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Hamid Rajabi Memari Ramakrishnan Nagasundara Ramanan Arbakariya B. Ariff 《Central European Journal of Biology》2010,5(4):446-455
The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower
mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression
of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay.
The amount of periplasmic expression in E. coli was 60 μg/L of culture, while the amount of nuclear expression in the plant was 4.46 μg/kg of fresh leaves. The result of
this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations
on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate
expression platform. 相似文献
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干扰素α-2b的聚乙二醇修饰 总被引:1,自引:0,他引:1
采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素α-2b(IFN α-2b),建立了修饰反应及分离纯化工艺.考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响,获得了优化的修饰反应条件,即在pH 6.5,20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中,干扰素α-2b的浓度为4 mg/mE,PEG与IFN α-2b的摩尔比为8:1,4℃时反应20 h;在优化的反应条件下,单修饰PEG-IFN α-2b的转化率达到55%.并且,采用离子交换层析对修饰产物进行分离纯化,单修饰产品纯度达到97%,体外活性保留达到未修饰干扰素α-2b的13.4%,其在SD大鼠体内的循环半袁期得到了较大的延长,且具有较好的水溶液稳定性. 相似文献
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目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。 相似文献
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人干扰素α—2b生产工艺的研究 总被引:6,自引:0,他引:6
人干扰素α-2b的简易、高效生产工艺研究,包括高效率、小型化的发酵技术,不用单克隆抗体亲和层析的纯化工序,以及大肠杆菌表达的人干扰素α-2b包涵体蛋白的天然构型复性等问题,从10L基因工程大肠杆菌发酵液所得1000g菌体中得人干扰素α-2b约500mg,效价为1×10~8u/mg,设备投资少,生产成本低,产品表现了高纯度、高效价。适宜大量生产,为广大人民群众提供廉价高质量药品创造了条件。 相似文献
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研究重组人干扰素α2b栓剂的稳定性。方法将重组人干扰素α2b栓在不同温度下保存不同时间,分别检验干扰素生物学活性、溶出度试验、融变时限、pH和微生物限度及外观的稳定性。结果重组人干扰素α2b栓在4~8℃条件下放置当时和放置48个月后的IFN生物学活性基本一致,未见下降;在20~25℃条件下放置24个月后IFN生物学活性未见下降,放置32个月后活性下降32%,36个月后IFN生物学活性下降55%;37℃条件下放置4个月后IFN生物学活性未见下降。结论重组人干扰素α2b栓剂的生物学活性及理化性质的稳定性好。 相似文献
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人干扰素α-2b生产工艺的研究 总被引:2,自引:0,他引:2
人干扰素α-2b的简易、高效生产工艺研究,包括高效率、小型化的发酵技术.不用单克隆抗体亲和层析的纯化工序.以及大肠杆菌表达的人干扰索α-2b包涵体蛋白的天然构型复性等问题.从10L基因工程大肠杆菌发酵液所得l000g菌体中得人干扰素α-2b约500mg,效价为l×103u/mg,设备投资少,生产成本低,产品表现了高纯度、高效价。适宜大量生产,为广大人民群众提供廉价高质量药品创造了条件。 相似文献
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基因工程人干扰素α2b和α2a亚型抗病毒效应的实验研究 总被引:4,自引:0,他引:4
本文采用鸡胚接种法和微量细胞病变抑制法研究了不同亚型α干扰素(α2aIFN和α2bIFN)抗流感病毒、脊髓灰质炎病毒以及麻疹病毒的作用。结果表明:两种亚型α干扰素均显示明显的抗病毒效应,其中(1)α2bIFN抗流感病毒作用略强于α2aIFN,(2)两亚型α干扰素抗脊髓灰质炎和麻疹病毒作用相似。 相似文献
