柱层析法制备乙型脑炎纯化疫苗的研究

为制备纯化乙型脑炎灭活疫苗,以地鼠肾细胞培养并经灭活的乙型脑炎病毒液,浓缩后上Sepharose 4FF凝胶层析柱,用紫外线280nm波长检测得到三个吸收峰。ELISA法证实第一峰为病毒抗原峰,另两个峰为杂蛋白峰。试验证明Seoharose 4FF凝胶过滤对于提纯乙型脑炎病毒是有效的,能去除99％的杂蛋白。     

兔出血症病毒四个分离株的病毒多肽和血清学关系研究

使用PEG-DS两相系统和超离心提纯的兔出血症病毒(RHDV)四个分离株病毒,再经Sepharose 4B柱层析进一步提纯后,得到较纯的病毒粒子,回收率可达70％以上。应用常规双向免疫扩散试验,交叉血凝抑制试验和酶联免疫吸附试验(ELISA)对四个不同地区分离株间的血清学关系进行了比较研究。结果表明实验中的四个分离株病毒均属同一血清型。SDS-PAGE结果表明,这四个分离株病毒均含有四条多肽,分子量为28—64KD。各株病毒多肽的分子量和各多肽在病毒粒子总蛋白中所占比例略有差异。因此四个分离株的RHDV在蛋白结构上可能存在地区差异。     

Matrex Cellufine Sulfate在重组HBsAg纯化中的应用

建立纯化重组CHO细胞HBsAg的新工艺。将含有HBsAg的重组CHO细胞培养收获液,采用Butyl S Sepharose疏水作用柱层析、Matrex Cellufine Sulfate亲和柱层析、Sepharose 4FF凝胶过滤柱层析进行纯化,得到HBsAg纯品,该HBsAg经检定合格。HBsAg回收率为72％。     

苜蓿花叶病毒提纯方法的改进*

用来自于白车根草（Trifolium repens）上的一个苜蓿花叶病毒分离物AMV－SY为材料,比较了3种以差速离心为主结合PEG沉淀和超速离心提纯病毒的方法,对提纯病毒进行紫外吸收测定、电镜检查和SDS－聚丙烯酰胺凝胶电泳检测的结果显示：以交替使用含有0.1mol/LEDTA和0.1mol/L MgSO4的磷酸缓冲液作为病毒悬浮介质的提纯程度最为理想,该方法提取苜蓿花叶病毒的得率为47.6mg/100g昆诺藜鲜病叶,该病毒分离物的外壳蛋白分子量为29kD。该方法的病毒得率较高、杂蛋白较少、病毒粒子完整,是比较理想的提纯方法。     

阴离子交换色谱纯化甲型肝炎病毒(YN5株)的研究

对阴离子交换色谱纯化HAV的合适条件进行了探索。先使用“试管法”研究DEAE Sepharose Fast Flow凝胶结合HAV及病毒解离的条件,然后分别使用线性阶段洗脱和阶段梯度洗脱在柱色谱上进行了HAV的纯化。结果表明经过阴离子交换色谱纯化得到的病毒保持有抗原性和免疫原性,HAV抗原回收率大于85％,杂蛋白去除率大于80％,纯化的病毒样品中的内毒素与宿主DNA的含量也大大降低,证明阴离子交换色谱可用于HAV疫苗的纯化。     

杨尺蠖核型多角体病毒蛋白质的特性

本文研究了杨尺蠖核型多角体病毒(AciNPV)蛋白质特性,用反复调等电点法,从AciNPV的多角体蛋白中分离到A,B两种蛋白,Sepharose 6B柱层析和超离心沉降分析表明,两者均为一个峰纯,沉降系数(S20w)分别为4.9和8.9,对A蛋白进行了16种氨基酸组成分析,Glu、Asp和Leu含量高于其它氨基酸,不含Cys,用SDS-聚丙烯酰胺凝胶电泳和免疫双扩散法分析表明,用两倍体积饱和硫酸铵沉淀法制备的多角体蛋白,与提纯的多角体A、B蛋白之间无差异,多角体蛋白结构多肽由6种组成,分子量在12500—54000道尔顿范围内,其中32000是多角体蛋白的主要多肽,病毒粒子结构多肽和核衣壳蛋白分别由19和7个多肽组成,分子量范围分别在89000～13000和34000～13000之间,间接试验结果表明,在AciNPV中有碱性蛋白酶活性存在。     

人二倍体甲型肝炎灭活疫苗纯化技术的研究

研究人二倍体细胞甲型肝炎灭活疫苗的纯化方法。 经人二倍体细胞培养的病毒液经澄清、浓缩后制成粗制疫苗,采用Sephacryl S 400、DEAE Sepharose FF、Sephadex G 10等柱层析手段进行纯化。试验证明甲型肝炎病毒粗制疫苗经以上3种层析柱纯化后,抗原组分单一,收率达到85%,杂蛋白去除率达85%以上,蛋白总含量、牛血清残留量各项指标符合中国药典要求,适用于大规模生产。     

