1种改良的高效单特异性兔多克隆抗体的制备方法

目的建立1种改良的高效单特异性兔多克隆抗体的制备方法。方法用RT-PCR方法获得bax保守N端1～123位氨基酸基因片段,并将其插入pET42a原核表达载体,诱导表达的Bax融合蛋白组合应用GST、His亲和层析技术获得免疫原(pET42a/Bax融合蛋白),HPLC鉴定纯度达95%。利用改良快速免疫法获得人Bax兔多克隆抗体,并经蛋白A柱亲和层析技术,抗原亲和纯化技术获得高效价高特异性的抗体。间接ELISA检测抗体滴度、Western blot和免疫组化试验检测抗体特异性,并与商业化抗体进行对比。结果通过快速免疫法得到人Bax兔多克隆抗体,经过Protein A柱纯化,再经抗原亲和纯化后,间接ELISA证明,抗体效价均达1:51 200;Western blot显示,只有经过抗原亲和纯化后的抗体特异性高,无其他杂带;免疫组化证明,在原发性肝癌组织中,人Bax兔多克隆抗体能特异地和内源性Bax结合,其高效高特异性已达国外Santa Cruse公司Bax商业化抗体水平。结论快速免疫法与抗原亲和纯化相结合,获得人Bax高效单特异性兔多克隆抗体,建立了1种改良的高效单特异性兔多克隆抗体的制备方法。     

a-N-乙酰半乳糖胺酶多克隆抗体的制备及特异性鉴定

目的制备a-N-乙酰半乳糖胺酶(NAGA)的高效价高特异性的兔多克隆抗体。方法利用pET22b-NA-GA/BL21(DE3)工程菌株表达NAGA,经Ni2+Sepharose 6 FF亲合层析,Phenyl Sepharose 6 FF疏水层析两步纯化,得到纯度95%的NAGA作为抗原免疫新西兰白兔,获得NAGA的兔抗血清,并经HiTrap rProtein A柱纯化获得高效价高特异性的抗体;用间接ELISA法检测抗体效价,用Western blotting评价抗体特异性。结果制备得到的NAGA兔多克隆抗体血清效价为1×106,经过rProtein A柱纯化后抗体蛋白纯度95%,效价为1×105;Western blotting显示:该NAGA兔多克隆抗体只与NAGA发生特异性反应。结论获得了NAGA的高效价高特异性的兔多克隆抗体。     

双亲和柱分离纯化高纯度抗体Fab酶切片段

目的建立利用抗原亲和柱及蛋白G亲和柱纯化抗体木瓜蛋白酶酶切产物中高纯度Fab片段的方法。方法以抗体对应的抗原蛋白与NHS-activatedSepharose4FastFlow活化偶联填料偶联制备抗原免疫亲和层析柱,将抗体木瓜蛋白酶酶切产物分剐通过蛋白G柱及抗原亲和柱制备抗体的Fab片段。通过sDS-PAGE电泳、ELISA瓣3定及临床类风湿因子阳性血清的干扰实验鉴定其制备效果。结果抗体木瓜蛋白酶酶切产物分别通过蛋白G柱及抗原亲和柱后可以依次去除抗体Fc片段及未切开的抗体、木瓜蛋白酶及失活的Fab抗体片段,最终获得高纯度、高活性的Fab抗体片段。SDS-PAGE电泳、ELISA测定抗体Fc段被完全去除,双抗夹心nIsA检测能消除类风湿因子阳性血清导致的假阳性。结论成功建立了双亲和柱分离纯化抗体Fab片段的方法,该方法适用于快速、高质量、大规模制备Fab抗体片段。     

用兔抗人分化抑制因子3抗体建立双抗体夹心ELISA法及初步临床应用

目的 制备兔抗人分化抑制因子3（Id3）多克隆抗体,建立检测血清Id3的双抗体夹心ELISA技术。 方法 用纯化的Id3重组蛋白免疫新西兰大白兔,制备兔抗Id3多克隆抗体,通过多肽亲和层析柱去除非特异性成分,免疫细胞化学染色、免疫双向扩散及间接ELISA法检测抗体特异性和效价。制备辣根过氧化物酶（HRP）标记抗Id3,以纯化Id3重组蛋白为标准抗原制备标准曲线,建立定量检测人Id3的双抗体夹心ELISA法。 结果 免疫细胞化学染色显示,制备的兔抗血清能与人Id3蛋白特异性结合,所得兔抗血清效价分别为1 ∶ 8（琼脂双向扩散）和1 ∶ 8 000（ELISA）。ELISA法的包被抗体和酶标记抗体的工作浓度分别为5 μg/ml和1 ∶ 4 000,标准曲线为Y= 0.079 3+0.018 8X,r=0.997。检测线性范围为2～64 U/ml,平均回收率为98.2%, 批内平均变异系数（CV）为4.4%, 批间平均CV为7.2%。该方法可用于人血清Id3含量的检测。 结论 兔抗人Id3多克隆抗体的制备及夹心ELISA法的建立为今后研究Id3蛋白的功能及其在临床和基础医学中的应用奠定了基础。     

