结核分枝杆菌FurB蛋白的表达及其单克隆抗体的制备

目的:制备针对结核分枝杆菌FurB蛋白的单克隆抗体(mAb)并分析其特性。方法:利用E.coli DH5α表达含有6×His的融合蛋白FurB;采用小鼠腹股沟皮下包埋硝酸纤维素膜的方法免疫小鼠,然后进行细胞融合、克隆化制备抗FurB mAb,用ELISA法初步鉴定其特异性位点和相对亲和力。结果:获得了高表达的融合蛋白,经SDS-PAGE分析,在相对分子质量15.0×10~3处有特异的目的蛋白条带。用该融合蛋白免疫小鼠后,获得了抗FurB mAb。结论:所获得的抗FurB mAb效价高、特异性强,为进一步研究FurB在结核分枝杆菌铁调控和致病过程中的作用提供了有效工具。     

抗结核分枝杆菌RpfB结构域单克隆抗体的制备及鉴定

目的:制备抗结核分枝杆菌Rpf B结构域单克隆抗体。方法:将p PRO-EXHT-Rpf B domain原核表达载体接种于大肠杆菌DH5中,用IPTG诱导表达Rpf B结构域蛋白,以纯化的Rpf B结构域蛋白作为免疫原,皮下包埋免疫小鼠3次,每次间隔2周;分离小鼠的脾细胞,与Sp2/0细胞融合,克隆化制备抗Rpf B结构域单抗,ELISA检测其效价,鉴定其特异性和相对亲和力,观察制备的抗Rpf B结构域单抗对Rpf家族其他蛋白的识别能力及其对结核分枝杆菌和藤黄微球菌的生长抑制作用。结果:制备了3株抗Rpf B结构域单抗,特异性高,亲和力较强,均能特异性识别Rpf B结构域。经小鼠腹腔注射制备腹水并纯化,获得了较高纯度的单抗,所制备的抗Rpf B结构域多肽的单克隆抗体可以识别多种Rpf样蛋白及其结构域蛋白。在抗体滴度为1∶1000时可有效抑制Rpf B结构域对结核分枝杆菌H37Ra和藤黄微球菌的生长促进作用,提示抗Rpf B结构域单抗可能会抑制进入机体内生长停滞或潜伏感染的结核分枝杆菌的再次激活,可能具有预防隐性感染复发的作用。结论:抗Rpf B结构域单抗的制备为进一步研究Rpf B结构域的生物学和免疫特性提供了实验工具。     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

结核分枝杆菌抗体检测蛋白芯片的制备及应用

目的:用重组结核分枝杆菌ESAT6、CFP10、M16和M38抗原制备相应的抗体检测蛋白芯片。方法:将制备的结核分枝杆菌抗原ESAT6、CFP10、M16和M38及购买的LAM抗原点于醛基化修饰的玻片上,制备成结核抗体检测蛋白芯片;使用该芯片对130例临床结核病患者和50例健康体检者血液样品进行检测,分析其敏感性和特异性,以及5项结核抗体的构成比。结果:结核杆菌抗体检测蛋白芯片的敏感性为90.8%(118/130),特异性为90%(45/50),LAM的检出率最高为91.5%。结论:用ESAT6、CFP10、M16和M38及LAM抗原制备的结核杆菌抗体检测蛋白芯片用于结核病辅助诊断的敏感性和特异性较高,可用于结核分枝杆菌抗体检测蛋白芯片试剂盒的开发。     

SARS冠状病毒囊膜E蛋白的重组表达与单克隆抗体制备

从基因库中调取SARS冠状病毒囊膜E蛋白基因序列,用连接PCR方法合成完整片段。测序确认后与人免疫球蛋白（IgG）序列Fc段连接并克隆至原核表达载体pPRO EX Hta中。从SDS—PAGE凝胶中回收表达产物后免疫BALB／c小鼠,经融合、筛选制备特异性单克隆抗体（mAb）。连接PCR扩增出231bp的DNA,测序结果与GenBank公布的序列一致,与人IgG序列Fc段基因连接后克隆至原核表达载体,在大肠杆菌中获得较好表达。表达产物经SDS—PAGE后,在相对分子质量（Mr）约37000处可见1条明显的诱导表达条带。Western印迹的结果表明,该电泳带与SARS患者的恢复期血清呈特异性免疫反应,,从SDS—PAGE凝胶中回收表达产物并免疫BALB／c小鼠,制备出2株抗E蛋白的mAb。这些mAb与SDS—PAGE凝胶上Mr约为37000的蛋白带也呈现很强的免疫反应。所获得的重组表达E蛋白及特异性mAb为进一步建立SARS病毒感染早期诊断的方法奠定了基础。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果：混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果:混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

鸡髓样分化因子88的原核表达及单克隆抗体制备

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。     

Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat-Shock Protein 60

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.     

结核分枝杆菌ESAT-6抗原鼠单克隆抗体的制备及初步鉴定

目的:制备重组结核分枝杆菌ESAT-6抗原鼠单克隆抗体.方法:采用杂交瘤技术,获得了12株针对结核分枝杆菌ESAT-6抗原的鼠单克隆抗体杂交瘤细胞株,对其中的5株进行了小鼠腹水的制备及相关鉴定.结果:5株单克隆抗体的腹水效价达到1:512 000～1:1 024 000,纯化后纯度高于90%,抗体亚类(型)均为IgGl...     

