啤酒泡沫活性蛋白质中Z4蛋白质的单克隆抗体制备鉴定(二)

目的：实验在前期研究的基础上,研究了啤酒泡沫活性蛋白质中Z4蛋白质的单克隆抗体制备（mAb）,对单抗特性进行鉴定,用于啤酒生产中泡沫活性蛋白质的鉴定与定量。方法：制备啤酒泡沫活性蛋白Z4蛋白的单克隆抗体,并测定单克隆抗体效价、单抗亚类鉴定及确定单抗识别位点,最后通用Western Blot免疫吸附法确定单克隆抗体的交叉反应性。结果：所得两株Z4蛋白质的单克隆抗体效价可达10^6以上,其均属于IgG1亚类,但具有不同的抗原识别表位,并且对泡沫活性蛋白均具有较好的反应特异性,与浑浊蛋白、LTP1和Z7无交叉反应。为快速鉴定啤酒泡沫蛋白质中Z4蛋白质研究提供了基础。     

大豆球蛋白A3酸性多肽的原核表达及B细胞表位分析

人工合成大豆球蛋白A3酸性多肽基因cDNA序列,构建原核表达载体pET30a-A3,将重组质粒转化大肠杆菌(E.coli)BL21后用IPTG诱导表达,经His亲和层析纯化,获得纯度约91%融合A3蛋白。Western blotting显示,所获得的A3融合蛋白可与对豆粕过敏的仔猪血清发生免疫反应,表明所得融合蛋白具有免疫活性。利用Lasergene7.0中Protean软件对A3蛋白氨基酸序列进行B细胞抗原表位预测和分析,推测其中97～113、156～160、169～176、186～193、271～284和295～312氨基酸区段是A3酸性多肽的B细胞抗原表位,其中169～176区段的平均抗原指数最高,表位抗原性最强。本结果为进一步研究该蛋白致敏机理及单抗制备奠定了初步基础。     

模拟肠液对Pen a1及其抗原表位免疫原性的影响

体外模拟人体肠液(SIF)消化分析Pen a1消化后免疫原性的变化规律。虾致敏蛋白Pen a1及其表位多肽经SIF消化后,用全抗体和表位特异性抗体检测Pen a1及其各抗原表位的免疫原性的变化,并测定表位多肽的SIF消化稳定性。结果表明,Pen a1的免疫原性在消化60 min内下降显著,在90 min后下降缓慢,生成的新片段仍具有免疫原性,但会逐步完全分解至SDS-PAGE无法检测出;Pen a1中5个抗原表位的免疫原性变化也类似。Western-blot表明五个表位抗体与生成的新蛋白结合程度不同,No.3、4、5抗体与20~30 ku处的新蛋白片段结合多于No.1、2。ELISA检测表明即使经过4 h的消化,免疫原性也只是降低了80%。Pen a1各表位消化稳定性为No.2No.1No.3No.4No.5;5个表位多肽的消化稳定性为No.3No.1No.4No.2No.5,与各表位上的酶切位点的个数呈负相关。可以得出Pen a1中No.2表位具有最高的消化稳定性,No.5表位表消化稳定性最差。     

柑橘PGIP的B细胞抗原表位分析和原核表达

柑橘是重要的经济园艺植物,但是部分人群在食用柑橘后会造成过敏现象,其中多聚半乳糖醛酸酶抑制蛋白(PGIP)是柑橘中重要的过敏原。本文利用Jameson-Wolf法、Kyte-Doolittle法、Emini法和Karplus-Schulz法对柑橘过敏原蛋白PGIP的抗原指数、亲水性、蛋白表面可及性及柔韧性进行分析,通过构建PET-28 a-PGIP载体转移到大肠杆菌(Escherichia coli)中进行原核表达。结果表明:当PGIP二级结构是β-转角和无规则卷曲,同时氨基酸序列的抗原指数0、柔韧性0、亲水性0和蛋白表面可及性1时的氨基酸时,过敏原蛋白PGIP的B细胞表位结合氨基酸的序列区域是在19~22、37~41、49~52、68~69、117~120、163~164、173~177、221~226、236~240。通过分析氨基酸的区域及原核表达,为寻找柑橘过敏原蛋白PGIP的B细胞抗原表位的最佳优势区域提供支持,同时也为消除柑橘过敏原蛋白PGIP对人体的影响的进一步研究提供理论基础。     