重组人tumstatin的原核生物表达和多克隆抗体制备

目的原核生物表达可溶性的tumstatin抗原并制备相应的多克隆抗体。方法利用原核表达载体pMAL-tumstatin在大肠埃希菌BL21中表达tumstatin,经Amylose Resin亲和层析柱和Q Sepharose Fast Flow柱纯化tumstatin,以纯化蛋白免疫新西兰兔,获得抗tumstatin多克隆抗体。利用western blot和ELISA法对多克隆抗体进行特异性和效价检测。结果tumstatin蛋白表达成功。SDS-PAGE分析表明其为可溶性表达。成功获得了tumstatin纯品及兔抗tumstatin多克隆抗体。ELISA法表明多克隆抗体效价>5 000。western blot检测证明多克隆抗体的特异性良好。结论成功表达、纯化tumstatin蛋白,并获得高特异性、高效价兔抗tumstatin多克隆抗体,为其在临床免疫学检测应用奠定了基础。     

CD26多克隆抗体的制备与鉴定

目的针对CD26酶催化结构域制备多克隆抗体。方法应用RT-PCR技术以人白细胞mRNA为模板,扩增获取编码CD26催化结构域的基因序列,克隆入原核表达载体PET32a后,转化BL21感受态细菌,经IPTG诱导表达得到his-CD26融合蛋白;亲和层析柱纯化并经Western-blot鉴定后,用此重组蛋白免疫2只新西兰纯种大白兔,获取免疫血清共180 ml,经蛋白A柱纯化及抗原抗体亲和纯化后,采用间接ELISA法检测抗体效价,Western-blot及免疫细胞化学染色进行抗体效价及特异性鉴定。结果构建出PET32a/CD26原核表达质粒,并在大肠杆菌BL21中获得高效表达,经his亲合层析纯化后蛋白量达2.3 mg/ml;制备的多抗血清纯化后效价达256 000,经Western-blot证明能特异性的识别CD26重组蛋白,免疫细胞化学染色显示能特异的结合于H9细胞。结论成功制备了能特异性识别CD26的多克隆抗体。     

沙眼衣原体LpxA蛋白的表达、纯化及多克隆抗体的制备

目的纯化沙眼衣原体LpxA蛋白并制备抗LpxA蛋白的多克隆抗体,为研究LpxA蛋白的功能奠定基础。方法 PCR扩增LpxA基因,以pET28a质粒为载体,构建表达质粒pET28a-LpxA,转化大肠埃希菌BL21,利用IPTG诱导含有6*His的融合蛋白表达,并使用Ni 2+亲和层析柱进行纯化。以纯化的His-LpxA蛋白作为免疫原,经背部皮下免疫新西兰兔,制备多克隆抗体,并使用免疫印迹法检测抗体与His-LpxA蛋白的反应以及使用ELISA法测定多克隆抗体的滴度。结果重组表达质粒pET28a-LpxA构建成功,融合His-LpxA蛋白的相对分子质量为32.8kDa,能够在大肠杆菌中高效表达,纯化后目的蛋白纯度约为95%,免疫新西兰兔后,抗血清能够识别重组的His-LpxA蛋白,其滴度大于1∶10 240。结论成功纯化了LpxA蛋白和制备了抗血清,为研究LpxA蛋白的功能提供实验基础。     

改良型TAT-VP3融合蛋白的原核表达纯化及多克隆抗体的制备

目的表达、纯化改良型TAT-VP3融合蛋白,并制备其多克隆抗体。方法利用原核表达载体PGEX-6P-1/改良型TAT-VP3在大肠杆菌Rosetta（DE3）中表达,经GST标签纯化树脂纯化蛋白,并用PreScission Protease酶切除标签,以纯化的蛋白免疫新西兰大白兔,获得改良型TAT-VP3融合蛋白的多克隆抗体。用ELISA进行效价检测,Western blotting鉴定其特异性。结果诱导表达并纯化了改良型TAT-VP3融合蛋白,纯度大于90%,蛋白浓度为1.2mg/ml。制备了该蛋白的多克隆抗体,ELISA表明多克隆抗体效价为1：32000,Western blotting证明多克隆抗体的特异性良好。结论成功纯化出改良型TAT-VP3融合蛋白,并制备出高效价、高特异性的多克隆抗体,为进一步研究该蛋白的体内外抗肿瘤活性及作用机制奠定了基础。     

