银鲫卵黄原的诱导、分离纯化及其生化鉴定

以雄性银鲫为实验材料,通过雌二醇（Estradiol-17β,E2）的多次诱导,使得E2诱导产物成为血清中的主要蛋白。而后,在快速蛋白液相色谱（FPLC）系统上,利用高交换量的阴离子交换层析Q柱,成功的从血清中提纯了与雌性特异蛋白相一致的E2诱导产物。糖、磷、脂蛋白分析表明,它是一类糖磷脂蛋白大分子。同时,它能被Mg^2 -Ethylenediamine tetraacetic acid(Mg^2 -EDTA)部分沉淀,这进一步证明,与雌性特异蛋白相一致的E2诱导产物就是卵黄原（Vitellogenin,VTG)。聚丙烯酰胺凝胶电泳表明,银鲫有两种VTG分子。     

猕猴和小鼠分泌片的分离纯化及某些生化特性

为了深入了解分泌片和ＩｇＡ在粘膜免疫系统中的作用,自猕猴和小鼠胆汁中提取和纯化分泌片,ＳＤＳ－ＰＡＧＥ结果表明猕猴和小鼠游离分泌片的分子量均约为６０ｋＤａ,但在非解聚型聚丙烯酰胺梯度凝胶电泳时分子量分别为７４ｋＤａ和６２ｋＤａ。采用ＬＫＢ－８１００等电聚焦柱测定其ＰＩ范围,猕猴分泌片为４．３－５．９,小鼠分泌为３．９－５．４。免疫双扩散证明,猕猴和小鼠胆汁中的分泌片均能与兔抗人分泌片免疫血清发生交     

快速提取、纯化叶绿体4.5SrRNA的方法

我们采用植物叶与热缓冲液、苯酚直接混合(约65℃)匀浆,离心抽提和乙醇沉淀后,得到植物叶总RNA。经聚丙烯酰胺凝胶电泳分离、纯化,即可得到叶绿体4.5S rRNA,此法不仅操作简单,而且得率高。 同时,经过对同一植物的不同组织或不同细胞组分,如根、细胞质、叶绿体和叶绿体核糖体小分子RNA的提取与鉴定,以简便的方法证明了4.5S rRNA是叶绿体核糖体成份,也证明了我们所采用的提取、纯化4.5SrRNA方法的可靠性。     

艾氏腹水癌患鼠腹水中一种高分子量DNA结合蛋白的分离

 本文利用免疫吸收法和免疫亲和层析法,从艾氏腹水癌患鼠腹水DNA结合蛋白中,分离得到了一种高分子量DNA结合蛋白。在免疫双扩散反应中,它与抗艾氏腹水癌患鼠血清DNA结合蛋白的兎抗血清反应形成一条沉淀线,但与正常小鼠血清DNA结合蛋白的兎抗血清不形成沉淀线。该DNA结合蛋白样品用2-巯基乙醇还原后,经SDC-PAGE分析,测得其分子量约为41000。     

超胶AcA_(34)柱层析法分离提纯人血清IgD

本文用超胶AcA_(34)柱层析法从IgD型骨髓瘤病人血清中分离提纯人血清IgD。经聚丙烯酰胺凝胶电泳、SDS聚丙烯酰胺凝胶电泳、免疫电泳和免疫双扩散等方法检查其纯度及活性均较满意。这个方法简单方便,时程短,效果好。此外,还用超薄层胶等电聚焦电泳法得到了IgD的等电聚焦图谱,薄层扫描为五条带,等电点在5.4—6.0。     

尿素改善SDS-PAGE分离小分子肽的效果

目的：探讨尿素对Tricine—SDS—PAGE系统分离小分子蛋白的影响。方法：利用常规SDS-PAGE和Tricine-SDS-PAGE分离小分子肽,比较不同组成的分离胶的分离效果。结果：调整聚丙烯酰胺凝胶的分子组成,使丙烯酰胺和甲叉双丙烯酰胺的交联度为5．05％,能够改善小分子肽的分离效果。加入36％的尿素比加入甘油可以更有效分离1kD的小肽。但尿素胶聚合太快影响分离效果。结论：尿素改善SDS—PAGE分离小分子肽的效果,制作尿素胶时,宜采用低浓度的AP和TEMED使凝胶慢速聚合。     

