人干扰素α-2b生产工艺的研究

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。     

重组人干扰素λ1的表达、纯化及生物活性研究

目的:利用大肠杆菌系统表达重组人Ⅲ型干扰素λ1(rhIFN-λ1),并进行纯化,以比较其与重组人干扰素-α2b(rhIFN-α2b)生物学活性的差异。方法:密码子优化的rhIFN-λ1基因与pET-44a载体连接构建原核表达质粒,在大肠杆菌BL21(DE3)中表达,表达产物经系列层析纯化;采用细胞病变抑制法比较纯化的rhIFN-λ1与rhIFN-α2b、市售rhIFN-λ1的抗病毒活性;采用MTS法比较这些干扰素的抗肿瘤细胞增殖活性。结果:基因优化后的rhIFN-λ1在大肠杆菌中得以高效表达,用所建立的纯化工艺可制备得到纯度达95.7%的rhIFN-λ1;rhIFN-λ1的抗病毒比活性为3.2×105 U/mg,比市售rhIFN-λ1的比活性(1.1×105 U/mg)略高,抗肿瘤细胞增殖活性为rhIFN-α2b的1.7倍。结论:获得的rhIFN-λ1具有剂量依赖的抗病毒活性和抗增殖活性,其抗病毒活性比市售rhIFN-λ1略高,抗细胞增殖活性高于rhIFN-α2b,提示rhIFN-λ1有较大的应用前景。     

重组人α2b型干扰素的纯化与鉴定

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。     

干扰素α-2b的聚乙二醇修饰

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.     

重组人干扰素α-2b在大肠杆菌中分泌表达

人干扰素α-2b原始基因在重组原核工程菌中表达量偏低,所以我们在不改变干扰素原有氨基酸组成的前提下,根据大肠杆菌密码子偏爱性使用定向突变技术对huIFNα-2b基因进行点突变。将大肠杆菌STⅡ信号肽基因与突变后huIFNα-2b基因融合并于信号肽5′端和huIFNα-2b基因3′端引入合适的酶切位点。融合基因克隆至载体pCSE,pET-22b和pPAK4L中,此3种载体分别含有组成型启动子、T7启动子和phoA启动子。融合基因在载体pCSE中表达量很低,其中约有50%的目标蛋白能够成功实现分泌。在E.coliBL21中,pET-22b经过IPTG诱导可以实现huIFNα-2b的高表达,但STⅡ信号肽不能被有效切除。含有phoA启动子的载体pPAK4L其在E.coliW3110中可以实现huIFNα-2b较高水平的分泌表达,经过低磷诱导其表达量最高可至20μg/mL(A550)菌液,约有30%的目标蛋白质信号肽能够被成功切除并分泌到胞间质中。     

疏水相互作用层析分离重组人干扰素α2b

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。     

聚乙二醇修饰重组人干扰素α-2b的碘染色法

目的：建立检测聚乙二醇位点特异性修饰重组人干扰素α-2b反应的方法。方法：采用分子量20000的甲氧基聚乙二醇马来酰亚胺修饰重组人干扰素α-2b,反应混合物样品经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后,碘染色法判断反应产物组成。结果：该修饰反应产物除含有单PEG化的干扰素α-2b外,还有不同修饰程度的多PEG化干扰素。结论：本方法方便快捷、分辨率高、特异性强,同时可用于其它聚乙二醇修饰蛋白质的分析研究。     

重组人干扰素α2b的制备研究

为了获得高活性高纯度的rh IFNα2b,对重组人干扰素α2b进行克隆、表达,并深入研究了其纯化工艺。采用重叠延伸PCR法合成了编码IFNα2b的基因,用DNA重组技术构建了原核表达载体p BV220-IFNα2b,获得了稳定的工程菌种。发酵产物通过破菌、洗涤获得包涵体,再经过变性、复性、离子交换层析和凝胶过滤层析的纯化,得到rh IFNα2b纯品,其比活可达1×10~8IU/mg。实验结果为进一步开展临床前研究和长效制剂奠定了基础。     

