重组人血白蛋白临床研究进展

人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。     

浅谈重组人血白蛋白的进展

人血白蛋白是临床上使用量最大的血液制品,但血浆来源的人血白蛋白由于来源受到限制,远不能满足市场的需要,而且人血浆有携带病毒的风险,利用基因重组等技术研制的重组人血白蛋白克服了这些不足,成为发展的趋势。表达系统是重组人血白蛋白生产过程中极其重要的环节,本文主要综述了重组人血白蛋白表达系统的研究进展和国内外产业化现状。     

人血白蛋白的分离及提纯研究进展

改进白蛋白工艺,加强产品稳定性及治疗对确保患者生命健康具有重要的意义,同时也节省企业资源,提高企业经济效益。目前国内血液制品发展面临较大的挑战,不仅受原材料血浆供应紧张的影响,同时缺乏自身产品多样性,因此对白蛋白分离及提纯工艺进行分析,提高人血白蛋白产品纯度,获得有效的血液制品是当前血液制品生产的重点及难点。本文将综述人血白蛋白分离及提纯的相关工艺要求,旨在为人血白蛋白的制备提供指导。     

国内人血白蛋白市场供需状况分析及应对策略

目的：解决国内人血白蛋白供需不平衡。方法：运用经济学中的供给与需求理论分析国内人血白蛋白市场供需状况,采用SWOT矩阵对整个人血白蛋白市场环境进行系统分析。结果与结论：由于原料供应短缺、需求旺盛等原因造成国内市场人血白蛋白供不应求,政府应在原料供应、价格干预、监管、宣传方面采取有效措施,同时企业应配合政府积极生产,加强技术创新等共同来协调人血白蛋白的供需平衡。     

小分子与血清白蛋白相互作用的研究

近年来,药物普遍应用的势态尤为明显,在这一势态之下,对小分子与血清白蛋白之间的相互作用机理进行研究便显得极为重要。因此对其进行研究,能够对药物运输及代谢过程有一定程度的了解,并能够为临床用药及新药研发提供依据。本课题重点分析了药物小分子与人血清白蛋白之间的相互作用。     

人血清白蛋白与金属离子作用的荧光光谱研究

通过金属离子对人血清白蛋白内源荧光的猝灭,探讨了金属离子Cu²⁺,Mn²⁺,Ni²⁺,Co²⁺,Cr³⁺等与人血清白蛋白的结合;基于Förster无辐射能量转移机理,得出了人血清白蛋白第一类Cu²⁺结合部位与214位色氨酸残基间的距离。     

胚胎发育早期接种人血清白蛋白诱导鸡的免疫耐受

外源基因的引入及表达常会引发宿主动物强烈的免疫应答,导致蛋白产量的下降或引起炎症反应。本研究试图通过在胚胎发育早期接种异源抗原诱导动物产生免疫耐受来解决这些问题。为了诱导鸡产生免疫耐受,将人血清白蛋白（Human serum albumin,HSA）通过胚胎血管微注射的方式接种到发育65—67h的鸡胚血管中,接种剂量为50μg;通过卵黄注射的方式接种到发育3—7d的鸡胚卵黄中,接种剂量为100μg。孵出的小鸡在3周龄时按照常规免疫方法再次接种同种抗原,收集不同时期的血清样本,用酶联免疫吸附试验（Enzyme-linked immunosorbent assay,ELISA）检测血清中抗-HSA抗体水平。结果表明,两种接种方式均能诱导小鸡产生免疫耐受：在65-67h的胚胎中通过血管微注射法接种抗原诱导小鸡产生免疫耐受的比率为64．52％;通过卵黄注射接种抗原诱导小鸡产生免疫耐受的最佳接种时间为发育的第6d,第5、7d接种对后期血清中抗-HSA抗体形成也有一定抑制作用,但是维持耐受的时间较短,第3、4d接种抗原对诱导小鸡免疫耐受的效果不明显[动物学报51（5）：845—851,2005]。     