甜菜坏死黄脉病毒的分离提纯及其生物化学特性的研究

本研究工作中,建立了一个有效的甜菜坏死黄脉病毒的分离提纯程序,解决了该病毒粒体易于聚集难以提纯的问题,其操作要点是,(1)通过Sepharose 2B柱层析代替超离心,有效地除去一些小分子量核酸杂质;(2)经PEG再次沉淀浓缩后,调整pH至酸牲(pH3.0),使病毒充分悬浮以减少凝聚;(3)在病毒等电点(pH4.8～4.9)条件下,进一步沉淀以纯化病毒。根据病毒提取物的OD260/OD280比值,算出核酸含量约4.5％。核酸电泳出现4条带,分子量分别为:2.25×10~(?),1.8×10~(?),1.05×10~(?),0.75×10~(?)道尔顿。病毒提取物经超速离心出现4个界面,沉淀系数分别为,200.8S,165S,125.8S,100S。说明甜菜坏死黄脉病毒可能是4组分病毒粒体。病毒粒体含一蛋白亚基,分子量约为2.05±0.05×10~4道尔顿,由16种共199个氨基酸组成。     

金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

云芝菌丝体多糖的分离纯化研究

利用热水抽提从云芝菌丝体中提取胞内多糖,初步纯化后,用DEAE-Sepharose CL-6B进行分离,从中分离出两个带电荷多糖组分即CVP-I和CVP-Ⅱ,对这两个组分分别用Sepharose CL-6B凝胶柱层析进行纯度鉴定,均出现单一峰,然后用紫外扫描发现这两个组分均出现蛋白多糖的特征吸收,从而可以判断这两个组分是蛋白结合多糖。     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli

Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM.     

The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

Large Scale Purification of Isoinhibitors of Trypsin from Swine Colostrum Using Zinc Chelate Chromatography and Chromatofocusing

Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography on zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (Nll₄)₂SO4, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose.

Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide qel electrophoresis in the presence of sodium dodecyl sulfate and e-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.     

Evidence of the importance of pH and salt concentration on column chromatography with Sepharose gel filtration in separation of proteins.

Gel filtration chromatography with Sepharose agarose gel has been widely applied in the purification of enzymes because of its capability to separate macromolecules according to molecular size. Although a wide range of pH and salt concentrations have been suggested for its use, we have found that the selectivity, or efficiency, of separation is strongly affected by the pH and salt concentrations actually used. Separation is best at neutral pH with low salt concentrations. Increasing the molarity of the buffer or salt content (such as ammonium sulfate) in the protein sample will either broaden protein peaks resulting in poor separation or displace the peaks to a position of much lower apparent hydrodynamic volume. Rabbit plasma monoamine oxidase (MAO), a protein of 150,000 MW, when combined with 1.3 m (NH₄)₂SO₄ at pH 5.4, was found to be retained in Sepharose 6B column until the very end and elute with ammonium sulfate molecules. This behavior was attributed to severe morphological changes on the gel surface at acidic pH leading to a loss of selectivity. Evidence for this interpretation is provided by parallel experiments with Sephadex columns under identical conditions which excludes the possibility of dissociation of MAO into subunits and by scanning electron microscopy which demonstrates the change of surface morphology of the gel. The necesslty of a careful selection of optimum conditions for Sepharose gel chromatographic separation is therefore suggested.     

重组人角质细胞生长因子-2基因克隆、表达、纯化与活性分析

 从人胚肺二倍体细胞KMB17中抽提总RNA ,经RT PCR扩增获得编码人角质细胞生长因子 2 (keratinocytegrowthfactor 2 ,KGF 2 )的cDNA .克隆于硫氧环蛋白表达载体pThioHisA ,序列分析表明与文献报道一致 .经IPTG诱导 ,在大肠杆菌BL2 1中实现高效表达 ,表达量可达菌体总蛋白 10 %～15 % .菌体超声破碎 ,上清经CM SepharoseFF阳离子交换 ,Heparin Sepharose亲和层析 ,Superdex 75凝胶过滤层析纯化得到重组人KGF 2 ,纯度高于 95 % .生物活性分析表明 ,它能够促进成纤维细胞NIH 3T3的增殖 ,诱导鸡胚背根神经结神经轴突的生长 ,促进鸡胚尿囊膜血管生成 .研究结果表明 ,获得了 95 %纯度的具有生物学活性的重组人KGF 2 ,为进一步的基础与应用研究提供了基础     

重组人GM－CSF／MCAF融合蛋白抗血清的制备与鉴定

重组人粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人单核细胞趋化激活因子（ＭＣＡＦ）融合蛋白经ＳｅｐｈａｄｅｘＧ－７５和ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ两步柱层析,获得了电泳纯的ＧＭ－ＣＳＦ／ＭＣＡＦ融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ试验表明,该抗血清效价高、特异性好,可分别与ＧＭ－ＣＳＦ／ＭＣＡＦ、ＧＭ－ＣＳＦ和ＭＣＡＦ发生反应。     

重组人GM－CSF／MCAF融合蛋白抗血清的制备与鉴定

重组人粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人单核细胞趋化激活因子（ＭＣＡＦ）融合蛋白经ＳｅｐｈａｄｅｘＧ－７５和ＣＭ－ＳｅｐｈａｒｏｓｅＦＦ两步柱层析,获得了电泳纯的ＧＭ－ＣＳＦ／ＭＣＡＦ融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ试验表明,该抗血清效价高、特异性好,可分别与ＧＭ－ＣＳＦ／ＭＣＡＦ、ＧＭ－ＣＳＦ和ＭＣＡＦ发生反应。