组蛋白与某些单克隆抗体发生非特异性免疫反应的研究—自身免疫性疾病发病机理探讨

本文描述了应用免疫双扩散技术检查组蛋白与某些单克隆抗体发生非特异性免疫反应。对抗人C—反应性蛋白(CRP)4株单克隆抗体进行了鉴定,它们识别了CRP上不同的抗原决定簇。组蛋白能够与CRP,纤维连接蛋白(FN),结核杆菌的单抗腹水起免疫沉淀反应,但不与其多克隆抗体及骨髓瘤病人血清起反应。组蛋白能与某些单抗起反应,初步解释了在自身免疫性疾病中抗组蛋白抗体产生的可能机制,说明组蛋白在机体免疫中具有一定的免疫调节作用。     

结核分枝杆菌抗原分析及免疫交叉反应研究

目的：筛选和鉴定结核分枝杆菌特异性和保护性抗原，研究结核杆菌的免疫反应特点，以探索结核病诊断和治疗的新途径。方法：采用超声破碎和滤膜抽滤的方法分别得到菌体蛋白和滤液蛋白，通过Western blot试验用结核杆菌的单克隆抗体及结核病人血清来检测蛋白样品，把发生阳性反应的蛋白在BECKMAN LF3200／多肽氨基酸序列测定仪上进行N末端序列分析，并用结核杆菌的单抗对自身抗原组蛋白进行了检测。结果：结核杆菌的31kD和30kD蛋白与结核杆菌的单抗及病人血清反应均呈阳性，但与正常小鼠血清和健康人血清反应呈阴性。31kD和30kD蛋白的N末端序列分别为：Ala Glu Val Asp Trp Leu Val Phe Ala Val和Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr。结核分枝杆菌的单抗与自身抗原组蛋白能发生免疫交叉反应。结论：结核杆菌的31kD和30kD蛋白是免疫保护性抗原，对免疫交叉反应分子基础的进一步研究必将增加对结核免疫机理的了解。     

组蛋白与空肠弯曲菌及ENA组份交叉反应性的研究

木文采用3株空弯菌与核抗原(ENA)交叉反应性单抗为材料,通过ELISA及竞争抑制试验证实:1株单抗可与组蛋白结合(酶指数EI=343,竞争抑制率35％),提示组蛋白与ENA及空弯菌之间存在分子空间结构模拟现象,可能是该菌诱发自身免疫综合征的一个重要原因。同时对动物模型的检测显示,抗组蛋白抗体的检测可能具有早期诊断意义。     

抗人Ⅳ型胶原单抗研制及其特性

本文用胃蛋白酶提取的人胎盘Ⅳ型胶原免疫Balb/c小鼠,免疫脾脏细胞与小鼠骨髓瘤细胞融合后,经有限稀释法克隆获得两株分泌抗人Ⅳ型胶原单抗的杂交瘤细胞株CO_5和CO_6。用小鼠腹水和杂交瘤细胞培养上清纯化制备的两种单抗均属小鼠IgGl,K键,分别针对Ⅳ型胶原分子上的两个不同抗原决定簇。两种单抗特异性强,与人Ⅰ型、Ⅱ型、Ⅲ型、Ⅴ型、Ⅵ型、Ⅷ型胶原、Ⅳ型胶原7S区及人白蛋白等相关蛋白无明显交叉反应,且单抗效价高,亲和力强。     

抗人心肌肌钙蛋白Ⅰ单克隆抗体制备中免疫抗原的选取

目的　获得与人骨骼肌肌钙蛋白Ⅰ没有交叉反应的抗人心肌肌钙蛋白Ⅰ (HcTnⅠ )特殊位点的单克隆抗体。方法　选择了包括牛心肌肌钙蛋白Ⅰ (BcTnⅠ )、根据HcTnⅠ氨基酸序列的亲水性设计的 4个钥孔虫戚血蓝蛋白 (KLH)偶联肽段和一个八倍体多肽分子 (MAP)等 6种代用抗原 ,通过免疫、杂交和筛选 ,获得了能分泌抗 (HcTnⅠ )的IgG型抗体的杂交瘤细胞株 ,并对得到的抗体进行了特异性鉴定。结果　用其中 4种抗原免疫得到了抗HcTnⅠ的IgG型单抗的细胞株 ,其中的 3种合成肽可以得到分泌抗HcTnⅠ特殊位点的IgG型单抗细胞株。结论　为临床上检测cTnⅠ以诊断急性心肌梗塞 ,提供了可能抗体     

