聚乙二醇干扰素α-2a和重组人干扰素α-2b治疗慢性丙肝的临床疗效观察

目的分析2种类型干扰素治疗慢性丙型肝炎的疗效对比。方法选取我院感染科自2008年6月至2010年12月慢性丙肝150例,随机分成分为2组,聚乙二醇干扰素α-2b(PEG-IFN-α-2b)组:PEG-IFN-α-2b1.5μg/kg体重,每周注射1次,疗程6个月;干扰素α-2b组:普通干扰素α-2b500万单位,隔日1次肌肉注射,疗程6个月。结果 PEG-IFN-α-2b组有13例ALT,3例接近正常,HCV-RNA12例恢复正常,2例接近正常,其余的均较治疗前有所下降。治疗前后有明显下降(P<0.05)。普通干扰素α-2b组ALT有8例恢复正常,1例接近正常,其余患者ALT治疗后仍高。HCV-RNA9例正常,1例接近正常,8例较治疗前下降,2例HCV-RNA定量与治疗前相同。(PEG-IFN-α-2b)组干扰素组完全应答者9例,部分应答者5例,无应答者6例,总应答率为70%。普通干扰素(α-2b)组完全应答者6例,部分应答者3例,无应答者11例,总应答率为45%。结论 (PEG-IFN-α-2b)组干扰素治疗慢性丙肝疗效优于普通干扰素α-2b。     

重组人干扰素α-2b重点专利及重要申请人技术发展分析

重组人干扰素α-2b具有抗病毒、抗肿瘤和免疫调节的作用,在临床上被广泛应用。本文以重组人干扰素α-2b的专利文献为基础,对该领域涉及的关键技术和重点专利进行了梳理,并重点综述了首次向市场推出重组干扰素α-2b产品的先灵公司(Schering)的技术发展路线,旨在为该领域的研发提供参考。     

聚乙二醇活性酯对于干扰素α2b化学修饰的初步研究

目的 探讨聚乙二醇活性酯（mPEG－ssMW5000和MW2000）修饰重组人干扰素α2b(rhIFNα2b）的最佳反应条件。方法 在不同反应条件下,用聚乙二醇活性酯修饰rhIFNα2b,采用VSV－Wish细胞病变抑制法测定修饰后rhIFNα2b的生物学活性。结果 随着PEG对rhINFα2b摩尔比的增加或修饰反应时间的延长,rhIFNα2b的修饰产物相对分子质量和不均一性增加,且随着rhIFNα2b修饰度的提高,其生物学活性逐渐降低。结论 初步确立了PEG修饰rhIFNα2b的反应条件,反应摩尔比与聚乙二醇活性酯的相对分子质量是影响反应的关键因素。     

重组人干扰素α_(2b)工程菌的生物学特性检测

目的 观察和了解重组人干扰素α2b（以下简称rhIFN-α2b）工程菌的生物学性质,保证产品质量稳定。方法 通过对rhIFN-α2b工程菌的微生物学、分子生物学和免疫学检测,全面了解工程菌的性质。结果rhIFN-α2b工程菌呈现典型的大肠杆菌形态,在甘油中可以贮存1年,在冻干条件下可以保存2年,其生物学特性比较稳定。结论 rhIFN-α2b工程菌生物学特性稳定不变。     

重组人干扰素α2b内毒素检查法的研究

目的研究鲎试剂法与家兔法检测重组人干扰素α2b内毒素结果的相关性,并参照家兔热原试验的阈值量,来确定本制品质量标准中内毒素的限量标准。方法分别用不同浓度的内毒素溶液和含不同浓度内毒素的供试品溶液进行家兔热原质试验,确定供试品内毒素限值,并在此限值下进行内毒素检测,方法均参照2000年版《中国药典》和2000年版《中国生物制品规程》进行。结果在含不同浓度内毒素溶液的家兔热原质试验中,致热阈值小于5.0EUml。在含不同浓度内毒素供试品溶液的家兔热原质试验中,致热阈值小于1.25EUml。以此确定本品内毒素限值,检测该制品的内毒素含量,结果均符合规定。结论本品在内毒素含量小于3.5EU500万单位dose的范围内,根据具体的对照实验及制品的用途来规定细菌内毒素限值,可用鲎试剂法代替家兔法检测注射用重组人干扰素α2b冻干制剂的热原质。     

