甲型副伤寒沙门菌鞭毛蛋白的分离、纯化和鉴定

目的提纯甲型副伤寒沙门菌鞭毛蛋白并鉴定。方法选用甲型副伤寒沙门菌,经自制的液体培养基扩大培养后,酸裂解法初步分离出鞭毛蛋白,用AKTAExplorer蛋白纯化系统除盐,弱阴离子交换层析纯化。纯化的蛋白用SDS-PAGE、Westernblot和磷钨酸负染扫描电子显微镜(scanningelectronmicrocopy,SEM)观察并摄片鉴定。考马斯亮蓝法测定所获得鞭毛蛋白的产量。结果SDS-PAGE提示纯化的鞭毛蛋白为一条相对分子质量(Mr)为52×103蛋白带;免疫印迹试验亦提示条带在Mr52×103处;SEM观察发现该鞭毛蛋白呈丝状;三者均证实该蛋白为同一均质蛋白。蛋白定量测得每克湿重的细菌可提取(4.8±0.5)mg鞭毛蛋白。结论经酸裂解法可获得高产量的鞭毛蛋白,且易于纯化和鉴定。     

syk真核表达载体的构建及其对人乳腺癌细胞MHC-Ⅰ类分子表达的影响

目的:构建非受体蛋白酪氨酸激酶Syk的真核表达载体,转染人乳腺癌细胞,使其在细胞中稳定表达,以研究Syk对人乳腺癌细胞MHC-Ⅰ类分子表达的影响.方法:以RTPCR法从人乳腺癌细胞株MDA-MB-468扩增出syk编码序列基因片段,将其克隆到真核表达载体pcDNA3.1D/V5-His-TOPO中.对重组质粒进行酶切、PCR及测序鉴定后,以脂质体介导法转染Syk缺失的人乳腺癌细胞MDA-MB-231,经G418筛选,构建syk基因稳定表达的细胞株,通过Western blot、RT-PCR和流式细胞术检测转染乳腺癌细胞Syk、MHC-Ⅰ及ICAM-Ⅰ的表达.结果:构建了hsyk真核表达载体pcDNA3.1D/V5-His-TOPO/hsyk,并在人乳腺癌细胞MDA-MB-231中获得稳定表达.表达Syk的MDA-MB-231细胞同时可以高表达MHC-Ⅰ和ICAM-Ⅰ.结论:成功地构建了syk真核表达载体并在真核细胞中表达,为进一步的肿瘤免疫研究奠定了实验基础.     

人胎儿血红蛋白的纯化及免疫活性鉴定

目的研究胎儿血红蛋白纯化的最佳方法,并对其进行免疫活性鉴定。方法采用生理盐水洗涤、四氯化碳萃取得到粗提胎儿血红蛋白,再经sephadexG50凝胶层析柱纯化。纯化后的胎儿血红蛋白利用SDS—PAGE电泳、蛋白质印迹鉴定其性质。结果获得了具有生物活性的胎儿血红蛋白．经SDS-PAGE电泳鉴定纯化蛋白杂带消失,蛋白单体相对分子质量为16000Mr,纯度为95％,蛋白质印迹结果显示纯化后的蛋白具有良好的抗原性。结论该方法成功获得了纯度较高的胎儿血红蛋白,且操作简便,纯化效果好。     

重阳木花粉过敏原的分析、鉴定与纯化

 【摘要】 目的　对重阳木花粉变应原蛋白进行分析、鉴定与纯化。方法　提取这重阳木花粉的粗提液,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS - PAGE)分离粗提液蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western - blotting)法鉴定其变应原成分,通过离子交换层析对重阳木花粉变应原进行初步纯化和免疫印迹鉴定。结果　重阳木花粉有18条主要蛋白带,12 000Mr和14 000Mr为重阳木花粉特异性变应原;通过离子交换层析方法纯化出重阳木花粉分子量为12 000Mr和14 000Mr的变应原主要分布在II峰中。结论　对重阳木花粉变应原进行了初步的分离、鉴定和纯化,为临床重阳木花粉过敏疾病的诊断和治疗奠定了基础。     