抗人心肌肌钙蛋白I单克隆抗体制备中免疫抗原的选取

目的 获得与人骨骼肌肌钙蛋白Ⅰ没有交叉反应的抗人心肌肌钙蛋白Ⅰ(HcTnⅠ)特殊位点的单克隆抗体。方法 选择了包括牛心肌肌钙蛋白Ⅰ(BcTnⅠ)、根据HcTnⅠ氨基酸序列的亲水性设计的4个钥孔虫戚血蓝蛋白(KLH)偶联肽段和一个八倍体多肽分子(MAP)等6种代用抗原，通过免疫、杂交和筛选，获得了能分泌抗(HcTnⅠ)的IgG型抗体的杂交瘤细胞株，并对得到的抗体进行了特异性鉴定。结果 用其中4种抗原免疫得到了抗HcTnⅠ的IgG型单抗的细胞株，其中的3种合成肽可以得到分泌抗HcTnⅠ特殊位点的IgG型单抗细胞株。结论 为临床上检测cTnⅠ以诊断急性心肌梗塞，提供了可能抗体。     

单克隆抗体斑点酶免疫试验检测粪便中血红蛋白的应用研究

应用抗人血红蛋白单克隆抗体(McAb)采用斑点酶免疫技术,对各种动物血及4842例健康人粪便进行潜血试验,研究结果表明抗人Hb-McAb斑点酶免疫试验最大特点是特异性强,不与羊、牛、猪、鸡、鸭鳝鱼的血发生交叉反应,此单抗仅与人Hb     

系列单克隆抗体分析癌胚抗原的决定簇图谱及生化特性

用7个抗癌胚抗原(CEA)、2个抗血型Lewis~v和H-2型抗原和2个抗上皮生长因子受体之系列单克隆抗体,分析了CEA的决定簇图谱、生化稳定性。结果表明,7个抗CEA的单抗有5个识别特异性抗原决定族,而另2个的决定簇与正常交叉抗原(NCA)有交叉。决定簇图谱分析表明,有2个单抗的对应决定簇是独立的,与其它的决定簇无交叉,且是识别CEA的特异决定簇;另5个单抗的决定簇彼此有交叉,但不完全一致。确定了CEA分子中包含血型前体抗原Lewis~y的决定簇。依据系列单抗对CEA的免疫学和生化稳定性分析,对其抗原决定簇进行了分类。     

空肠弯曲菌与可提取核抗原的分子交叉反应

取空肠弯曲菌（ＣＪ－Ｓ１３１）免疫的Ｂａｌｂ／ｃ小鼠脾细胞与骨髓瘤细胞ＳＰ２／Ｏ融合，制备出４种能与空肠弯曲菌和可提取核抗原（ＥＮＡ）起反应的单抗。以Ｈｅｐ－２细胞作基质的免疫荧光染色表明，４种单抗均呈阳性结果，证实为抗核抗体。当单抗腹水被空肠弯曲菌菌体吸收后，其抗ＥＮＡ的活性明显降低，证明这４种单抗与空肠弯曲菌ＣＪ－Ｓ１３１和ＥＮＡ存在交叉反应性。免疫印渍试验表明，４种单抗均与空肠弯曲菌ＣＪ－Ｓ１３１的４３ｋＤ外膜蛋白反应。这一结果提示：空肠弯曲菌ＣＪ－Ｓ１３１的４３ｋＤ外膜蛋白与ＥＮＡ之间可能存在相似的分子构象，这为自身免疫病发机制的分子模拟假说提供了实验的依据。     

用e抗原阳性血清制备抗-HBe单克隆抗体及其鉴定

目的：为了解决多克隆抗HBe抗体的来源困难和单克隆抗体的配对问题。方法：用人的e抗原阳性血清，经过提纯后免疫BALB／c小鼠，用杂交瘤技术获得8株能分泌抗HBe单抗的细胞株。结果：7株单抗为IgG1,1株11E8为IgG2a，8株单抗标酶后能与基因工程抗原制备的包被单抗配对的只有11E8，11E8酶标单抗的效价为1∶6400，并且仅与HBeAg起反应，与HBsAg，HBcAg，正常人血清均不发生交叉反应，用于临床标本检测中与多克隆抗HBe酶标抗体的结果一致。结论：血源性的单克隆抗HBe-HRP，可以替代多克隆抗HBe-HRP制备诊断试剂盒，克服了多克隆抗HBe来源困难问题。     

Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein

Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.     