花生过敏原Ara h1免疫显性抗原决定簇的鉴别

为探索花生过敏原致敏机理,采用固相合成肽技术合成Ara h1的23条多肽。以花生过敏患者血清为抗体,鉴别和定位Ara h1的抗原决定簇。结果表明:Ara h1第21～34位,第89～98位,第393～403位,第498～507位,第594～605位氨基酸序列识别率在60%以上,为Arah1的抗原决定簇,其中第498～507位的多肽识别率为100%,是显性抗原决定簇,其氨基酸序列为RRYTARLKEG。用丙氨酸依次取代显性抗原决定簇的每个氨基酸,结果抗原决定簇的致敏性增强或丧失,说明Ara h1第499位和第503位的精氨酸和第502位的丙氨酸为降低Ara h1致敏性的关键氨基酸。     

花生主要过敏原Ara h 1线性B细胞表位的预测及鉴定

花生过敏由于其高致敏率和致敏严重性而引起了人们的广泛关注。Ara h 1是花生中的主要过敏原,属于Cupin超家族,通过抗原表位识别结合免疫球蛋白E引发花生过敏。本研究采用生物信息学分析花生主要过敏原Cupin超家族Ara h 1线性B细胞表位氨基酸组成,及其与Ara h 1二级、三级结构之间关系,通过质谱分析Ara h 1氨基酸序列中的抗消化肽段,并分析抗消化肽段与预测线性B细胞表位的关系。结果表明,Ara h 1的线性B细胞表位富含亲水性氨基酸和带电氨基酸;其二级结构没有明显的分布规律,具有一定的回转折叠结构;分析Ara h 1三级结构发现,表位主要位于单体之间的疏水相互作用区域,部分表位埋入三聚体构象内部;Ara h 1抗消化序列与表位之间存在部分重叠。综上,质谱检测体外模拟胃肠道消化肽段并结合表位生物信息学分析可以作为鉴定Cupin超家族线性B细胞表位的新方法。     

草莓轻型黄边病毒外壳蛋白抗原表位预测、克隆、表达及其免疫原性分析

应用IEDB、DNAStar、DNAMAN和Snap Gene等生物信息学工具对草莓轻型黄边病毒外壳蛋白的氨基酸残基序列进行分析。选择一段抗原性较强的肽段(位于外壳蛋白27~38氨基酸残基处),依据大肠杆菌(Escherichia coli)的密码子偏好性化学合成了该肽段的DNA编码序列(AE)。AE片段克隆至E.coli表达载体pET32a(+)的Eco RⅠ和XhoⅠ位点,获得重组质粒pET32a(+)-AE。含AE片段的开放阅读框长561 bp,编码了一个187氨基酸残基的重组融合蛋白,理论分子质量为20.29kD。重组质粒pET32a(+)-AE分别在E.coli BL21(DE3)和E.coli Rosetta中进行了诱导表达和条件优化。重组融合蛋白在E.coli Rosetta中的最佳表达条件为1.5 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)、35℃诱导表达2 h,产率为13.22 mg/L;在E.coli BL21(DE3)中的最佳表达条件是为0.5 mmol/L IPTG、30℃诱导表达2 h,产率为9.55 mg/L。重组融合蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离、质谱鉴定表明,其含有所预测的草莓轻型黄边病毒外壳蛋白抗原表位肽段AE。AE重组融合蛋白经Ni~(2+)亲和色谱纯化、EK酶切、分子筛除去融合蛋白标签后,免疫产蛋鸡。从免疫鸡所产鸡蛋中提取鸡卵黄免疫球蛋白(immunoglobulin of yolk,Ig Y),Dot-Blot检测抗体结合活性。结果表明,所提取的IgY可特异性识别AE肽段和SMYEV外壳蛋白。这一结果表明所预测的AE肽段具有较好的免疫原性。     

花生过敏原Ara h 2.02 二级结构和B 细胞抗原表位预测

为预测花生过敏原Ara h 2.02 的二级结构和B 细胞抗原表位,通过Genbank 数据库搜索到Ara h 2.02 基因,并推导出其相应的氨基酸序列,采用SOPMA 和DNAStar 软件预测该蛋白质的二级结构以及通过Jameson-Wolf 法、Kyte-Doolittle 法、Emini 法和Karplus-Schulz 法分别预测其抗原指数、亲水性、表面可性、柔韧性等参数,并综合分析预测该蛋白的B 细胞抗原表位。结果表明：在序列28～31、56～73、76～90、92、148、156～158、168～169 区域最可能是Ara h 2.02 蛋白的B 细胞抗原表位的优势区域。该结果将为寻找Ara h 2.02 的最佳优势表位提供支持,同时为该蛋白单克隆抗体的制备等研究提供理论依据。     