重组人干扰素α2b喷雾剂的研制

目的重组人干扰素α2b(以下简称为rIFNα2b)喷雾剂的试制及临床前实验。方法将重组人干扰素α2b原液进行稀释,调整pH和渗透压,加入适宜稳定剂,除菌过滤后分装于带有定量喷雾装置的棕色玻璃瓶。对配制好的制品分别进行理化和生物学试验、稳定性试验、鼻腔局部刺激性试验、急性毒性试验、长期毒性试验和动物体内抗病毒试验等。结果连续3批制品经检定各项指标均合格,2～8℃保存24个月后生物学活性无明显下降,使用安全,无不良反应。其他各项检定指标均符合现行《中国药典》的要求。结论研制的rIFNα2b喷雾剂安全、稳定,具有一定的抗病毒作用和潜在的临床应用价值。     

干扰素α-2b的全球专利技术路线分析

干扰素α-2b是一种重要的广谱抗病毒、抗肿瘤治疗药物。本文针对干扰素α-2b存在的稳定性差、半衰期短、毒副作用等问题,分别从构建突变体、与人血清白蛋白HSA融合、与Fc融合、PEG修饰等方面对干扰素α-2b技术发展脉络进行专利分析。     

聚乙二醇-重组人干扰素α2b结合物冻干保护剂的筛选

目的筛选聚乙二醇-重组人干扰素α2b(PEG-rhIFNα2b)结合物的冻干保护剂。方法采用两步筛选法,以氨基酸、甘露醇和糖类作为冻干保护剂,与PEG-rhIFNα2b在适宜条件下制备成冻干制剂,分别于40、25和4℃放置不同时间取样,检测其理化性质和生物学稳定性,筛选最佳保护剂配比。结果通过6～12个月的试验,证实该冻干制剂外观、pH无明显改变,但再溶解速度略慢于以人血白蛋白作保护剂的冻干制剂;采用15mmol/L精氨酸+10mmol/L谷氨酸+4%甘露醇+1%蔗糖/海藻糖的冻干保护剂配方,在4℃和25℃放置12个月后,抗病毒活性仍较好,相当于人血白蛋白的冻干保护效果。结论已筛选出不含人血白蛋白的PEG-rhIFNα2b结合物的冻干保护剂。     

重组干扰素α-2b联合阿德福韦治疗慢性乙型肝炎临床研究

目的评价重组干扰素α-2b联合阿德福韦治疗慢性乙型肝炎患者的临床疗效和安全性,探讨两者联合治疗的协同作用。方法治疗组服用阿德福韦10mg,每天1次口服。重组干扰素α-2b注射液500万单位,每周2次,皮下注射。对照组单用阿德福韦10mg,每天1次口服,疗程均为6个月。结果治疗满6个月后,治疗组和对照组ALT复常率差异无显著性意义;HBeAg/抗HBe血清转换率HBVDNA转阴率差异有显著性意义,停药随访6个月后,2组ALT复常率、HBeAg/抗HBe血清转换率HBVDNA转阴率差异有显著性意义。结论重组干扰素α-2b联合阿德福韦治疗慢性乙型肝炎疗效优于单一用药,是慢性乙型肝炎患者安全有效的治疗方法。     

微波联合重组ɑ-2b人白细胞干扰素凝胶治疗尖锐湿疣疗效观察

目的探讨微波联合重组ɑ-2b人白细胞干扰素凝胶治疗尖锐湿疣疗效。方法对198例患者分组治疗,治疗组局部外用重组ɑ-2b人白细胞干扰素凝胶,对照组则不使用任何药物。结果治疗组中痊愈86例(86%),复发14例(14%);对照组治愈67例(68.37%),复发31例(31.63%)。两组复发率比较χ2=7.88,P<0.005,有显著差异性。结论微波联合重组ɑ-2b人白细胞干扰素凝胶外用治疗尖锐湿疣,治疗组的复发率明显低于对照组。     

稀释复性法体外复性重组牛凝血酶原-2包涵体

对重组牛凝血酶原-2包涵体的体外重折叠复性进行了研究。凝血酶原-2是α-凝血酶生成过程中的一个最小的单链中间前体。重组牛凝血酶原-2在E.coli中高效表达并形成包涵体,经2.5mol?L?1脲和0.7%Triton X-100洗涤,再用8mol?L?1脲和0.3mol?L?1DTT溶解后,得到了包涵体裂解液。研究优化了体外复性溶液,其组成为含2.7mol?L?1脲、0.6mmol?L?1GSSG、GSSG/GSH0.9、0.1%PEG6000和0.5mol?L?1L-Arg、pH7.4Na3PO4缓冲液。牛凝血酶原-2复性后经透析,再被Echis Carinatus Snake Venom激活,转化为有活性的α-凝血酶,其总活力可达2948U?mL?1。结果表明稀释复性法可用于重组牛凝血酶原-2包涵体的体外重折叠复性。     

