从EDTA处理的重组E.coli中分离纯化重组人SOD包涵体

以金属离子螯合剂乙二胺四乙酸（EDTA）代替超声法使重组大肠杆菌菌体释放,并提取重组人超氧化物歧化酶包涵体,纯度为65％。采用透析法复性包涵体,经凝胶色谱纯化后,目的蛋白纯度约为90％,酶比活力为5400u／（mg蛋白）。     

重组人白细胞介素-6包涵体精制工艺研究

目的 建立重组人白细胞介素-6(recombinant human interleukin-6,rhIL-6)包涵体精制工艺,获取高纯度的包涵体蛋白.方法 采用超声破碎法和生物酶解法破碎细胞;摸索洗涤缓冲液中尿素的适当浓度,经多次洗涤获取高纯度的包涵体蛋白.结果 成功建立了rhIL-6包涵体蛋白的精制工艺,其中尿素浓度为2.0 mol/L,溶菌酶浓度达到0.8 mg/g菌体.包涵体蛋白洗涤后纯度达到90%以上.结论 建立合理的精制工艺,包涵体蛋白纯度达到90%以上.     

重组人血管内皮抑素的阳离子交换色谱复性与纯化条件研究

对rhEndostatin包涵体蛋白的色谱复性条件进行研究,摸索出最佳的复性、纯化条件,解决其在大肠杆菌中表达提取时遇到的蛋白变性问题。按实验条件对表达的rhEndostatin包涵体蛋白进行提取、洗涤、变性;利用正交法设计重组蛋白的色谱复性、纯化实验;利用SP-Sepharose Fast Flow阳离子交换柱对rhEndostatin包涵体蛋白进行了色谱复性、纯化。最佳复性条件为rhEndostatin上样浓度为2.5 mg/mL,洗脱体积流量为1/20床体积/min(3.5 mL/min),NaCl浓度0.1 mol/L,GSH/GSSG的浓度比为3/0.3 mmol/L。在此条件下纯度约97%,蛋白回收率为90%,重组蛋白的活性最高,IC50达到5μg/mL。     

基因重组人血红蛋白的纯化

目的 :探索重组人血红蛋白的纯化方法。方法 :将α、β珠蛋白串联基因克隆进 pBV2 2 0表达载体 ,获得了高效表达 ,表达产物达细菌总蛋白的 2 0 %左右。该表达产物以包涵体形式存在 ,包涵体经洗涤后 ,用 8mol/L尿素溶解 ,先用Q SepharoseFastFlow阴离子交换纯化后 ,又经Sephacryl 10 0凝胶过滤纯化。 结果 :经两步纯化后重组人血红蛋白的纯度达 90 %左右。纯化产物经复性 ,具有与氧结合的能力。结论 :成功地得到人重组血红蛋白的纯化方法     

梅毒螺旋体重组蛋白Tp0463的表达与抗原性鉴定

目的表达梅毒螺旋体(Tp)重组蛋白Tp0463(rTp0463)并鉴定其抗原性,为进一步探讨其在梅毒血清学诊断和免疫保护中的作用奠定基础。方法 PCR扩增Tp0463基因,构建原核表达重组体pET-28a(+)/Tp0463,转化宿主菌诱导表达重组蛋白,Ni-NTA亲和层析法纯化蛋白,Westernblot鉴定表达产物的抗原性。结果成功构建了原核表达重组体pET-28a(+)/Tp0463,经诱导高效表达了一分子量大约为16KDa的重组蛋白,以包涵体表达形式为主,纯化蛋白的纯度>90%;Westernblot结果显示纯化蛋白能被梅毒患者混合血清特异性识别。结论高效表达了rTp0463,该重组抗原有良好的抗原性。     

重组猪促肾上腺皮质激素在大肠埃希菌中的高效表达与分离纯化

本研究建立了一种以大肠埃希菌作为宿主,表达重组猪促肾上腺皮质激素(pig adrenocorticotropic hormone,pACTH)的新方法。pACTH在N末端与经典猪瘟病毒N末端自切蛋白酶Npro突变体EDDIE融合,然后在大肠埃希菌胞浆中以包涵体形式表达,包涵体约占菌体湿重的36.2%。经过复性后的包涵体可以促使EDDIE自酶切,从而获得pACTH。通过优化复性条件,EDDIE-pACTH蛋白的自切率从25%提高至60%。复性液通过酸沉淀,能够去除杂蛋白,经反相色谱纯化后获得纯度为99.56%的pACTH。     