用自制的GFP血清抗体做免疫印迹

介绍如何用自制的GFP血清抗体为本科生开展免疫印迹实验。实验包括大肠杆菌表达的GFP的超声破碎抽提、GFP的非变性PAGE制备电泳和SDS-PAGE制备电泳、GFP血清抗体的快速亲和纯化及最后的免疫印迹分析等实验组成的完整系列。学生通过本实验的训练在蛋白质免疫印迹的操作上有明显的进步。     

鲫鱼血清凝集素的生物学性质研究

采用硫酸铵盐析和梯度聚丙烯酰胺凝胶电泳技术,从鲫鱼血清中分离出一种天然凝集素,对其生物学性质进行研究。结果表明,该凝集素能够识别和凝集多种细菌,红细胞和酵母,不被透析,耐热,耐碱,抗DNase,RNase,SDS,对胰蛋白酶,糖苷酶和β-巯基乙醇敏感等特性,是一种分子量为67kD,等电点为6.2的糖蛋白分子,其活性能被乳糖、半乳糖,甘露糖和重金属离子所抑制,能显著促进巨噬细胞的吞噬,杀菌能力,是一种重要的免疫调节分子。     

现有溶酶体分离纯化技术的对比、分析和应用

溶酶体作为真核细胞中重要的细胞器,不仅是降解内外源物质的场所,也是细胞能量感知和调节的中心,能够协调细胞物质的转运、代谢和分泌。溶酶体稳态失衡会导致许多疾病,如溶酶体贮积症、肿瘤、免疫缺陷和神经退行性疾病。获得完整、高纯度的溶酶体是研究其微观结构、稳态调控和相关分子功能的重要前提。目前常用的溶酶体分离纯化技术包括离心分离纯化、荧光辅助细胞器分选、亲和免疫分离纯化、磁性纳米颗粒分离纯化和Lyso-IP等。该文综述现有溶酶体分离纯化技术的原理、特点和应用,并对它们进行对比分析。     

人血浆LDL的分离纯化及兔抗人apoB-100抗体的制备

目的：为分离得到较高纯度的人血载脂蛋白B-100（apoB-100）及制备高效的兔抗人apoB-100抗体。方法：采用两步密度梯度离心分离人血浆得到LDL,产物经6％琼脂糖凝胶（Sepharose-6B）分子筛分离纯化后,再由透析浓缩获得纯化产物即apoB-100,同时用5％聚丙烯酰胺凝胶（PAGE）电泳鉴定为一条带。用分离纯化得到的apoB-100与完全及不完全福氏佐剂混合,以此作为免疫原经4-3-2—2-1（周）免疫新西兰大白兔,随后心脏取血制备兔抗人apoB-100抗体。结论：分离纯化apoB-100浓度为1．4625mg／ml,制备的兔抗体经免疫双扩散测定其效价为1：32。     

Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid

Many studies employ bronchoalveolar lavage fluid for assessment of biologically active substances secreted from the lung. However, investigators continue to search for a useful reference standard to correct for the inevitable but variable degree of dilution of this fluid. The glycoprotein, soluble secretory component of IgA, may serve as a valid reference protein. We report a simplified method for the purification of secretory component from colostrum. Soluble secretory component was isolated from human colostrum using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography. Secretory component rich fractions were assayed by enzyme immunoassay. They were also evaluated for total amino acid content and distribution and sequence determination with satisfactory agreement with published results. We then demonstrated that soluble secretory component concentration in tracheal aspirate fluid did not correlate with either albumin or with total protein measured in the same samples. Therefore, we conclude that the secretory component of IgA serves as a useful reference marker because its use may avoid errors resulting from leakage of plasma proteins into epithelial lining fluid. Advantages of this method for establishing a standard for secretory component include ready availability of soluble secretory component, simplicity of the method and relative rapidity of the techniques.     