Impact of the heterogenicity of amino acid sequence on the immunoreactivity of an antigenic epitopic complex localized within amino acids 1192-1456 of protein NS3 protein of hepatitis C virus

Recombinant proteins were used to study the effect of heterogenicity of the primary structure of NS3 protein of hepatitis C (HCV) on the immunoreactivity of a complex of antigenic epitopes located within the amino acid sequences 1192-1456. Six genes encoding for the above fragment NS3 from different genotypes were collected from synthetic oligonucleotides and expressed in E. coli cells, by using the polymerase chain reaction. The homology of amino acid sequences of antigens ranged from 78.4 to 92.2%. All the antigens showed a higher coefficient of their reactivity with antibodies in the sera samples from patients infected by HCV of a respective genotype; however, there was no strong genotype-specific immunoreactivity. The findings lead to the conclusion that the primary structure of antigens has an impact on their immunoreactivity. Selection of variants of the primary structures of antigens is essential in developing a diagnostic test.     

Cloning studies on the gene coding for L-(+)-lactate dehydrogenase of Plasmodium falciparum

We show that the L-(+)-lactate dehydrogenase (EC 1.1.1.27;L-lactate: NAD+-oxidoreductase) of Plasmodium falciparum (LDH-P) is encoded in the parasite genome. A monoclonal antibody (McAb 7.2) has been shown to bind the LDH-P subunit which has an apparent molecular mass of 35 kDa. A polyclonal antiserum raised against affinity purified LDH-P has been used to isolate cDNA clones containing LDH-P epitopes from a lambda gt11Tn5 expression library. DNA sequence analysis of one clone, lambda LDH-P.1, reveals a single open reading frame which shows a degree of homology to the N-terminal domain of known LDH amino acid sequences.     

Comparative sequence of the helper component (HC) region of potato virus Y and a HC-defective strain, potato virus C

Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code for a protein (PVC-HC) which is similar in molecular weight and immunological reactivity to the helper component protein of PVY (PVY-HC). PVC-HC, however, was inactive with respect to its ability to effect aphid transmission of either PVC or PVY. The 5'-terminal 2.7-kb regions of PVC and PVY were sequenced. Within the HC region there was 92% nucleotide homology between the two strains; comparison of the derived amino acid sequences revealed 24 amino acid differences. Comparison of the PVC-HC sequence with that of five potyviruses revealed 2 amino acid changes which were specific to PVC-HC. These amino acids are prime targets for mutational analysis of HC activity.     

Complete nucleotide sequence of the structural gene for alkaline proteinase from Pseudomonas aeruginosa IFO 3455.

The DNA-encoding alkaline proteinase (AP) of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, the gene-incorporated bacteria expressed high levels of both AP activity and AP antigens. The amino acid sequence deduced from the nucleotide sequence revealed that the mature AP consists of 467 amino acids with a relative molecular weight of 49,507. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified AP reported previously. The amino acid sequence analysis revealed that both the N-terminal side sequence of the purified AP and several internal lysyl peptide fragments were identical to the deduced amino acid sequences. The percent homology of amino acid sequences between AP and Serratia protease was about 55%. The zinc ligands and an active site of the AP were predicted by comparing the structure of the enzyme with of Serratia protease, thermolysin, Bacillus subtilis neutral protease, and Pseudomonas elastase.     

Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-biphosphate carboxylase share a common antigenic determinant

An immunological relationship was detected between the coat protein of the common (U1) strain of tobacco mosaic virus (TMV) and the large subunit of the ubiquitous CO2-fixing host enzyme, ribulose-1,5-biphosphate carboxylase (RuBisCo). When assayed by Western immunoblotting or indirect ELISA, polyclonal antisera to TMV coat protein and to RuBisCo reacted with both antigens. In addition, a monoclonal antibody specific for the C-terminal antigenic determinant of TMV coat protein reacted with RuBisCo. Conversely, several monoclonal antibodies generated to the large subunit of RuBisCo reacted with TMV coat protein. This cross-reactivity was verified by an examination of the amino acid sequences of both proteins. A region of homology was found between the carboxy proximal portion of coat protein and the sequence 60-73 residues from the amino terminus of RuBisCo large subunit. This homology was not mirrored at the nucleic acid level because of different codon usages for the two proteins.     

The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies

To characterize the 40-kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS-polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non-carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40-kD allergen. Nucleotide sequence determination of the two lambda gt11 cDNA clones obtained showed that the 40-kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.     

SLE患者血清中病原体相关噬菌体7肽的检测

目的用噬菌体7肽库筛选系统性红斑狼疮（SLE）患者血清特异性抗体,测序分析其实际意义。方法先用30例正常人混合血清与噬菌体7肽库反应,未与正常人白清结合的7肽再与30例SLE患者混合血清结合,获得SLE血清特异性结合的噬菌体克隆。用患者混合血清进行Dot—ELISA实验鉴定获得的噬菌体克隆,进一步用SLE患者及正常人血清各12例筛选阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况,并对最终鉴定的噬菌体克隆进行测序与比对分析。结果混合的阳性噬菌体克隆与SLE患者个体血清反应阳性率明显高于其与正常人血清的反应率;序列分析显示阳性噬菌体克隆的抗原表位与杆菌、球菌、弧菌等微生物有同源性,与裂殖酵母属、链球菌属、立克次（氏）体属等有100％同源性,与人类抗原表位无关。结论SLE患者血清中存在与病原体抗原表位结合的抗体成分,提示SLE可能与病原体感染有关。     

Genetic polymorphism of the gene encoding the outer surface protein A (OspA) of Borrelia burgdorferi

The genes coding for the outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme borreliosis have been cloned and sequenced. Two German strains (skin isolate PKo and cerebrospinal fluid isolate PBi) have been analyzed. Using an OspA-specific monoclonal antibody (L32 2E7) for immunological screening of a genomic pUC 18 library of B. burgdorferi strain PKo, an OspA-producing clone was detected and subclones containing the open reading frame were constructed. The gene coding for the OspA protein of B. burgdorferi strain PBi was amplified using polymerase chain reaction (PCR) and cloned in pUC8. The open reading frame of both ospA genes consists of 822 nucleotides corresponding to a protein of 273 amino acids. Both proteins have a calculated molecular mass of 29.6 kDa. Molecular analysis revealed significant differences between each other and to already-published sequences of ospA of B. burgdorferi strains B31, ZS7 and N40 (the ospA genes of B31, ZS7 and N40 are nearly identical). The deduced amino acid sequences of the OspA protein of strains PKo and PBi showed a homology of 83% to each other and 77% and 80%, respectively, to OspA protein of strain B 31. The three proteins contain a variable middle region, whereas the N and the C terminus are conserved. This unexpected high dissimilarity of the ospA genes may be important in respect to vaccination studies and diagnostic procedures (i.e., development of PCR primers or serodiagnostic antigens). Moreover, the molecular heterogeneity of OspA confirms three out of seven immunologically defined OspA serotypes of a recently proposed OspA serotyping system.     

Sequence analysis of the Streptococcus mutans scrB gene.

The complete nucleotide sequence of the Streptococcus mutans GS-5 scrB gene coding for sucrose-6-phosphate hydrolase activity was determined. A potential ribosome-binding site as well as promoter sequences were identified upstream from the gene. The deduced amino acid sequence of the enzyme suggested a molecular weight of 51,750, which is similar to that estimated for the enzyme isolated from strain GS-5. The enzyme is slightly acidic, with a pI of 5.9, and is a relatively hydrophilic protein. The nucleotide and amino acid sequences of the enzyme showed significant homology with those of the sacA protein from Bacillus subtilis. In addition, a region of amino acid homology with the S. mutans fructosyltransferase and B. subtilis levansucrase proteins was also detected.