Study of renaturation condition of anti-TNF-α recombinant antibodies

Recolnbinant Protein of antibody empressed in foreignhosts (generaly Eschewhia colt) may accumulate in insoluble fonnS--inclusion bodies, which has not any biological activity. In most cases, insoluble inclusion bodiestend to Predondnate at higher expression levels. Recentstudies have revealed that the refolding of protein as wellas dissolving inclusion bodies into functional Protein mayl)e associated with denatu~ and renatllrant. In this paperl we used two eXPression productS as objects, w…     

Effect of Yisui Shengxue Granule (益髓生血颗粒) on the Oxidative Damage of Erythrocytes from Patients with Hemoglobin H Disease

<正>Objective:To investigate the effect of Yisui Shengxue Granule(益髓生血颗粒,YSSXG),a complex Chinese medicine,on the oxidative damage of erythrocytes from patients with hemoglobin H(HbH) disease. Methods:Twenty-two patients with HbH disease and 22 healthy volunteers were observed.YSSXG was given to patients with HbH disease for 3 months.Before and after the 3-month treatment,blood parameters[hemoglobin (Hb),red blood cells(RBCs),and reticulocyte percent(Ret)]were examined;inclusion bodies in erythrocytes were observed by transmission electron microscopy(TEM);activities of antioxidant defense enzymes [superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(Cat)]and erythrocyte membrane malondialdehyde(MDA) concentrations were determined.Results:In patients with HbH disease,measured values of RBC and Hb obtained from the first to the third months after treatment with YSSXG were significantly higher than before treatment(P0.01).Measured values of Ret from the second to the third months after treatment were significantly lower than before treatment(P0.05 and P0.01,respectively).Prior to treatment with YSSXG,TEM images of RBCs showed the presence of numerous inclusion bodies.After treatment with YSSXG,the amount and volume of inclusion bodies decreased.Treatment with YSSXG also led to a significant increase in SOD activity(P0.01),a decrease in Cat activity(P0.01),and no significant differences in GSHPx activity(P0.05) or MDA concentration(P0.05).However,compared with the healthy counterparts,SOD, GSH-Px,and Cat activities presented at high levels(P0.01) both before and after treatment.Conclusions: YSSXG could improve the degree of hemolysis and anemia in patients with HbH disease.The mechanism may be related to its antioxidative effects,which could elevate the activity of total SOD in erythrocytes and efficiently inhibit the oxidative precipitation ofβ-globin chains.     

RADIOGRAPHIC MEASUREMENTS OF THE HEIGHTS OF VERTEBRAL BODIES IN THORACIC AND LUMBAR SPINE

Radiographic measurements was performed on 124 normal adults for anterior, posterior and middle heights of the vertebral bodies in thoracic and lumbar spine. The normal ratios of vertebral height in one vertebral body and one with the adjacent bodies were presented. The method for measurement and its diagnostic value to osteoporodc vertebral fractures were discussed.     

Purification and immunity analysis of recombinant 6His-HPT protein expressed in E. coli

Objective To obtain HPT protein (Hygromycin B Phosphotmnsferase), a kind of plant selective maker gene product expressed from E.coli and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into E.coli DH5α for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E.coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from E.coli by a relatively simple method, which has high immunity activity.     

Expression, Purification, and Refolding of Recombinant Fusion Protein hlL-2/mGM-CSF

Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.     

DETERMINATION OF POLYAMINES IN SKIN LESIONS AND URINE OF PSORIATIC PATIENTS AND ITS CLINICAL SIGNIFICANCE

From April, 1991 to Marth, 1992, according to the inclusion criteria, skin lesion biopsy specimens from 23 cases and morning urine from 60 caese of psoriasts in out-patient department of dermatology were collerted. Determination of polyamine levels in skin and urine specimens was carried out through high pressure liquid chromatography and ultraviolet spectrophotometry. Meanwhile, clinical manifestations were recorded in desinged form. Finally, all the variables were applied to a computor for the analysis of variance and linear correlation with a computer.     