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We have evaluated the effect of intraperitoneal (IP) injection of human recombinant interferon-2α (IFN-α) on Bleomycin-induced pulmonary injury in hamsters. Pulmonary injury was induced by a single intratracheal (IT) instillation of Bleomycin (Bleo). Six groups of male Syrian hamsters were treated as follows: 1) IT Bleo and daily IP injections of low-dose interferon-α (2 × 104 U), 2) IT Bleo and daily IP injections of high-dose interferon-α (105 U), 3) IT Bleo and IP injections of saline, 4) IT saline and IP low-dose IFN-α, 5) IT saline and IP high-dose IFN-α, 6) IT saline and IP saline. Animals were sacrificed 28 days after IT treatment. Lung injury was evaluated histologically and biochemically. Treatment of hamsters with low-dose but not high-dose IFN-α significantly augmented the Bleo-induced lung injury, as determined by a semiquantitative morphological index. Lung hydroxyproline measurements were highest in Bleo-low-dose-IFN-α followed by Bleo-high-dose-IFN-α and Bleo-Sal as compared to Sal-Sal and Sal-IFN-α controls. These results suggest that IFN-α augments Bleo-induced lung injury but that this effect is complex and does not follow a simple-dose-response pattern. 相似文献
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采用鼻腔给药方法,随机选取昆明小鼠60只,每组20只,共分3组,第1组为病毒对照组,第2组为感染前给药组即感染病毒前24h给药,第3组为同时给药组即病毒感染与用药同时进行,小鼠每天滴鼻给药1次,IFNα2b滴鼻剂剂量为10000IU/只,病毒浓度为10个EID50,对照组给予生理盐水,连续给药6d后处死小鼠,观察小鼠肺部病变,并采用血凝滴定法检测鼠肺中流感病毒含量。观察重组人干扰素α2b(IFNα2b)抗流感病毒效果,小鼠肺部病变严重程度依次为1组>3组>2组,血凝滴定结果表明,1、2与3组GMT平均值分别为4.141,2.000,2.070,重组人干扰素α2b滴鼻剂对流感病毒有明显抑制作用。 相似文献
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研究基因工程α2b干扰素脂质体抗鼠肺敏感株流感病毒A/FM/1/47的作用。取9日龄SPF种蛋鸡胚随机分3组,2组实验,1组对照,按组分别接种0.2 mL干扰素、干扰素脂质体及生理盐水,35℃孵育6 h后接种EID50剂量的0.2 mL流感病毒,继续孵育72 h后取每个鸡胚的尿囊液,测血凝效价,比较抗病毒作用。然后,取小鼠随机分3组,2组实验,1组对照,按组每天滴鼻接种干扰素、干扰素脂质体及生理盐水,3 d后每组分别取一半滴鼻感染LD50剂量的流感病毒,另一半滴6 d后,滴鼻感染LD50剂量的流感病毒,比较抗病毒作用。鸡胚感染病毒组的二个实验组血凝效价均降低,干扰素组的血凝效价低于对照组,干扰素脂质体组最低。小鼠鼻腔接种干扰素组的小鼠生存情况好于对照组,干扰素脂质体组小鼠生存情况最好。干扰素脂质体有明显的抗流感病毒作用,并且干扰素脂质体组优于干扰素组。 相似文献
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目的:制备重组人干扰素α2b阴道泡腾片,并对其质量和局部用药安全性进行研究。方法:将重组人干扰素α2b经制粒干燥,用酸、碱分开制粒法制备重组人干扰素α2b阴道泡腾片,按照《中国药典》对其性状、重量差异、崩解时限、pH、干扰素效价、水份含量、稳定性以及局部刺激性进行了考察。结果:本品处方、工艺合理,制备过程对干扰素生物活性影响较小;制剂质量符合规定,在4℃低温条件下保存具有较好的稳定性;局部刺激试验对家兔阴道组织无明显的刺激性。结论:该制剂质量稳定、可控,具有良好的稳定性和安全性。 相似文献
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重组人干扰素α2b的提取和纯化 总被引:1,自引:0,他引:1
为了提高重组人干扰素α2b的质量 ,增加产量 ,降低成本 ,通过破菌 ,包涵体提取 ,离子交换层析 ,凝胶过滤等纯化方法制备干扰素纯品。结果表明干扰素纯度可达 95%以上 ,比活达到 1.2× 10 8IU/mg以上。结果提示 ,采用本方法收率高 ,纯度好 ,工艺稳定 ,适合大规模生产 相似文献
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《Process Biochemistry》2010,45(2):196-202
The osmotic shock process for the release of periplasmic recombinant human interferon-α2b from Escherichia coli was optimized using response surface method (RSM). The process parameters such as pH, buffer concentration and sucrose concentration in hypertonic solution, cell concentration to hypertonic solution, contact time of cells with hypertonic solution, temperature of hypertonic solution, cell concentration to hypotonic solution, contact time of cells with hypotonic