引起重组白蛋白电泳检测假性结果的研究

目的:建立SDS-PAGE结合pH值变化对重组人血白蛋白电泳检测假性结果的分析方法。方法:采用还原SDS-PAGE(12.5%)和Native-PAGE(8%～25%),将包含重组人血白蛋白的发酵上清液的pH值分别调为4.0、5.0、6.0、6.5、7.5、8.0、9.0和10.0进行电泳分析。结果:在不同的pH值条件下,重组人血白蛋白会出现不同程度的降解。结论:包含重组人血白蛋白的发酵液中存在不同种类的蛋白酶,导致重组人血白蛋白的假性降解。     

脊髓灰质炎减毒活疫苗的热稳定性试验

本文对国产三种剂型OPV进行了热稳定性试验,在所试温度中能保持疫苗有效服用剂的时间为:4℃ 4个月以上,10℃ 2个月,22℃ 7天,34℃ 2天,36℃ 1天。试验发现液体苗对热的稳定性要比糖丸苗相对好些。     

重组人血清白蛋白在汉逊酵母中的表达与纯化

优化人血清白蛋白（Human Serum Albumin, HSA）基因的密码子,合成基因连接到用于汉逊酵母（Hansenula polymorpha）表达的表达载体上,构建成重组人血清白蛋白（recombinant Human Serum Albumin, rHSA）表达质粒,转化汉逊酵母细胞,筛选得到的rHSA高表达细胞株HP-rHSA－C,30L发酵罐批式发酵表达量可达1.033g/L,经Streamline SP,Phenyl Bio-Sep 6FF,DEAE Sepharose层析分离获得纯化蛋白,除菌过滤后稀释,进行小鼠免疫原性分析,结果与人血清中提取的人白蛋白具有相同的抗原、抗体反应特性。     

Effect of genetic variation on the thermal stability of human serum albumin

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

The participation of human serum albumin domains in chemical and thermal unfolding

Fluorescence spectroscopy and differential scanning calorimetry were used to follow local and global changes in human serum albumin domains during chemical and thermal denaturation of this protein. Results suggests that thermal and chemical treatments involved an unfolding pathway of at least two steps and that domain IIA is not homogeneous. Unfolding at site I exposes a larger hydrophobic area to the solvent than at site II. The bilirubin-binding site showed atypical behavior: a significant increase in the hydrophobic area was exposed to the solvent when its binding site was denatured by guanidine hydrochloride. This result might be due to the high specificity of the bilirubin-binding site, whose binding makes an extensive conformational change in the environment of this site.     

Effect of citraconylation on the thermal aggregation of human serum albumin

Treatment of human serum albumin with citraconic anhydride permitted the introduction of up to approx. 20 citraconyl groups per monomer molecule of the protein. This caused an increase in the apparent Stokey radius as determined by gel filtration and viscosity measurements, although there was no significant change in helix content as measured by optical rotary dispersion. Citraconylation prevented the precipitation of albumin on heating, and significantly slowed the ate of formation of soluble aggregates, even though a large number of the citraconyl groups were lost during the period of high temperature.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Aggregation of human serum albumin during a thermal viral inactivation step

A terminal pasteurization step has been used for some plasma-derived protein products such as human serum albumin (HSA), which consists of heating the protein in solution at 60 °C for 10 h. Native and denaturing SDS-PAGE and dynamic light scattering were used to follow the stability of HSA during this process. It appears that a thermally unstable fraction, comprised primarily of haptoglobin, is involved in the formation of soluble aggregates of HSA. Therefore, it appears that aggregation during heat treatment is not due to conformational instability of HSA itself, but arises from unfolding of a thermally labile protein impurity. As haptoglobin aggregates, it entraps some HSA, which is present at much higher concentrations. This study emphasizes that, in a complex mixture of naturally occurring proteins, one thermally labile species can trigger aggregation of more stable proteins.     

A theoretical analysis of the binding of palmitate by human serum albumin

The distribution of palmitate between the form bound by human serum albumin and the free form in plasma was calculated by use of 12 stepwise equilibrium constants and a computer program. Computations were carried out for molar ratios of palmitate to serum albumin of 0.5, 1,2,3, and 4. At most 0.0003% of the palmitate would be in the unbound form, and the remainder distributed among different complexes with albumin. At low molar ratios, the complexes with 1 to 2 moles of palmitate/albumin would predominate while at the highest ratio the complexes of 3 and 4 moles of palmitate/albumin would be most abundant. In the delivery of palmitate to tissues the relative contribution of the different complexes would change, as the molar ratio of fatty acid to albumin changed.