金樱子多糖的分离纯化及组成分析

对金樱子多糖的分离纯化方法作了探讨,筛选出较佳的工艺条件,通过紫外分光光度法,红外光谱以及气相色谱法分别对金樱子多糖纯品LP2的纯度,分子构型,单糖组成进行了分析;并通过了DEAE-纤维素（氯型）柱层析得一葡聚糖。     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.     

幽门螺杆菌尿素酶的纯化及其特性

本文利用Ｓｅｐｈａｃｒｙ１ Ｓ－２００和Ｑ－Ｓｅｐｈａｒｏｓｅ两步层析,从ＨＰ蒸馏水浸液中提取纯化尿素酶,并对其特性进行了测定,证明ＨＰ尿素酶各含一个６６ｋＤ和２９．５ｋＤ的亚单位,并以６个分子聚合成６２５ｋＤ大分子蛋白,有很好的抗原性,并显示特异的血清学反应。用ＨＰ超声浸液抗原和尿素酶,加入２μｇ霍乱毒素粘膜佐剂,给ＳＰＦ ＢＡＬＢ／Ｃ小鼠口服免疫,可使８０％小鼠抵抗ＨＰ活菌的攻击,证明尿素酶是一种抗ＨＰ的保护性抗原。     

长裙竹荪抑菌活性物质的分离纯化及其抑菌效果

长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明：该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。     

The binding of human milk lactoferrin to immunoglobulin A

To define the step at which translational initiation factor IF1 exercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor. It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5-fold the limiting Vmax of the 'pre-ternary complex'----ternary complex transition which represents the rate-limiting step in 30 S initiation complex formation. This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.     

烘炒法分离提取半乳甘露聚糖型种子胶

种子多糖胶分离提取工艺试验表明,根据种子胚乳坚硬的物理性能,用机械分离方法分离种子胚乳,其多糖胶抽提率大于80％。烘炒法适用于种皮厚而硬一类种子胚乳的分离,正交试验表明烘炒温度对种子多糖胶抽提率和粘度指标影响最大;皂荚胚乳的最优化分离条件是种子在80℃下预热60min,然后在160℃烘炒机中烘炒4min,再进入开片、选片和筛选工序;野皂荚胚乳的分离条件是种子80℃时预热30min,130℃下烘炒3min。     

PEG蛋白质分离纯化的新方法

ＰＥＧ化蛋白质的分离纯化比较困难,本工作发现ＰＥＧ化蛋白质仍能被硫酸铵盐析,据此可以简单地将ＩＬ－２及ＰＥＧ化ＩＬ－２沉淀出来,而将有毒的活化ＰＥＧ等副产物留在溶液中。此方法效果理想而又十分简便。文献中未见报道。此外,实验还发现,在ＰＥＧ－ＩＬ－２与ＩＬ－２的混和液中加入一定量的ＰＥＧ后,二者之间溶解度差别增大,当用ＳｅｐｈａｃｒｙⅠＳ－２００柱分离时,先用含１０％ＰＥＧ,０．２５ｍｏｌ／ＬＮａＣｌ的缓冲液平衡洗脱ＰＥＧ－ＩＬ－２,再降低盐浓度洗下ＩＬ－２,即可使二者完全分开。过去要将ＩＬ－２与ＰＥＧ－ＩＬ－２分离开非常困难,本工作解决了这个问题,这点亦未见文献报道。     

Transforming growth factor-β enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells

Transforming growth factor-β (TGF-β) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-β₁ on the expression of various plasma membrane determinants. TGF-β₁ induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-β₁-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-β₁-treated cells with an anti-human β-galactosyltransferase (β-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-β-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, β-GT. These results indicate that TGF-β₁ may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.