用于肿瘤靶向治疗的EGF-IL-18融合蛋白在昆虫细胞中的表达及活性鉴定

目的：制备能用于肿瘤靶向治疗的重组表皮生长因子受体干扰序列-白细胞介素18(EGF-IL-18)融合蛋白，并鉴定其生物学活性。方法：利用Bac-to-Bae表达系统在昆虫细胞Sf9中表达融合蛋白，用离子交换层析、疏水层析、凝胶过滤层析等方法纯化表达产物，并以IFN-γ诱导实验和EGFR竞争结合实验初步评价融合蛋白的生物活性。结果：经SDS-PAGE和Westem blot检测，Sf9细胞中表达的重组蛋白与预期的EGF-IL-18融合蛋白分子量一致，纯化后融合蛋白具有较高纯度和良好的免疫原性。IFN-γ诱导实验和EGFR竞争结合实验显示，该融合蛋白具有肿瘤导向性和抗肿瘤活性。结论：成功表达并纯化了具有肿瘤导向性的抗肿瘤蛋白——EGF-IL-18融合蛋白，该蛋白具有较好的肿瘤导向性和抗肿瘤活性，可用于肿瘤的生物治疗研究。     

兔抗Syk抗体制备和在乳腺癌诊断中的应用

目的：制备兔抗小鼠脾酪氨酸激酶(Syk)抗体，分析其在乳腺癌诊断中的价值。方法：应用重组小鼠Syk蛋白，常规免疫雄性家兔制备抗Syk抗体。用免疫印迹法检测Syk抗体的纯度和特异性。应用免疫组化法，检测乳腺组织中Syk蛋白的表达。结果：用重组小鼠Syk蛋白免疫家兔6wk后，静脉血中抗Syk抗体效价为1：6400(A450=1．03)。将Syk经SDS-PAGE分离，可见1条相对分子质量(Mr)为72000的蛋白条带．与理论值相符。用抗Syk血清做免疫印迹证实，在Mr为72000处有1条明显的带，证明是抗Syk特异性抗体。乳腺组织切片经抗Syk抗体免疫组化染色，在正常乳腺组织细胞胞浆中可见棕黄色颗粒，而浸润性乳腺导管癌组织的细胞浆不着色。结论：制备了高效价、特异性的兔抗Syk抗体。用该抗体检测证实，Syk在正常乳腺组织细胞中呈高表达，在乳腺癌组织细胞中表达缺失。有可能用于乳腺癌的临床诊断。     

利用聚丙烯酰胺凝胶电泳纯化的微量蛋白制备抗血清

目的　以SDS PAGE纯化的微量蛋白制备抗人层粘连蛋白受体前体蛋白 (LRP)的血清。方法　以SDS PAGE分离纯化原核表达的人LRP与 6×组氨酸的融合蛋白 ,切下并粉碎融合蛋白所在凝胶块。将含 10 μg融合蛋白的凝胶颗粒与弗氏佐剂混匀后 ,免疫BALB/c小鼠。以Westernblot检测所获抗血清的滴度和特异性。结果　以胃癌细胞总蛋白为靶抗原时 ,Westernblot显示 ,所获抗血清仅识别 1条Mr 约 4 2 0 0 0的蛋白带 ,将抗血清做 1∶2 0 0 0稀释后 ,仍能清晰地显示其靶抗原所在位置。结论　以聚丙烯酰胺凝胶颗粒为载体 ,用微量蛋白即可成功地制备抗血清     

艾蒿花粉主要变应原的分离、纯化与鉴定

目的　对我国蒿属花粉中常见、重要的变应原艾蒿花粉进行分离、鉴定与纯化。方法采用不同的提取液得到艾蒿花粉粗浸液 ,经饱和 (NH4 ) 2 SO4 分级沉淀后用聚丙烯酰胺凝胶电泳 (SDS PAGE)分离蛋白质组分 ,并用凝胶成像系统测定各组分的相对分子质量 (Mr) ;采用Westernblot鉴定其主要及次要变应原 ;通过DEAE CelluloseDE 32离子交换层析 (ionexchangechromatography ,IEC)和SephadexG 75凝胶层析 (gelchromatography)对艾蒿花粉变应原进行纯化。结果　分离后得到 2 0多种蛋白质组分 ,其中Mr 为 5 8× 1 0 3、38× 1 0 3、2 5× 1 0 3、2 0× 1 0 3、1 6× 1 0 3等 5个条带蛋白含量最丰富 ;分离到的蛋白质组分中有 9种蛋白能与确诊的蒿属花粉过敏患者血清中蒿属花粉特异性IgE结合 ,其中Mr 为 6 2× 1 0 3、4 3× 1 0 3、38× 1 0 3的蛋白条带的结合率最高 ;经纯化后仅得到Mr 为 6 2× 1 0 3的主要变应原。结论　艾蒿花粉的主要变应原Mr 分别为 6 2× 1 0 3、4 3× 1 0 3和 38× 1 0 3,层析技术可以对Mr 为6 2× 1 0 3的主要变应原成分进行纯化。     