利用捕获法建立筛选鸭坦布苏病毒适配单抗的 表面离子共振方法

目的以表面离子共振(surface plasmon resonance,SPR)系统为技术平台,利用捕获法建立检测鸭坦布苏病毒适配单抗的SPR方法,并对抗坦布苏病毒单克隆抗体的亲和力及抗原抗体结合的动力学进行研究。方法将鼠抗抗体偶联于CM5芯片上,采用Kinetics程序将HBS缓冲液梯度稀释的不同浓度的单克隆抗体进行捕获反应,拟合解离曲线计算不同单抗的K_D值并确立检测灵敏度低限并对其他不同亚型及5种常见禽类病毒抗体进行特异性检测试验。结果测得3株单抗的K_D值分别为1.50×10~(-13)、1.16×10~(-11)、7.85×10~(-12),建立的表面离子共振方法在检测其他禽源病毒时无阳性响应信号,坦布苏重组E蛋白的检测极限达0.625nmol/L。结论该方法可用于快速筛选检测坦布苏病毒的稳定适配单抗,并可有效获知其亲和力数据,为大规模推广建立免疫筛查方法奠定基础。     

采用狂犬病毒G基因原核表达的间接ELISA方法

为获得狂犬病毒G基因并在大肠杆菌中进行表达,进而初步建立用于评价狂犬疫苗免疫效果的方法,根据狂犬病病毒RV(rabies virus)ERA株G基因序列设计引物,从病毒中提取出RNA,经逆转录并扩增得到RVG基因;将该基因片段定向克隆到原核表达载体pET28a(+)中,构建重组质粒PET-RVG并转化到大肠杆菌BL21(DE3)gold中,经IPTG诱导表达并确定表达的最佳条件;Ni亲和层析柱纯化获得重组G蛋白,并以G蛋白为抗原建立检测狂犬病毒抗体的间接ELISA方法.检测结果表明,经灰度分析纯化后纯度可达96%.     

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.     

LOCALIZED CONFORMATIONAL CHANGES OCCURRING IN MYOGLOBIN UPON HEATING AS REVEALED BY USE OF MONOCLONAL ANTIBODIES

Six rat monoclonal antibodies (mAbs) to beef myoglobin, which recognize characterized epitopes of the protein, were used as macromolecular probes to detect irreversible conformational alterations occurring in myoglobin heated over the range 55–85C for 60 min. Light scattering measurements indicated the formation of small amounts of aggregated protein between 55–75C and increasing aggregation above 75C. To exclude an increase of immunoreactivity for the polymerized molecules in ELISA tests, all heated samples were filtered. No oligomers were detected in these samples by size exclusion chromatography using an HPLC system. Using competitive and sandwich ELISA the denaturation midpoint was observed around 79C. However, four mAbs that recognize the heme pocket region demonstrated an increased immunoreactivity over the range 55–75C. These results indicate that the heme pocket region undergoes an irreversible conformational change upon heating at temperatures as low as 55–60C, far below the denaturation midpoint. At these same temperatures, no irreversible conformational changes could be detected in the other regions of myoglobin recognized by the corresponding mAbs.     

STRESS-70 PROTEINS IN PLASMA FROM STRESSED AND UNSTRESSED PIGS

Blood protein patterns from stressed and unstressed pigs were compared by denaturing and reducing gel electrophoresis and immunological detection by Western blotting and ELISA. No substantial differences in protein patterns or immunodetection with commercial anti-pig antibody were seen. However, in both stressed and unstressed pigs, the presence of a 70 kDa stress protein was found utilizing two anti-heat shock protein (HSP) antisera: an anti-70 kDa E. coli and an anti-recombinant pea HSP 70. Analysis of denatured and reduced proteins from the cellular fraction by Western blot using the anti-HSP antibody did not detect cross-reacting material from either, stressed or unstressed animals. In contrast, in plasma, both antibodies recognized 2 polypeptides of approximately 70 kDa. Analysis of the native plasma proteins by ELISA detected cross-reactivity only with the anti-E. coli HSP antiserum. These results indicate that stress-70 proteins are present in pig plasma and contain epitopes common to bacteria and plant stress-70 proteins.     