A Comparative Study on Proteomics between LNCap and DU145 Cells by Quantitative Detection and SELDI Analysis

The differences in intracellular and extracellular protein expressions between human prostate cancer lines LNCap and DU145 were examined, The proteins of the two cell lines were extracted and condensed by using protein extraction kits. And the intracellular and extracellular proteins were quantitatively detected on a micro-plate reader by using bicinchoninic acid （BCA） method. The proteins in cell culture fluid were qualitatively assayed by SELDI-TOF-MS, The results showed that the intracellular protein contents of LNCap cells were extremely higher than those of DU145 cells. After serum-free culture, both intracellular and extracellular protein contents of LNCap and DU145 were decreased to some extent. And the intracellular proteins were decreased by 5% in LNCap and by 36% in DU145 respectively, while the extracellular proteins were decreased by 89% in LNCap and 96% in DU145 respectively. SELDI assay revealed that there were 5 marker proteins in LNCap and 6 in DU145. Although both LNCap and DU145 cell lines originated from human prostate cancer, they had some differences in protein expression.     

Characteristics of immunocomplex in autopsy tissues of hemorrhagic fever with renal syndrome.

Using repeated PAP or repeated PAP and ABC immunocytochemical methods, we were able to demonstrate viral antigens, Ig and CIq in the tissues of 20 autopsy materials that had been preserved for 3-30 years. Serial paraffin sections were stained with the first antibodies against both viral antigens (G2 and Np) and human IgG, IgM, C3 and Clq. Immunocomplex, composed of viral antigen, IgG and Clq were found diffusely in the cells of each organ and extracellalarly in the sera, various secretions and exudates. When stained by A25 etc, coarse granular antigen or inclusion bodies were found without demonstrable Ig and Clq. It was concluded that the immunocomplex in tissues of epidemic hemorrhagic fever in Shaanxi Province, China was both intracellular and extracellular and was perhaps soluble due to antigens in excess, with characteristics quite different from that of other immune diseases.     

Synergistic Effects of Chuanxiong-Chishao Herb-Pair on Promoting Angiogenesis at Network Pharmacological and Pharmacodynamic Levels

Objective: To investigate the synergistic effects of Chuanxiong-Chishao herb-pair(CCHP) on promoting angiogenesis in silico and in vivo. Methods: The mechanisms of action of an herb-pair, ChuanxiongChishao, were investigated using the network pharmacological and pharmacodynamic strategies involving computational drug target prediction and network analysis, and experimental validation. A set of network pharmacology methods were created to study the herbs in the context of targets and diseases networks, including prediction of target profiles and pharmacological actions of main active compounds in Chuanxiong and Chishao. Furthermore, the therapeutic effects and putative molecular mechanisms of Chuanxiong-Chishao actions were experimentally validated in a chemical-induced vascular insufficiency model of transgenic zebrafish in vivo. The m RNA expression of the predicted targets were further analyzed by real-time polymerase chain reaction(RT-PCR). Results: The computational prediction results found that the compounds in Chuanxiong have antithrombotic, antihypertensive, antiarrhythmic, and antiatherosclerotic activities, which were closely related to protecting against hypoxic-ischemic encephalopathy, ischemic stroke, myocardial infarction and heart failure. In addition, compounds in Chishao were found to participate in anti-inflammatory effect and analgesics. Particularly, estrogen receptor α(ESRα) and hypoxia-inducible factor 1-α(HIF-1α) were the most important potential protein targets in the predicted results. In vivo experimental validation showed that post-treatment of tetramethylpyrazine hydrochloride(TMP·HCl) and paeoniflorin(PF) promoted the regeneration of new blood vessels in zebrafish involving up-regulating ESRα m RNA expression. Co-treatment of TMP·HCl and PF could enhance the vessel sprouting in chemical-induced vascular insufficiency zebrafish at the optimal compatibility proportion of PF 10 μmol/L with TMP·HCl 1 μmol/L. Conclusions: The network pharmacological strategies combining drug target prediction and network analysis identified some putative targets of CCHP. Moreover, the transgenic zebrafish experiments demonstrated that the Chuanxiong-Chishao combination synergistically promoted angiogenic activity, probably involving ESRα signaling pathway.     