人表皮丝集蛋白的纯化和鉴定

丝集蛋白是类风湿关节炎相关的自身抗体。利用乙醇沉淀、HiPrep 16/10 QXL凝胶结合高效液相色谱离子交换层析(HPLC)纯化人表皮丝集蛋白,聚丙烯酰胺凝胶电泳确定相对分子质量,并采用免疫印迹法(Western blot)、用抗丝集蛋白单克隆抗体进行鉴定。所获得的表皮细胞丝集蛋白相对分子质量44 000,纯度达到电泳纯,经免疫印迹法鉴定,抗原性良好。表明利用乙醇沉淀法结合HPLC系统,可高效、简便、快速地分离和纯化人表皮丝集蛋白。     

重组人Ⅱ型胶原250-270多肽的表达及活性研究

目的　表达、分离、纯化重组人Ⅱ型胶原 2 5 0 2 70多肽 (recombinanthumancollagentypeⅡpeptide 2 5 0 2 70 ,rhCⅡ 2 5 0 2 70 )并研究其对类风湿关节炎 (rheumatoidarthritis ,RA)患者外周血单个核细胞 (PBMC)和关节滑液单个核细胞 (SFMC)中特异性T细胞的活化作用。方法　利用DNA合成、聚合酶链式反应、重组DNA等基因工程的方法构建含有人Ⅱ型胶原功能性多肽CⅡ 2 5 0 2 70多聚基因的表达载体pGEX 4T 1,转化E .coliBL2 1,融合表达目的蛋白 ,经GlutathioneSepharose○R4B亲和层析柱分离纯化 ,12 %聚丙烯酰胺凝胶电泳 (SDS PAGE)和凝胶薄层扫描测量其相对分子质量 (Mr)和纯度。以不同浓度 (0 .1、1.0、10、10 0、10 0 0 μg/ml)的 6聚rhCⅡ 2 5 0 2 70和化学合成的CⅡ 2 5 0 2 70刺激体外培养的RA患者PBMC和SFMC ,等浓度的GST蛋白和PBS作为阴性对照 ,流式细胞仪测定刺激前后 2组培养细胞中T细胞CD6 9的表达率 ,以分析其对特异性T细胞的活化作用。结果　融合表达的目的产物经SDS PAGE分析 ,相对分子质量 (Mr)大小约为 43× 10 3,与预期相符。分离纯化的 6聚rhCⅡ 2 5 0 2 70经SDS PAGE凝胶薄层扫描 ,纯度为 89% ,浓度为 2 .1mg/ml。FACS分析 :经 10 0 μg/ml浓度的 6×rhCⅡ2 5 0 2 70刺激 3d     

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.     

Nuclear antigens: Characterization of the acidic nuclear ribonucleoprotein-Sm-antigen complex by analytical and preparative isoelectric focusing

The soluble ribonucleoprotein nuclear antigen (RNP), associated with the Sm antigen and present in extracts of rabbit thymus aceton powder, was purified by ion-exchange chromatography. Analytical isoelectric focussing (IEF) showed two bands at pH 5.2 and two bands at pH 5.4. After treatment by RNAse these 4 bands disappeared, and a new band with a pI of 6.1, representing the intact protein moiety, appeared. During preparative IEF in granulated gels the antigenicity of RNP was lost. Only the Sm antigen could be detected by counter-immunoelectrophoresis. Fractions with Sm antigenicity refocussed as two intense bands (pI 6.8 and 7.2) and one or two fainter bands (pI 6.1). On reducing SDS-PAGE the SM antigen revealed two bands of apparent molecular weights of about 110, 000 and 28, 500, indicating a total molecular weight of 138, 500.     

人脂联素球状结构域基因在昆虫杆状病毒表达系统中的表达

目的利用杆状病毒表达系统表达人脂联素球状结构域（gAd）基因。方法以人基因组为模板,PCR法扩增人gad基因,将gAd基因与供体质粒pFastBacHTB连接,转化含有穿梭载体Bacmid的大肠杆菌DH10Bac。筛选转座成功的重组穿梭载体Bacmid—gAd,通过脂质体介导,转染昆虫细胞Sf9,经SDS-PAGE、免疫印迹法检测表达产物。结果重组杆状病毒感染的Sf9细胞形态变化明显,SDS-PAGE电泳结果显示,BacmidgAd组和对照组相比．在相对分子质量15000～25000之间多出一清晰的蛋白条带．免疫印迹法证实该条带能与相应的抗His标签抗体结合。结论人gAd基因成功在真核细胞中表达,为后续的实验研究奠定了基础。     