重组人肝细胞生长因子α的纯化与活性测定

目的纯化重组人肝细胞生长因子α(rhHGFα)包涵体,复性并测定其活性。方法大肠杆菌中表达的rhHGF琢以包涵体形式存在,表达量约占菌体总蛋白的25%。离心分离包涵体,用8mol/L尿素溶解,梯度稀释复性。经SephadexG75凝胶过滤和POROSHQ阴离子交换纯化,噻唑蓝法(MTT)测定活性。结果rhHGF琢纯度达到90%以上,其刺激肝细胞生长的活性达到rhHGF标准品的80%。结论建立了纯化rhHGF琢的技术路线,rhHGF琢具有刺激原代培养大鼠肝细胞生长的作用。     

大肠杆菌表达重组人粒细胞—巨噬细胞集落刺激因子的中试纯化

重组人粒细胞 -巨噬细胞集落刺激因子 (rHuGM -CSF)表达产物在大肠杆菌中以包涵体的形式存在。方法　包涵体高压匀浆破菌抽提后 ,经凝胶过滤层析、复性、离子交换层析及凝胶过滤层析等纯化步骤。结果终产物纯度达 97% ,比活性达 1 2× 10 7U/mg蛋白 ,测定N端 2 0个氨基酸系列与其DNA系列推导的氨基酸系列完全一致。     

重组人胰岛素pET28a-IHI下游工艺研究

目的对含有重组人胰岛素原基因的大肠杆菌工程菌pET28a-I HI摇瓶发酵,并探讨采用磺酸化方法对包涵体蛋白纯化复性的工艺。方法工程菌pET28a-I HI经摇瓶发酵、机械破碎得到包涵体,包涵体经过磺酸化、等电点沉淀、DEAE-52柱层析三步纯化后采用15%SDS-PAGE电泳检测,经过复性,酶切,DEAE-52纯化后,冷冻干燥,样品进行HPLC检测以及整体生物学活性实验。结果工程菌表达量约在30%左右,经过三步纯化,包涵体纯度可达98%以上,酶切之后经质谱检测,与人胰岛素标准品相对分子质量一致,而且动物实验降血糖效果明显,1 L发酵液最终可得到人胰岛素140mg。结论该工艺耗时短,效果好,成本较低。     

重组人干扰素α2b包涵体纯化方法的研究

目的研究重组人干扰素α 2b包涵体的纯化方法。方法超声破碎菌体 ,干扰素α 2b包涵体经洗涤液洗涤后 ,SDS -PAGE检测其纯度 ,VSV -WISH细胞系统测定干扰素活性。结果差速离心很难完全将包涵体和细菌碎片分开。超声破碎 6次和 9次的干扰素α 2b包涵体纯度和活性相近 ,均明显高于 3次超声破碎处理的包涵体。用含2～ 4mol/L尿素、1%TritonX - 10 0的洗涤液对干扰素α 2b包涵体洗涤 ,能提高包涵体纯度。结论建议超声破碎 6次 ,用含 2～ 4mol/L尿素、1%TritonX - 10 0的洗涤液纯化干扰素α 2b包涵体     

Recombinant human interferons

The genes for several human interferons (IFN) alpha, of IFN beta and IFN gamma have been cloned and expressed in bacterial cells. Recombinant interferons have been purified from these cells and shown to be as biologically active as their natural counterparts. The expression systems, purification schemes and the biochemical properties of these interferons are discussed.     

Recombinant Interleukin-2

Recombinant interleukin (IL)-2 is a newly approved immunoregulatory protein produced by lymphocytes that exhibits a wide range of immunologic effects. It is a true biologic response modifier in that is has no known direct antitumor activity, but mediates its cytotoxicity through activation of effector cells including T cells, natural killer cells, and lymphokine-activated killer cells. Recombinant IL-2 has demonstrated activity in patients with renal cell carcinoma and melanoma, with objective response rates of approximately 15–20%. The median duration of response in renal cell carcinoma is 23 months. Toxicity experienced with high-dose IL-2 can be significant. The most common dose-limiting toxicities are hypertension, weight gain, oliguria, respiratory insufficiency, and neurotoxicity. These effects are generally manageable and reversible on discontinuation of therapy. Administration of low-dose IL-2 has emerged as a means of substantially reducing toxicity. At least in renal cell carcinoma, it appears that the response rate to low-dose IL-2 is comparable to that with higher dosages.     