大肠埃希菌表达的含PTD穿膜序列的融合蛋白3种复性方法比较

目的:探索融合蛋白PTD-NFATminiDBD-eGFP纯化以及复性的条件,以提高其复性效率,为后续融合蛋白生物学功能的研究做准备。方法:表达、提取并纯化包涵体形式的融合蛋白PTD-NFATminiDBD-eGFP,分别用稀释复性、透析复性、柱上复性的方法,对融合蛋白进行复性,分析不同方法的特点及包涵体复性率,采用流式细胞术检测融合蛋白的穿膜活性。结果:3种复性方法得到的融合蛋白复性率依次为:柱上复性得率最高,其次是透析复性,稀释复性得率最低。柱上复性后蛋白的穿膜效率最高,相对较低的是稀释复性,而未复性的融合蛋白穿膜效率极低。结论:3种复性方法均能对PTD穿膜蛋白复性,但以柱上复性最佳。     

恶性疟原虫谷氨酸脱氢酶融合蛋白复性及纯化的研究

目的 建立恶性疟原虫谷氨酸脱氢酶(GDH)融合蛋白质的复性和纯化方法。方法 将含有恶性疟原虫GDH全长基因的融合蛋白表达质粒GDH／pGEX—4T—1转化到宿主菌BL21，该GDH／GST融合蛋白在IPTG的诱导下获得高水平的表达，经SDS—PAGE分析表达产物主要为不溶性的包涵体。经超声酶解法、离心、变性剂／去污剂洗涤后获得包涵体。包涵体经8mol／L尿素变性后分别采用分子筛柱层析、透析和稀释3种方法复性，并通过浊度分析、非还原SDS—PAGE等方法比较、优化复性方法和条件。复性后的GDH／GST融合蛋白经过不同柱层析方法进行纯化。结果 经SDS—PAGE分析表明，GDH／GST融合蛋白表达量可占到菌体总蛋白的25％左右；浊度分析表明：稀释复性法优于其它两种方法，同时20mmol／L，Tris—HCI、1mmoH，EDTA、pH8．5及GSSG／GSH=1：10为该融合蛋白稀释复性的最佳条件，其复性回收率可达90％以上。复性产物采用二步阴离子交换层析可获得较好的纯化效果。结论 通过稀释复性，二步阴离子交换层析可获得高纯度、具有天然空间构象的重组GDH／GST融合蛋白。     

大肠杆菌表达的人血管内皮细胞生长因子的复性与纯化研究

以包涵体的形式在大肠杆菌中表达人血管内皮细胞生长因子（VEGF121）,采用发酵罐进行工程菌的高密度发酵,得到菌干重46g／L,VEGF121包涵体4．5g／L。包涵体经洗涤、溶解、超滤法复性,得率为81％。复性后的目标蛋白经离子交换色谱及SepharcryS-100凝胶过滤色谱纯化,目标蛋白的纯度达到95％,目标蛋白的总得率31％。采用该工艺制备的VEGF121能刺激人脐静脉血管内皮细胞增殖,具有天然VEGF生物学活性。     

重组THANK诱导表达和变性、复性及纯化

目的 :获得较纯的具有生物学活性的 THANK蛋白。方法 :THANK基因在大肠杆菌中高效表达 ,菌体超声破碎后 ,以洗涤剂反复洗涤包涵体。包涵体经 8mol/L尿素变性溶解后 ,再用 Sephacryl S- 2 0 0凝胶过滤层析初步纯化 ,对重组蛋白浓度、氧化还原剂等复性参数进行优化和选择 ,将蛋白稀释复性。复性后组分经 Q Sapharose Fast Flow离子交换层析再次纯化 ,最后以 Sep hadex G- 2 5脱盐。 结果 :得到了纯度 >97%、具有一定生物学活性的 THANK蛋白。 结论 :THANK蛋白变性、复性及纯化方法的建立 ,为 THANK活性蛋白的制备提供了依据     