Galectin-7多克隆抗体的制备与鉴定

目的 制备鼠抗重组半乳糖凝集素7(Galectin-7)多克隆抗体及其在膀胱癌组织中特异性鉴定。方法 PCR法从人包皮组织中扩增Galectin-7基因,并构建到pET28a表达载体中,使用IPTG诱导重组蛋白的表达,通过镍柱亲和层析纯化获得Galectin-7蛋白。以纯化的Galectin-7蛋白混合福氏完全佐剂免疫Balb/C小鼠制备抗体,并用ELISA,Western blot及免疫组织化学法检测抗体。结果 成功表达、纯化了Galectin-7重组蛋白,ELISA检测Galectin-7蛋白免疫的小鼠血清效价为1:32 000,Western blot显示该抗体能与天然的Galectin-7特异结合,免疫组织化学法检测结果表明该抗体识别人膀胱肿瘤组织的天然Galectin-7。结论 通过制备重组Galectin-7蛋白为免疫原,免疫家兔,成功地制备了效价高、特异性好的抗Galectin-7多克隆抗体。     

唾液酸结合性免疫球蛋白凝集素6的生物信息学分析及多克隆抗体制备

目的 表达并制备唾液酸结合性免疫球蛋白凝集素6(Siglec-6)的多克隆抗体,并对其特异性进行鉴定。为下一步探讨Siglec-6在介导细胞信号转导和调节造血细胞及免疫细胞的功能,研究其在免疫调节和炎症反应中的作用奠定基础。方法 通过生物信息学分析方法预测Siglec-6蛋白的跨膜结构、理化特性、疏水性等因素,设计出两个多肽,两个多肽各自与血蓝蛋白(KLH)交联后对新西兰兔进行免疫,结果发现这两个多肽均可作为抗原免疫生物试剂,并成功获得高效价的抗Siglec-6多克隆抗体。通过酶联免疫吸附试验(ELISA)法检测其效价,经过亲和层析纯化,用Western blotting测定抗体的特异度。结果 通过ELISA检测其效价分别为1∶51 200和1∶12 800,Western blot结果表明该方法所制备的抗体具有特异度。结论 该研究通过生物信息学方法成功预测了抗原表位,并据此预测成功制备了高效价、高特异度的抗Siglec-6多克隆抗体。     

光损伤诱导视网膜上皮细胞半胱天冬酶-3表达与促红细胞生成素的干预效应

背景:有实验表明,促红细胞生成素对视网膜的光化学损伤有保护作用,该作用与光化学损伤视网膜色素上皮细胞中半胱天冬酶-3表达的变化有关? 目的:采用光刺激诱导人视网膜色素上皮细胞凋亡,观察不同剂量促红细胞生成素对细胞半胱天冬酶-3表达的影响.设计:观察对比实验.单位:青岛大学医学院.材料:成人视网膜色素上皮-19细胞株购自美国细胞培养收集公司.DMEM/F12混合培养基、新生牛血清、胰蛋白酶购自GIBCO生物技术公司.重组人促红细胞生成素购自Sigma生物技术公司.人半胱天冬酶-3定量EIA试剂盒购自上海西唐生物科技有限公司.半胱天冬酶-3单克隆抗体购自美国Santa Cruz 公司;PV6001免疫组织化学试剂盒和DAB显色试剂盒购自北京中山生物技术公司.方法:实验于2006-05/2007-01在青岛大学医学院病理生理教研室完成.取成人视网膜色素上皮-19细胞株传代培养的2～5代细胞建立光损伤模型,传代细胞随机分为7组,每组4孔,①正常对照组:不加光照,不加促红细胞生成素药物干预.②光损伤模型组:光照12 h,不加促红细胞生成素药物干预.③光损伤 10 000U/L 促红细胞生成素组、光损伤 20 000 U/L 促红细胞生成素组及光损伤 40000 U/L 促红细胞生成素组:光照12 h,分别加10 000、20 000及40 000 U/L促红细胞生成素.④光损伤 40 000 U/L促红细胞生成素组 AG490组:光照12 h,加促红细胞生成素 40 000 U/L及Jak2激酶抑制剂 50 000 U/L.⑤光损伤 40 000 U/L促红细胞生成素组 蛋白激酶B特异性抑制剂组:光照12 h,加促红细胞生成素 40 000 U/L及蛋白激酶B特异性抑制剂100 μmol/L.主要观察指标:采用酶联免疫吸附实验和免疫组织化学法定量检测不同含量促红细胞生成素干预治疗前后视网膜色素上皮细胞半胱天冬酶-3表达的变化.结果:正常对照组半胱天冬酶-3无明显表达;光损伤模型组半胱天冬酶-3表达于视网膜色素上皮细胞核上,呈大量特异性黄色着色;光损伤不同剂量促红细胞生成素组半胱天冬酶-3表达的特异性黄色着色随促红细胞生成素含量增加而减弱,以光损伤 40 000 U/L 促红细胞生成素组最弱光损伤 40 000 U/L促红细胞生成素组 AG490组半胱天冬酶-3呈强阳性表达,光损伤 40 000 U/L促红细胞生成素组 蛋白激酶B特异性抑制剂组半胱天冬酶-3表达仍呈阳性,但较光损伤模型组稍弱.结论:重组人促红细胞生成素可减少视网膜色素上皮细胞的光损伤后半胱天冬酶-3的表达.重组人促红细胞生成素抗光损伤诱导的视网膜色素上皮细胞凋亡作用机制之一可能是抑制半胱天冬酶-3的表达.     