蛋白激酶PKR的克隆表达及与其相互作用的蛋白质的亲和纯化

目的:克隆并表达双链RNA调节的蛋白激酶(PKR)基因,以纯化分离与PKR相互作用的蛋白质。方法:设计特异性引物,PCR扩增分别得到FLAG-PKR-HA和HA-PKR-FLAG基因片段,克隆至表达载体pSG5中。将重组质粒通过脂质体法转染PKR稳定沉默(PKRkd)的HeLa细胞,Western blot检测PKR及标签表达情况;最后,利用HA、FLAG串联亲和纯化系统(TAP)纯化分离与PKR相互作用的蛋白,Western blot技术及银染SDS-PAGE验证PKR蛋白复合物的纯化和分离情况。结果:成功构建了PKR融合蛋白表达载体及FLAG、HA亲和纯化系统,SDS-PAGE和银染结果显示TAP系统分离得到了PKR蛋白带和两条可能与PKR相互作用的蛋白质片段。结论:成功纯化和分离了与PKR相互作用的蛋白质,为进一步研究奠定了基础。     

人乳头状瘤病毒16型结构蛋白L1和L2在大肠埃希菌 …

目的　研究人乳头状瘤病毒(HPV)主要结构蛋白L1(HPV16L1)和次要结构蛋白L2(HPV16L2)在原核系统的表达。方法　采用pET30a载体分别构建pET30aL1和pET30aL2质粒,并分别转入大肠埃希菌BL21菌株,经IPTG(异丙基硫代βD半乳糖苷)诱导,表达融合蛋白6×HisL1和6×HisL2,用SDSPAGE和Westernblot方法进行检测。结果　6×HisL1和6×HisL2融合蛋白在BL21菌中高效表达,约2～3mgml,其相对分子质量分别约为60000和97000;6×HisL1降解较6×HisL2多。结论　HPV16结构蛋白L1和L2能在原核表达系统高效表达;6×HisL2稳定性高于6×HisL1融合蛋白。     

C6/36细胞感染登革Ⅱ型病毒前后差异表达蛋白的初步鉴定

目的 了解C6/36细胞感染登革病毒前后相关表达蛋白的变化情况。方法 分别提取C6/36细胞和登革Ⅱ型病毒感染后的C6/36细胞的可溶性蛋白质,SDS-PAGE凝胶电泳对比分析C6/36细胞感染登革病毒前后电泳图谱的差异;分别以正常C6/36细胞总蛋白质、C6/36细胞感染登革病毒后的SDS-PAGE凝胶图谱中的差异条带蛋白免疫Balb/c小鼠的免疫血清、Santa-Cruz公司登革病毒单抗作一抗,Western-blot鉴定C6/36细胞感染登革病毒前后SDS-PAGE凝胶电泳图中的差异条带。结果 SDS-PAGE凝胶电泳图显示约40 000 Mr和70 000 Mr处,C6/36细胞感染登革病毒后的蛋白条带比正常C6/36细胞的蛋白条带明显粗亮;Western-blot鉴定确认差异条带不是登革病毒在细胞表达的蛋白质,而是C6/36细胞本身表达的蛋白质。感染前后的40 000 Mr和70 000 Mr蛋白条带同源。结论 C6/36细胞感染登革病毒前后分子量为40 000 Mr和70 000 Mr蛋白表达有差异。     

Studies on allergens of Dermatophagoides pteronyssinus by direct and indirect RAST.

Dermatophagoides pteronyssinus (DP) extract was fractionated by Sephadex G-75 and liquid isoelectric focusing (IEF) and the allergenic activity of different fractions was monitored by direct and indirect RAST. The fractionation on Sephadex G-75 showed that the allergenic activity of DP extract was related to wide molecular weight spectrum components, even though the maximum amount was recovered in effluent that contained protein with a molecular weight ranging between 25,000 and 12,500 daltons. By fractionation of the mite extract on IEF, three main peaks of allergenic activity (pI less than 3.0; pI = 4.3 +/- 0.25; pI = 6.4 +/- 0.25) were found. Cross-inhibition experiments showed a high degree of cross-reactivity between allergenic material eluted in very distant regions of molecular weight or isoelectric point. The allergenic activity of unfractionated mite extract and of its IEF fractions was destroyed by pronase - but not by neuraminidase - treatment. These results suggest that DP extract probably contains one main allergen existing in multiple molecular forms rather than several distinct allergens and that a protein moiety of the allergen is necessary for the combination with IgE.     

Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan

A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori, or to another microbial HSBP.