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

重组人G-CSF大肠杆菌表达产物的纯化及鉴定

将RT－PCR扩增的人粒细胞集落刺激因子（hG－CSF）结构基因与表达载体pJGW1重组，并在大肠杆菌中高效表达。经SDS－PAGE分析，rhG－CSF表达量占菌体可溶性蛋白的25％以上。将其复性后，经CM52离子交换层析纯化，产物经SDS－PAGE分子量测定、肽谱分析、等电点测定表明与天然hG－CSF具有相同性质。免疫印迹证实rhG－CSF抗原特异性良好。用小鼠骨髓细胞集落形成法测定活性达1×108U／mg。     

Recovery of Human Interferon Alpha-2b from Recombinant Escherichia coli by Aqueous Two-Phase System

Recovery of periplasmic human recombinant interferon alpha-2b (IFN-α2b) from Escherichia coli rosetta-gami2 (DE3) using a single-step polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system (ATPS) was investigated in this study. The influences of system parameters including PEG molecular weight, tie-line length, volume ratio, crude stock loading, system pH, and sodium chloride (NaCl) concentration (%, w/w) were studied. The results showed that the optimum condition to obtain the high purification factor of IFN-α2b in a single step was achieved by ATPS composed of 4% (w/w) PEG 8000, 13% (w/w) potassium phosphate, 0.5% (w/w) NaCl, 10% (w/w) crude stock, and a system pH of 6.5. A purification factor of 26.3 and recovery yield of 40.7% were obtained from optimized ATPS.     

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.     

肿瘤坏死因子相关凋亡诱导配体的可溶性表达及纯化条件的优化

以人胎盘cDNA为模板,通过PCR技术特异性扩增出约500bp的肿瘤坏死因子相关凋亡诱导配体的可溶性片段(Soluble TNF-related apoptosis-inducing ligand,sTRAIL)基因。经双酶切后插入到原核表达载体pTASH的Eco RⅠ和HindⅢ之间,转化大肠杆菌JM109菌株。宿主菌主要以可溶形式表达sTRAIL,可溶形式sTRAIL蛋白达到60%。采用两种方法纯化sTRAIL,比较两者对目的蛋白的影响。SDS-PAGE结果显示,破碎上清经过SP-Sepharose FF和RP-HPLC两步纯化后,纯化的蛋白为19kD单一条带,纯度达98%以上;而经过SP-Sepharose FF和Q-Sepharose FF两步纯化的蛋白,纯度只达到93%。Western blot鉴定表明纯化产物为sTRAIL。用HepG2细胞测定sTRAIL的生物学活性,发现重组蛋白在体外能明显诱导肿瘤细胞凋亡。     

大肠杆菌表达的vMIP-Ⅱ包涵体的纯化与复性研究

目的纯化、复性大肠杆菌表达的vMIPⅡ包涵体蛋白。方法用8molL尿素缓冲液变性溶解vMIPⅡ包涵体,然后加入十二烷基磺酸钠(SDS)进一步促溶,利用金属亲和层析和分子筛层析纯化,再利用柱层析去除SDS,纯化后透析复性;通过荧光和单核淋巴细胞趋化抑制实验检测复性蛋白的结构和抑制单核淋巴细胞趋化的活性。结果重组vMIPⅡ纯化和复性后,仍具有完整的高级结构和抑制单核细胞趋化的活性。结论已获得了重组vMIPⅡ活性蛋白,为进一步研究应用奠定基础。     

Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble β-Glucans

β-Glucan is widely distributed in various plants and microorganisms and is composed of β-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with β-1,6- or β-1,4-linkage. Numerous studies have investigated different β-glucans and revealed their bioactivities. To understand the structure-function relationship of β-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain β-1,6-branched β-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived β-1,3-glucan recognition protein and fungal endo-β-1,6-glucanase (Neg1)-derived β-1,6-glucan recognition protein, respectively. In this approach, two β-glucan recognition proteins bound to β-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of β-glucan. To test the applicability of this assay, β-glucan and two β-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a β-1,3-glucan with a long polymer of β-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between β-1,3-glucan and long-chain β-1,6-branched β-1,3-glucan in various soluble and insoluble β-glucans.     

在大肠杆菌中表达蝮蛇毒类凝血酶基因gloshedobin

将大连蛇岛蝮蛇毒腺中的类凝血酶基因gloshedobin克隆到pET-32a(+)上,构建了T7启动子控制的大肠杆菌表达质粒,并考察了质粒的稳定性. 采用IPTG诱导,以融合蛋白的形式进行表达,得到以包涵体形式存在的类凝血酶. 通过实验对诱导时间、诱导温度及诱导剂浓度进行了优化,并采用正交实验考察了金属离子对表达的影响,结果表明Fe3＋, Co2＋对类凝血酶的表达有促进作用.