重组人α2a干扰素的高效表达与纯化

构建了人α2a型干扰素的表达载体,并在大肠杆菌中获得了高效表达,表达量平均为10~ IU/L菌液。还对其表达产物进行了纯化。经盐酸胍裂解菌体、硫酸铵沉淀、酸化处理,再经阴、阳离子交换层析和单克隆抗体亲和层析,使表达产物纯化了1072倍,比活性达1.2×IO~ IU/mg蛋白,达到序列纯,回收率达54％。表达产物N端21个氨基酸序列分析结果与IFN-α2a cDNA推导的序列相符合。     

重组人干扰素α1b抗新型冠状病毒的基础和临床研究进展

干扰素是机体抗病毒的第一道天然防线,干扰素α1b是中国人干扰素家族中主要的抗病毒表达亚型。SARS-CoV-2通过多种途径抑制先天免疫关键分子干扰素的产生。已上市多年的重组人干扰素α1b显示出强大的体外抗SARS-CoV-2病毒活性。初步临床研究显示,包括重组人干扰素α1b在内的I型干扰素对COVID-19显示出积极的治疗和预防作用。全球多个国家正在开展干扰素治疗COVID-19的临床试验,我国自主知识产权的重组人干扰素α1b率先开展了验证性临床试验。     

干扰素a-2b的聚乙二醇修饰

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a-2b(IFN a-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a-2b的浓度为4 mg/mL, PEG与IFN a-2b的摩尔比为8:1, 4oC时反应20 h; 在优化的反应条件下, 单修饰PEG-IFN a-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a-2b的13.4%, 其在SD大鼠体内的循环半衰期得到了较大的延长, 且具有较好的水溶液稳定性。     

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.     

扁担塘螺类生产力的研究Ⅱ、纹沼螺的周年生产量

采用四种方法对扁担塘纹沼螺的周年生产量进行了测算,结果表明,四种方法得到的生产量吻合极好。生产量的去壳干重和带壳湿重分别是体长频度法,178.07mg     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Development and scale-up of a fed-batch process for the production of capsular polysaccharide from Haemophilus influenzae

The production of the capsular polysaccharide, polyribosylribitolphosphate, from Haemophilus influenzae type b is important for the production of effective conjugate vaccines. Factors limiting the production of this polysaccharide from H. influenzae type b in liquid culture were investigated. A fed-batch fermentation was developed that increased cell density and PRP titer approximately four fold when compared to the batch fermentation. This fed-batch process was successfully scaled from the 1.5 l development scale to the 500 l manufacturing scale. The maximum cell density in the 500 l fermentation was 6 g dry cell weight per liter and the PRP concentration was 1.3 g l(-1).     

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9)IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha2b for both research and industrial purpose.     

人血清白蛋白和人干扰素α2b的融合蛋白在毕赤酵母中的表达

为了延长IFNα2b在血浆中的半衰期,构建了编码HSA和hIFNα2b的融合基因并在毕赤酵母中获得高效表达,工程菌经5L发酵罐培养后获得的含融合蛋白的培养液经超滤浓缩、蓝色葡聚糖凝胶层析、疏水柱层析以及阴离子柱层析,融合蛋白的纯度达到95%以上。该融合蛋白能与干扰素抗体和人血清白蛋白抗体结合,并表现出与重组干扰素α2b相似的抗病毒活性。以猕猴为动物模型,分别从静脉和皮下单剂量给药,给药浓度为90μg/kg时,在336h后血浆中仍可检测到融合蛋白。其静脉注射的血浆半衰期为101h,皮下注射的半衰期为68.2h。皮下注射的生物利用度为67.9%。IFNα2b与HSA融合后,明显的延长了血浆半衰期,显现了其良好的临床应用前景。     

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.