Absorption of enteral recombinant human erythropoietin by neonates

BACKGROUND: Erythropoietin (EPO) is present in amniotic fluid, colostrum, and human milk. One possible function of ingested EPO might be to stimulate erythropoiesis. However, it is unclear whether human neonates absorb recombinant human erythropoietin (rhEPO) after oral administration. OBJECTIVE: To determine whether circulating EPO concentrations increase following enteral administration of rhEPO to neonates. METHODS: The study was designed as a 2-center prospective, blinded, randomized, 2 x 2 crossover study, with each infant receiving 1 dose of rhEPO 1000 units/kg and 1 dose of placebo. Serum EPO concentrations were measured at baseline, 2, and 4 hours following study drug administration. The rhEPO and placebo dosing were separated by a mean of 72 hours. Analysis was stratified by gestational age (< or =35 wk, >35 wk) and feeding type (human milk, infant formula). RESULTS: No significant change in serum EPO concentration was identified at 2 or 4 hours following enteral administration of rhEPO. CONCLUSIONS: Enteral administration of a large dose of rhEPO to neonates <4 months of age did not result in increased circulating EPO concentrations at 2 or 4 hours following dosing, regardless of whether it was administered in human milk or infant formula.     

Ratio of baseline erythropoietin (EPO) level and corrected reticulocyte count as an indicator for a favourable response to recombinant human erythropoietin (rhEPO) therapy in anaemic cancer patients

PURPOSE: Recently, recombinant human erythropoietin (rhEPO) was introduced for the management of anemia in malignancy. To identify an indicator for a favourable response to rhEPO, 28 anaemic cancer patients undergoing chemotherapy and treated with rhEPO were evaluated. METHODS: Patients were classified into responder (16 of 28, 57%) and nonresponder (12 of 28, 43%) groups according to their responses to rhEPO therapy (response being defined as an increase in Hb level of > 2 g/dl from baseline without blood transfusion). RESULTS: Treatment with rhEPO showed significant improvements in the red blood cell (RBC) count, haemoglobin (Hb), packed cell volume (PCV), and reticulocyte count (ret. count) after 4 weeks. Upon analysing the baseline value of the EPO level and the corrected ret.count in these two groups, we found that the ratio of the EPO level and the corrected ret.count (EPO/ret.count) demonstrated a statistical significance (P = 0.03) in the prediction of response to rhEPO therapy. This ratio showed a sensitivity of 87.5%, specificity of 66.7%, and overall accuracy of 78.6%. CONCLUSION: Our study suggested that the baseline ratio of EPO/ret.count should be used as an indicator for a favourable response to rhEPO therapy.     

重组人红细胞生成素在肿瘤相关贫血中的应用

Itiswellknowthatrecombinanthumanerythropoietin（rhEPO）caneffectivelymanagechronicrenalfailure－relatedanemia．rhEPOcorrectedsymptoms，improvedqualityoflifeofanemiapatientsandreducedrisksassociatedwithheterotransfusion．Intheendof１９８０＇sandbeginningof１９９０＇s，someresearchersstudiedtherapeuticeffectofrhEPOinthetreatmentofanemiaduetootherdiseasesespeciallycancer－relatedanemiasuchaschemotherapyorradiotherapyinducedanemiaandneoplasticanemia．Manyfactorssuchasbleeding，infec－tion，hemo…     

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.     