RNase等10种标准蛋白质在高效疏水作用色谱上的复性

目的: 研究变性蛋白质在高效疏水作用色谱(HPHIC)上的保留行为及复性效率. 方法: 分别用盐酸胍(GuHCl)、尿素(Urea)和十二烷基磺酸钠(SDS)将核糖核酸酶(Rnase)等10种标准蛋白质变性,然后在流动相为硫酸铵的条件下,用HPHIC对变性蛋白质进行复性,并测定其保留时间、Z值、质量及活性回收率. 结果: 在10种变性蛋白质中有5种在HPHIC上得到复性,其保留时间、峰形、Z值与天然蛋白基本一致,其中溶菌酶和核糖核酸酶的质量和活性回收率高,复性效果好. 结论: 用HPHIC对变性的标准蛋白质进行复性,得到较好的复性效果.     

抗胃癌单链抗体免疫毒素的制备及活性检测

目的 对抗胃癌单链抗体免疫毒素基因重组质粒进行表达,进而获得有活性的单链抗体免疫毒素,方法 应用IPTG诱导大肠杆菌表达系统进行单链抗体免疫毒素基因重组质粒的表达。并对产生的不溶性包涵体进行纯化,经变性、复性后,观察其生物学活性。结果 诱导表达了约６６ＫＤ的蛋白,分子量符合预计大小,以包涵体的形式存在于菌体中,经包涵体纯化、变性、复性,使其折叠为正确的空间结构,经验测可与胃癌细胞ＭＧＣ８０３特异结合,并有一定的细胞毒性。结论 获得了可与胃癌细胞系ＭＧＣ８０３特异结合。并有一定的细胞毒性的抗胃癌单链抗体免疫毒素。     

Purification and renaturation of recombinant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4GLU)) fusion protein.]

OBJECTIVE: To evaluate the effect of a novel approach for purification and renaturation of recombinant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4Glu)) fusion protein. METHODS: A novel purification method established in our laboratory was adopted for the purification of the inclusion body, and after renaturation, recombinant human IL2-PE66(4Glu) fusion protein was purified by DEAE-Sepharose FF ion-exchange chromatography. RESULTS: The purity of the fusion protein that retain its biological activity was as high as 95%, and a recovery rate over 80% of the refolded IL2-PE66(4Glu) fusion protein was achieved. CONCLUSION: The purification and refolding method for inclusion body adopted in this study is simple and practical, which lays the foundation for a large-scale production of the fusion protein.     

内皮抑素在大肠杆菌中表达、纯化及复性的研究

目的 探讨重组人内皮抑素(recombinant human endostatin,rhES)表达、纯化及复性的最佳生产方案.方法 活化rhES工程菌,诱导表达,提取并洗涤包涵体,然后变性、复性、浓缩.通过鸡胚尿囊膜实验(chorioallantoic membrane,CAM)检测rhES活性.结果 rhES产品纯度超过95%.CAM中rhES显著抑制新血管生长.结论 本实验方法可以提取到高活性、高纯度的可溶性rhES,为大规模生产rhES奠定了基础.     

抗胃癌鼠单抗3H11 scFv的包含体表达及柱上在位复性

目的:在大肠杆菌高效表达抗胃癌单抗3H0011的scFv.方法:利用PCR技术将3H11 scFv基因从分泌型表达载体转移至高效表达载体,获得3H11 scFv包含体的表达,经超声裂解、洗涤、变性后,尝试透析复性和凝胶过滤色谱柱上在位复性,结果:透析复性未能得到有活性的3H11 scFv,而柱上在位复性效果良好,获得有功能性的3H11 scFv.结论:成功进行了3H11 scFv的柱上复性,不同scFv在表达和复性中显示出明显差异,在基因工程抗体研制中值得注意.     

血管抑素3A工程蛋白的柱复性与离子交换纯化

由大肠杆菌以包涵体形式表达的一种抗内皮细胞生长工程蛋白(A nti-ang iogen ic agent,简称3A)经变性,Sephacry l S-100 HR柱复性,SP Sepharose FF离子交换吸附纯化,SephadexG-25脱盐,获得复性率为53.47%,HPLC纯度为92.52%的3A活性蛋白。以猪髋动脉内皮细胞为受检细胞,表明纯化蛋白具有抑制内皮细胞生长的特性。