎病毒prM蛋白单克隆抗体的制备

目的 克隆日本乙型脑炎病毒(Japanese encephalitis virus,JEV)前膜蛋白(prM)编码基因,原核表达、纯化prM蛋白,以纯化产物为免疫原制备单克隆抗体(mAb);方法从感染JEV病毒的鼠脑中克隆编码JEV prM蛋白的基因并将其克隆入原核表达载体pET32a,转化大肠埃希菌BL21(DE3)LvsS后以IPTG诱导表达.表达产物经Ni-NTA纯化后进行SDS-PAGE分析.用纯化的蛋白免疫BALB/c小鼠,经细胞融合和亚克隆后筛选出能分泌识别prM蛋白的mAb的杂交瘤细胞株.用Western Blot和免疫组化方法检测单克隆抗体的特异性.结果 从鼠脑中克隆出约500 bp的JEV prM基因,将其克隆入原核表达载体中,在大肠埃希菌中获得了较好表达.纯化的prM蛋白免疫BALB/c小鼠,经传统细胞融合及筛选方法制备出单克隆抗体,抗体滴度为105.ELISA、Western Blot和免疫组化检测结果证实该株单抗具有较好的特异性.结论 成功的表达和纯化了日本乙型脑炎病毒的prM蛋白,并完成了单克隆抗体的制备,为乙型脑炎病毒感染的早期诊断及预防的研究奠定了良好的基础.     

Herpes simplex virus type 1 ICP-0 induces reactivation of pseudorabies virus from latently infected trigeminal ganglia explant cultures

Summary.  Pseudorabies virus (PrV), like other alphaherpesviruses, is a neurotropic virus that can establish a latent infection in swine. Reactivation of PrV from latency may occur spontaneously or after induction with corticosteroids. The mechanisms involved in the establishment of latency and reactivation are currently unknown. Here, we examined gene-specific reactivation of PrV by herpes simplex virus type 1 (HSV-1) immediate early protein, ICP-0. Primary neuronal cell cultures established from the trigeminal ganglia of latently infected swine were superinfected with recombinant adenoviruses expressing ICP-0. Reactivation of PrV occurred in cultures that were superinfected with two different ICP-0-expressing adenovirus recombinants, but not in cultures that were either mock-infected, or superinfected with wild-type adenovirus, or recombinant adenoviruses not expressing ICP-0. Infectious PrV was detected between 4 and 7 days postinfection, regardless of the promoter driving expression of ICP-0. Results from these experiments show that HSV-1 ICP-0, a homologue of PrV EP0, can induce PrV reactivation from explanted trigeminal ganglia of latently infected swine. Accepted October 14, 1997 Received August 4, 1997     

Role of glycoprotein gD in the adhesion of pseudorabies virus infected cells and subsequent cell-associated virus spread

Summary Pseudorabies virus (PrV) infected cells in suspension are able to adhere to a monolayer of uninfected cells by means of PrV glycoproteins expressed at the outer cell membrane, with gB and gC playing a major role as ligands and a heparinlike substance as receptor. In order to investigate the role of gD in this process and subsequent transmission of infectivity to contact cells, experiments with a gD deletion mutant, heparin and a monoclonal antibody (Mab) against gD were performed. The first indication that gD is active during cell adhesion was found by the observation that the binding of gD^– PrV infected cells was five times weaker than that of wild type (WT) PrV infected cells. Further evidence was given by the use of a Mab against gD. Preincubation of WT PrV infected cells with this Mab led to a reduction of the percentage adhering cells from 69% to 49%. The same Mab inhibited the heparin independent and heparin resistant binding of WT PrV infected cells indicating that gD is important during both processes. Furthermore, it was demonstrated in a plaque assay that, after contact with a monolayer, gD^– PrV infected cells in suspension were able to induce plaques with an efficiency of 1%. In conclusion, we can state that beside the interaction of the ligands gB and gC with a heparinlike receptor also the interaction of gD with a receptor which differs from a heparinlike substance mediates the binding of WT PrV infected cells to uninfected cells and that gD is not essential for the subsequent cell-to-cell spread of the virus.     

Monoclonal antibodies derived during graft-versus-host reaction. II. Antibodies detect unique determinants common to many MCF viruses

Hybridoma cell lines were recovered from the spleens of 6-week-old (B6 X D2)F1 mice undergoing graft-versus-host reaction induced by the transfer of 5-week-old B6 parental spleen cells. These cell lines produced antibodies reactive with envelope polypeptides of a variety of MuLV. The viral specificity assessed by membrane immunofluorescence and virus-binding radioimmunoassay indicated that the reactivity of these antibodies was distinctly different from monoclonal antibodies recovered from (B6 X D2)F1 recipients of D2 spleen cells reported previously (Portis et al., Virology 118, 181-190, 1982). Ten out of 17 monoclonal antibodies in the current study reacted exclusively with MCF viruses and three of these antibodies detected envelope determinants which were shared by a broad panel of MCF viruses of diverse origin. These common MCF determinants were expressed by the gp70 molecule as determined by Western blot analysis. The production of these antibodies by young mice in the absence of exogenous virus inoculation suggests that these antigens may be encoded by endogenous MCF-like sequences.     

Preparation and partial characterization of monoclonal antibodies specific for nuclear protein of avian influenza virus]

AIM: To prepare the monoclonal antibodies (mAbs) specific for nuclear protein (NP) of avian influenza virus (AIV) and identify their biological properties. METHODS: BALB/c mice were immunized with AIV (formaldehyde-inactivated AIV H9N2, Triton X-100-lysed H9N2 and AIV NP expressed in E.coli, respectively). Hybridoma cell lines secreting anti-AIV NP mAbs were developed through cell fusion, screening and cloning. The mAb's titer was determined by indirect ELISA. Specificity of mAbs was identified by cross-reaction test and indirect immuno-fluorescence assay (IFA). RESULTS: 6 hybridoma cell lines secreting anti-AIV NP mAbs were obtained, designated 4F4, 1C3, 1G11, 1C2, 1D10 and 2F7. ELISA detection showed that the titers of two mAbs (1G11 and 1D10) out of 6 mAbs were the highest (2(-13) and 2(-14), respectively) and their specificity was also better than that of the others, confirmed by cross-reaction test and IFA. CONCLUSION: In this study 6 mAbs against AIV NP were obtained. The mAbs 1G11 and 1D10 perform the best in titer and specificity. This work paves the way for AIV study and development of method for rapid detection of AIV.