Lack of acute cardioprotective effect from preischaemic erythropoietin administration in a porcine coronary occlusion model

BACKGROUND: Recombinant human erythropoietin (rhEPO) has been proposed to possess important tissue protective, apart from haematopoietic, effects. Cardioprotective effects have thus been reported in rodents exposed to myocardial ischaemia. Pathways common to the mediation of ischaemic preconditioning may be involved. Before clinical testing such possible cardioprotective effects needs assessment in an experimental large animal model with closer similarity to human ischaemic pathophysiology. METHODS: A control group and two rhEPO groups were studied. EPO1 pigs were given EPO corresponding to the early window and EPO2 pigs to the early and late window of ischaemic preconditioning in a closed chest, catheter-based, porcine coronary occlusion model (45 min of occlusion of the left anterior descending artery). Infarct size as a proportion of the ischaemic area (IS/AAR) was measured in vivo by myocardial perfusion imaging (MPI) and postmortem by a histochemical procedure (at 150 min of reperfusion). RESULTS: IS/AAR did not differ significantly between control (C), EPO1 and EPO2 groups, neither measured by MPI (mean+/-SD for C: 0.87+/-0.13; EPO1: 0.92+/-0.08; EPO2: 0.87+/-0.11), nor histochemically (mean+/-SD for C: 0.64+/-0.20; EPO1: 0.75+/-0.17; EPO2: 0.80+/-0.07). In the EPO2 group mean arterial pulmonary pressure and dP/dtmax were increased compared with control group. CONCLUSION: Despite promising results from studies in rodents, rhEPO did not reduce infarct size measured after 2.5 h of reperfusion in our porcine model.     

Comparison of carbamylated erythropoietin-FC fusion protein and recombinant human erythropoietin during porcine aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury

Purpose  

Recombinant human erythropoietin (rhEPO) attenuated ischemia/reperfusion (I/R) injury-induced spinal cord damage. Since carbamylated EPO derivatives are stated to be devoid of rhEPO side effects, we tested the hypothesis that a newly developed carbamylated EPO-FC fusion protein (cEPO-FC) would compare favorably with rhEPO.     

Erythropoietin and myocardial protection: what's new?

Erythropoietin (EPO), the principal hematopoietic cytokine produced by the kidney and the liver in fetuses, regulates mammalian erythropoiesis and exhibits diverse cellular effects in non-hematopoietic tissues. The introduction of recombinant human EPO (rhEPO) has marked a significant advance in the management of anemia associated with chronic renal failure. At the same time, experimental studies have unveiled its potential cardioprotective actions. As with other preconditioning agents, administration of exogenous rhEPO can confer myocardial protection against ischemia-reperfusion injury, in terms of reduction in cellular apoptosis and necrosis as well as improvement in myocardial functional recovery. The purpose of this study is to review current information regarding the various protocols used to investigate the effects of rhEPO administration as well as its cardioprotective properties. We also address the potential mechanisms underlying the protective effects of EPO. A better understanding of these mechanisms is essential for the development of clinical applications and the design of novel therapeutical strategies.     

促红细胞生成素对氯化钴诱导的PC12细胞凋亡的拮抗作用

目的 观察促红细胞生成素(EPO)对氯化钴(CoCl2)诱导的PC12细胞缺氧损伤凋亡的影响并探讨其作用机制.方法 将PC12细胞分为四组:空白对照组、CoCl2处理组、重组人促红细胞生成素(rhEPO)处理组、rhEPO+CoCl2处理组.应用MTT,乳酸脱氢酶(LDH),流式细胞术(FCM)及Hoechst33258染色法检测rhEPO对氯化钴诱导的细胞活性下降及凋亡的影响.通过逆转录PCR(RT-PCR)法检测Survivin表达情况.结果 MTT结果显示rhEPO预处理能够提高PC12细胞活力,0.6 mmol/L CoCl2单独处理组细胞存活率仅为(43.07±5.9)%,rhEPO处理24 h后细胞存活率上升明显至(77.89±3.4)%(P<0.01).LDH检测,FCM及Hoechst33258结果表明CoCl2处理能诱导PC12细胞损伤及凋亡,而预先采用2 U rhEPO处理PC12细胞可抑制CoCl2的细胞毒性作用,减少细胞缺氧性损伤及凋亡.RT-PCR显示rhEPO处理组PC12细胞Survivin表达较CoCl2处理组明显升高.结论 EPO处理能抑制CoCl2诱导的PC12细胞凋亡,其细胞保护作用的机制可能与上调PC12细胞的Survivin表达有关.