重组L-门冬酰胺酶前体脂质体与重组L-门冬酰胺酶对小鼠毒性作用比较

为了考查L-门冬酰胺酶(L-Asparaginase,L-ASNase)前体脂质体(Proliposomes)( L-ASNasePL) 与L-ASNase的急性毒性及对外周血中白细胞总数及血小板数的作用.脂质体组小鼠静脉注射给药(L-ASNasePL),给药剂量为给以最大药物浓度(4000KU/m1),最大静脉注射体积(0.5m1/20g);L-ASNase 组给药体积为0.4ml/20g,给药一次,给药后每天观察记录小鼠活动、死亡情况,共观察14d.结果L-ASNasePL 的毒性与L-ASNase 相比明显降低,其小鼠iv的L-ASNasePL的最大耐受量为10.0×105KU/kg,相当于每日人临床拟用剂量的5000倍.对正常小鼠体重、外周血中白细胞总数及血小板数的影响:L-ASNase高剂量组(1000KU/kg)有显著降低小鼠外周血中白细胞总数及血小板数的作用(P<0.05),而L-ASNasePL对小鼠外周血中白细胞总数及血小板数无明显影响.L-ASNasePL组及L-ASNase组对小鼠体重增长均无明显影响.说明L-门冬酰胺酶前体脂质体急性毒性明显小于L-门冬酰胺酶,L-ASNasePL对小鼠外周血中白细胞总数及血小板数无明显影响.     

重组融合蛋白Atacicept

Atacicept（TACI—Ig,TACE—Fc5,sTACI）是由默克雪兰诺（Merck Serono）公司开发的一种含有TACI受体胞外BAFF／APRIL结合区域以及人IgG的Fc区域的可溶性重组融合蛋白。在B细胞恶性肿瘤及自身免疫性疾病中,在B细胞激活中起重要作用的肿瘤坏死因子（TNF）超家族成员——     

Recombinant human serum albumin

Human serum albumin (HSA) is responsible for 80% of the colloid osmotic pressure of plasma (25-33 mmHg). Its main clinical use is in maintaining colloid oncotic pressure and increasing circulating plasma volume with the typical dosage in excess of 10 g per dose. HSA is isolated by fractionating human plasma, which entails possible contamination by viruses or prions. Recombinant HSA (rHSA) has been successfully produced using a methylotrophic yeast, Pichia pastoris. Due to the fact that the clinical usage of HSA infusion often exceeds 10 g, rHSA preparation requires a high level of purity. rHSA purified by means of Streamline technology is identical to plasma-derived HSA (pdHSA) with no detectable mannan component from P. pastoris. The structural and functional properties of rHSA are similar to those of pdHSA. Preclinical and clinical trials have confirmed the safety and efficacy of using this rHSA preparation in different disease conditions, such as hemorrhagic shock, cirrhosis with ascites, and other critical clinical conditions related to plasma volume and oncotic pressure. In addition to its use as a plasma expander, rHSA has great potential as a biomaterial for other medical and pharmaceutically related applications.     

Recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) is a promising vector for somatic gene therapy due to the ability to transduce terminally-differentiated and non-dividing cells, the lack of any apparent pathogenicity, a low immunogenicity, a relatively high stability of transgene expression, and the potential for targeted integration. Improved methods of rAAV packaging allow the generation of concentrated and highly purified rAAV for clinical trials. Preclinical studies with rAAV are currently in progress for the treatment of a variety of inherited monogenic defects, but also for acquired diseases like HIV infection and cancer. Because of the broad host range of wild type AAV, rAAV is able to transduce a variety of human tissues, preferentially those of epitheloid origin, with high efficiency and therefore may be used for various clinical applications. Whilst several issues including safety, tissue tropism and methods to achieve site-specific integration need further improvement, rAAV certainly has a sufficient number of advantages to be seriously considered as a gene therapy vector.     

Recombinant vaccines against leptospirosis

Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine.     

重组L-天冬酰胺酶的鉴定

目的 :对重组L 天冬酰胺酶进行鉴定。方法 :用基因测序、氨基酸组成分析、N 末端氨基酸测序、胰肽图谱、IEF、分子量、紫外扫描等各种实验手段对重组L 天冬酰胺酶及其天然品进行了全面比较。结果 :重组产品与天然品具有同质性。结论 :重组L 天冬酰胺酶具有较好的应用前景。