人免疫缺陷病毒I型Vif蛋白的原核表达、纯化及单克隆抗体制备

目的：克隆、表达、纯化人免疫缺陷病毒I型（HIV-1）Vif蛋白,制备其单克隆抗体。方法：提取感染了HIV的细胞基因组DNA,PCR扩增vif基因,插入表达载体pET32a,转化大肠杆菌BL21（DE3）获得工程菌株,IPTG诱导蛋白表达,Western印迹鉴定目的蛋白,亲和层析纯化目的蛋白;免疫BALB／c小鼠,制备单克隆抗体。结果：构建了Vif蛋白的原核表达载体vif-pET32a,并在大肠杆菌中获得高表达,目的蛋白以包涵体形式存在;纯化获得高纯度的重组Vif蛋白,蛋白浓度可达0．56mg／mL;建立了抗Vif蛋白单克隆抗体细胞株,制备了腹水,滴度可达1：16×10^6,抗体纯化后保持了活性和特异性。结论：在原核表达系统中表达、纯化了重组Vif蛋白,制备了针对Vif蛋白的单克隆抗体,为研究Vif蛋白的功能和抗原性奠定了基础。     

抗果蝇MRJ蛋白单克隆抗体的制备和鉴定

制备抗果蝇MRJ蛋白单克隆抗体可用于研究果蝇mrj的生物学功能,使用IPTG诱导重组质粒pET28a-mrj在大肠杆菌Rosetta中表达,重组蛋白经过Ni-IDA凝胶柱亲和纯化后免疫BALB/c小鼠。然后取免疫好的小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合,经克隆和筛选获得了能分泌抗果蝇MRJ蛋白单克隆抗体的杂交瘤细胞株。腹水制备后获得单克隆抗体,通过ELISA和Western Blot对所获得的抗体进行鉴定,结果表明所制备单克隆抗体能够特异性结合于原核及真核细胞表达的MRJ蛋白,可用于研究mrj基因的生物学功能。     

抗人线粒体核糖体小亚基蛋白17单克隆抗体的制备和鉴定

以纯化人线粒体核糖体小亚基蛋白17(MRPS17)免疫BALB/c小鼠,经细胞融合和ELISA法筛选成功获得1株抗MRPS17杂交瘤细胞。以所获特异性单抗作为一抗,使用Western印迹、免疫组化和免疫荧光等方法检测标本中MRPS17。结果显示:Western印迹检测人骨骼肌组织、黑素瘤组织和体外培养HeLa细胞提取蛋白质,在分子量约13kDa处有一特异性条带,与阳性对照纯化MRPS17相一致;免疫组化检测石蜡切片标本显示人骨骼肌细胞和恶性黑素瘤细胞胞浆中强阳性着色;细胞免疫荧光检测于培养的HeLa细胞,可见细胞核周围胞浆部位颗粒状绿色荧光,其分布与线粒体特异性荧光探针(MitoTrackerRedCM-H2XRos)的荧光分布一致。说明成功制备了具有高度特异性并可适用于多种检测方法的抗人MRPS17单抗,应用该单克隆抗体对人MRPS17进行了亚细胞水平定位,为线粒体生物学相关研究提供了新的研究工具。     

单克隆抗体在植物研究中的应用

单克隆抗体是现代生命科学研究的重要工具。随着分子生物学的发展,单克隆抗体在植物研究中发挥着越来越重要的作用。本文综述了单克隆抗体在蛋白表达、蛋白定位、蛋白相互作用、植物成分的定性与定量、植物成分纯化、植物病害检测、标签抗体等方面研究中的应用。     

Enzyme-Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody

Two enzyme-linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP-specific antigenic determinant. One ELISA is a four-layer system working in the concentration range 5-600 ng GFAP/ml. The other ELISA is a five-layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5-60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2-14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.     

GST-HAI-1融合蛋白的表达及抗人HAI-1单克隆抗体的制备

制备抗人肝细胞生长因子激活物抑制因子（HAI-1）单克隆抗体,为对HAI-1进行进一步的研究打下基础。将人HAI-1 cDNA分段克隆,构建GST-HAI-1融合蛋白原核表达载体,转化大肠杆菌后加IPTG诱导融合蛋白表达,经制备型SDS-PAGE法分离表达的GST-HAI-1融合蛋白,通过割胶、电洗脱回收融合蛋白,并以此为抗原免疫BALB/c小鼠,应用细胞融合技术制备产生抗人HAI-1单克隆抗体的杂交瘤,以ELISA、Western blot和免疫组织化学染色进行鉴定。最终获得抗人HAI-1单克隆抗体杂交瘤细胞株ZMC6,产生的单克隆抗体可特异性地与表达的GST-HAI-1融合蛋白反应,并可识别大肠组织中的膜型及脱落型HAI-1蛋白。该单克隆抗体的制备成功,为深入研究HAI-1的功能提供了有力工具。     

Comparative profiles of intramacrophage behavior of Mycobacterium tuberculosis and Mycobacterium avium complex with different levels of virulence

Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) strains with different levels of virulence in mice were examined for profiles of interaction with murine peritoneal macrophages (Mphis). Their growth rates in Mphis were in these orders: H37Ra strain (attenuated) > H37Rv strain (virulent) for MTB, and N-260 strain (moderate virulence) > MAC N-444 strain (low virulence) for MAC. MTB but not MAC caused the necrotic death of host Mphis in terms of increased release of lactate dehydrogenase from infected Mphis. The MTB H37Ra strain induced a greater production of reactive nitrogen intermediates (RNI) by Mphis than the MTB H37Rv strain did. However, this phenomenon was not observed with MAC, implying less important roles of RNI in the expression of Mphi antimicrobial activity against MAC organisms.     

Identification of a Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein Which Is Recognized by a Monoclonal Antibody 5-2-26

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of ³²P-labeled N protein showed that the protein contained phosphoserine and phospho-threonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354–389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.     

Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

单克隆卵黄抗体技术研究进展

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