Assessment of the gluten toxicity of wheat and naan in Xinjiang Uyghur Autonomous Region,China

Xinjiang is a high-risk area for celiac disease (CD) regardless of genetic or environmental factors. However, no case has been reported yet in people living in Xinjiang. This study aims to explore the potential connection between diet and occurrence of CD in the Xinjiang population. To this end, the levels of T-cell stimulatory epitopes in 164 accessions of Xinjiang wheat were tested by using Western blot with monoclonal antibodies against α-gliadin epitopes Glia-α9 and Glia-α20. Three wheat varieties with remarkably low amounts of T-cell stimulatory epitopes were obtained. Western blot and R5 competitive ELISA were performed for the assessment of potential toxicity related to CD of naan. Results showed a reduction of gluten toxicity after wheat flour was processed into naan, suggesting it may have the potential to help to reduce the risk of CD for the genetically predisposed individuals.     

Preparation and characterization of monoclonal antibodies directed against bovine β-casein

Using hydridoma technology, three monoclonal antibodies (mAbs) specific for β-casein were isolated. ELISA and immunoblot analysis with β-casein and some of its purified and partially purified proteolytic fragments, identified the epitope of the mAbs in the second half of the molecule between residues 105 and 193. Our data differ from those of Kobayashi et al. (Biochim. Biophys. Acta, 1077, (1991) 11–18) who recently identified the epitope of an anti-bovine β-casein monoclonal antibody at residues 193–202 of the β-casein molecule.     

Immunological characteristics of monoclonal antibodies against shellfish major allergen tropomyosin

Two types of murine monoclonal antibodies (MAbs) against American lobster (Homarus americanus) were generated and characterized. Three purified MAbs were characterized to be specific to the shellfish major allergen tropomyosin. MAbs 5G5E1 and 1A3A7 were reactive to tropomyosin from crustacean species only, whereas MAb 2A7H6 was reactive to both crustacean and mollusk tropomyosins. None of the antibodies reacted to vertebrate tropomyosins. Competitive ELISA indicated that the antigenic epitopes recognized by the two types of MAbs were different from each other. In addition, competitive immunoblot results showed that the binding of shellfish-allergic patient IgEs to lobster tropomyosin was inhibited by the MAb 2A7H6 only. This finding suggests that the antigenic epitope for the 2A7H6 antibody might be similar or close to the allergenic epitope shared by crustaceans and mollusks. Consequently, the MAbs recognizing the different common antigenic epiotopes obtained in the present study would not only facilitate the allergen characterization of shellfish, but may also be useful for the development of specific and sensitive immunoassays for allergen quantification or epitope mapping.     

Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in Escherichia coli trxB and gor double mutant

The dengue virus is currently the most important flavivirus causing human diseases in the tropical and subtropical regions of the world. The envelope protein domain III of dengue virus type 2 (D2EIII), which induces protective and neutralizing antibodies, was expressed as an N-terminal fusion to a hexa-histidine tag in Escherichia coli. The expression of recombinant D2EIII of 103 amino acids in the soluble form can be achieved using suitable host strains, such as Origami, at a low induction temperature of 18 degrees C. The enhanced production of the soluble protein could be attributed to the thioredoxin reductase (trxB) and glutathione reductase (gor) double mutations in the Origami genome. The soluble and refolded D2EIII proteins were recognized by different antibodies including human patient antiserum. The immunization of rats with soluble D2EIII protein elicited the production of antibodies that could recognize the D2EIII protein in the D2EIII precursor protein and in C-terminal truncated dengue envelope protein type 1-4. Thus, this protein production system is suitable for the production of authentic recombinant dengue proteins that may be used in the diagnosis of the dengue virus infection or in vaccine development.     

TBa过敏原表位区段的融合表达及免疫活性分析

为了确定苦荞过敏原TBa(tartarybuckwheatallergen)的抗原表位及进一步了解荞麦过敏反应机制,本实验以苦荞过敏原TBacDNA序列为模板,设计引物,克隆苦荞过敏原TBa表位区段基因,分别构建TBa及两个表位区段原核表达载体,在大肠杆菌BL21(DE3)中进行表达并纯化,采用竞争ELISA对其免疫活性进行分析与比较。SDS-PAGE及WesternBlot鉴定和检测结果表明,目的蛋白在E.coliBL21(DE3)中可高效表达,其N端带有6个组氨酸标签。Ni2+-NTA琼脂糖柱亲和纯化得到了纯度较高的目的片段。ELISA实验结果显示,表达产物与荞麦食品过敏病人血清中的IgE具有特异的结合活性。本研究为揭示苦荞过敏蛋白结构与功能的关系奠定了基础。     

Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae

Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura⁺ phenotype. Finally, selection for Ura^? cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has ‘popped out’ the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.