solution and temperature of hypotonic solution were initially screened using Plackett Burman design. Further optimization was carried out using central composite design (one of the design in RSM) for sucrose concentration in hypertonic solution as well as cell concentration to hypertonic and hypotonic solutions. The optimal cell concentration was 0.05 g/mL in hypertonic solution and 0.2 g/mL in hypotonic solution. The use of hypertonic solution containing 18% sucrose with a combination of 100 mM Tris and 2.5 mM EDTA buffer (pH 8.0 and 25 °C) and cold water (4 °C) as a hypotonic solution gave the optimum release of interferon-α2b. Increased product concentration in the final solution resulted from the optimized process would reduce the downstream steps during purification. The concept of reuse of hypertonic solution was also demonstrated. 相似文献
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Shih-Hua Fang Ming-Yang Lai Lin-Hwa Hwang Pei-Ming Yang Pei-Jer Chen Dr. Bor-Luen Chiang Ding-Shinn Chen 《Journal of biomedical science》2001,8(6):484-491
Some patients with chronic hepatitis C respond to interferon (IFN)-alpha treatment, and the efficiency can be improved by combining it with ribavirin. The mechanism of this improvement is unknown. To investigate the effects of these two regimens on the immune responses in 51 patients with chronic hepatitis C, we examined the hepatitis C core antigen-specific proliferative response and cytokine production profiles, natural killer (NK) cell cytotoxicity and cytotoxic T cell function during treatment. The results are as follows: (1) both viral clearance and biochemical normalization occurred more frequently in patients receiving combination therapy; (2) the function of NK cells increased after treatment in the responders of both groups (p < 0.05); (3) the level of IFN-gamma produced by hepatitis C core antigen-stimulated peripheral blood mononuclear cells was higher in patients receiving combination therapy, especially in responders; (4) the core antigen-specific proliferative response decreased after treatment, and (5) in addition, the core-specific cytotoxic T cell activities of five responder patients also increased significantly after therapy. In conclusion, enhancement of immune responses, especially those related to type-1 T helper cell activity, may contribute to better efficacy in combining ribavirin with IFN-alpha for treatment of chronic hepatitis C. 相似文献
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重组人α2b型干扰素的纯化与鉴定 总被引:4,自引:0,他引:4
采用单克隆抗体亲和层析一步纯化法对大肠杆菌表达的重组人a2b型干扰素(IFN-α2b)进行了纯化,然后利用凝胶过滤高压液相层析、SDS-聚丙烯酰胺凝胶电泳和N端25个氨基酸的序列测定等方法对纯化产品进行了鉴定,表明一步纯化的IFN—α2b达到95%以上的纯度,其比活性为2.54×108U/mg但重组IFN—α2b产品N端不均一,含有约30%的去Met分子和约70%的带Met分子。从蛋白质序列的水平上证实,本实验室用定位诱变法构建的IFN—α2b基因在大肠杆菌系统中得到了正确的表达。 相似文献