抗体药物分离纯化中的层析技术及进展

抗体,特别是单克隆抗体是一类重要的生物技术药物,具有巨大的市场前景。目前抗体药物生产的瓶颈已由抗体表达转移到抗体的分离纯化中,其中层析是最关键的技术。典型的抗体纯化过程首先通过蛋白A亲和层析对抗体进行捕获,然后进一步精制,主要方法包括离子交换层析、疏水相互作用层析以及羟基磷灰石层析等,这些传统的层析方法不可避免存在一定的局限性。为此,研究者致力于开发合适的新方法,如疏水性电荷诱导层析和短肽仿生层析等,很好地弥补了传统层析方法的不足。本文根据近年来国内外在单抗药物分离纯化方面所取得的研究进展,着重介绍了在单抗分离纯化中的常用的层析技术及研究进展。     

水解牡蛎粉基本成分分析及其辅助降血糖功能的评价

本论文从刺参中分离纯化得到一种海参肽,并研究其对NIH/3T3细胞增殖和胶原蛋白分泌的影响。采用活性追踪的方法从新鲜刺参中通过离子交换层析、凝胶层析分离纯化得到海参肽SP12,采用MTT法检测SP12对NIH/3T3细胞的增殖作用;采用流式细胞术检测SP12对NIH/3T3细胞细胞周期的影响;采用羟脯氨酸测定试剂盒的方法检测SP12对NIH/3T3细胞胶原蛋白分泌的影响;采用Western Blot及RT-PCR的方法,在蛋白水平及基因水平上检测SP12对NIH/3T3细胞I型胶原蛋白、MMP-1及TIMP-1表达的影响。结果表明SP12能显著提高NIH/3T3细胞的增殖率,能促进NIH/3T3细胞由G1期向S期转变,增加其胶原蛋白的分泌量,且在0~20 μg/mL范围内均呈剂量依赖性。在蛋白水平和基因水平,SP12均能显著促进I型胶原蛋白及TIMP-1的表达,抑制MMP-1的表达,这可能是SP12促进NIH/3T3细胞胶原蛋白分泌的作用机制。     

Effect of extrusion process on antioxidant activity, total phenolics and β-glucan content of extrudates developed from barley-fruit and vegetable by-products

The aim of this study was to determine the effects of extrusion processing variables on antioxidant activity (AA), total phenolic content (TP) and β-glucan content (BG) of extrudates. Products were prepared by extrusion cooking of barley flour, barley flour–tomato pomace and barley flour–grape pomace blends. Antioxidant activity as measured by the DPPH method was 43.17 ± 0.362, 27.57 ± 0.120 and 82.23 ± 0.785% while TPs, expressed as ferulic acid equivalents, were 5.29 ± 0.126, 4.66 ± 0.023 and 9.15 ± 0.015 mg g⁻¹ dry sample in the extracts obtained from barley flour, tomato and grape pomaces, respectively. Extrusion cooking decreased AA and TP of barley, barley–tomato pomace and barley–grape pomace extrudates. Temperature and screw speed had significant ( P  <   0.05) effect on BGs of barley flour and barley–grape pomace extrudates. However, BG of tomato pomace blend extrudates had significantly ( P  <   0.05) influenced from pomace level only. Results indicated that the content of β-glucan is higher in barley flour than in extrudates of barley flour and pomace blends.     

Effects of sowing and harvesting dates on yield and some quality characteristics of crops in sugar beet/cereal rotation system

BACKGROUND: Cereals are the main crops in rotation following sugar beet harvest. However, a delay in planting winter cereals as a result of late sugar beet harvest is a disadvantage in sugar beet/cereal rotations. In this study, field trials were carried out to investigate the effects of sowing and harvesting dates on the yield and quality of sugar beet (Beta vulgaris L.) and cereals (wheat, Triticum aestivum L., and six‐rowed barley, Hordeum vulgare L.) during two consecutive years. Beet was sown on three dates and harvested on four dates, followed by cereals which were sown on four dates. Root yield and digestion ratio (sugar beet) and grain yield, 1000‐kernel weight, test weight and protein content (wheat and barley) were determined. RESULTS: The highest root yield (52.53 t ha^?1) was obtained from early sown (5 April) and late harvested (1 November) beet, while the highest digestion ratio (17.4%) was obtained from late sown (3 May) and late harvested (1 November) beet. Lower cereal grain yields were obtained from later sowings. The highest grain yields (5.87 and 6.02 t ha^?1) were obtained from earlier sown wheat. Protein content was also affected by sowing date, which appeared to be very important under unstable climatic conditions. CONCLUSION: It is essential to consider the timing of sowing of cereal crops when unstable climatic conditions are present. Hence sowing date recommendations in regular climatic conditions may be misleading for countries dependent on winter rainfall for cereal production. Copyright © 2007 Society of Chemical Industry     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Performance of Husked,Acid Dehusked and Hull‐less Barley and Malt in Relation to Alcohol Production

Studies carried out on normal husked barley, normal hull‐less (naked) barley, acid dehusked barley and acid dehusked hull‐less barley, as well as the malts derived from them, showed that when acid dehusked barley samples (obtained from either husked or hull‐less barley), were processed using commercial enzyme preparations, they produced more alcohol when compared with the alcohol yield obtained from the barley samples from which the acid dehusked samples were derived. When the husked (Optic) control, acid dehusked and hull‐less barley samples were malted, Optic control barley produced malt that gave higher dextrinising units (DU) and diastatic power (DP), whilst acid dehusked Optic and hull‐less barley produced malts that gave similar DU results on day 5 of the germination time. When mashed, acid dehusked (Optic) barley malt produced wort that filtered faster than the wort obtained from the malt made from hull‐less barley. This observation is very important because it shows that the husk of the barley is not the only factor that determines the filtration performance of the malted barley, since both the malt samples made from husked and acid dehusked barley had similar filtration rates on day 5 of the germination time. The slow filtration rate observed for the wort made from hull‐less barley suggests that other factors play some role during the filtration of the mash made from hull‐less barley malt. Although hull‐less malt appeared to develop lower DU and DP enzyme activities, when compared with the values obtained for the Optic control, hull‐less barley malted faster and produced optimum predicted spirit yield (PSY) at day 4 of the germination time. In contrast, the control husked Optic barley malt that had higher DU and DP produced equivalent (optimum) predicted spirit yield one day later at 5 days germination time. This is an advantage for hull‐less barley, both in terms of time and energy saving during the malting of barley. Although the acid dehusked Optic barley produced more alcohol than the husked control when commercial enzyme preparation was used to process barley, it was surprising that when the derived malt was assessed, it gave a lower predicted spirit yield than the husked control, even though it produced a higher amount of hot water extract (HWE). The higher extract yield and lower predicted spirit yield obtained from the malt made from acid dehusked malt confirmed that high extract yield is not necessarily associated with high fermentable extract.     

Engineering electrospun nanofibrillar surfaces for spinal cord repair: a discussion

BACKGROUND: The design of implants comprised of biodegradable electrospun nanofibers for the purpose of bridging injuries in damaged spinal cord is discussed. Electrospun nanofibers structurally mimic the extracellular matrix on which neurons and other cell types grow in vivo. This property has created great interest for their use in tissue engineering applications. However, their employment as biomimetic surfaces for such in vivo applications is still in its infancy. RESULTS: A nonwoven fabric comprised of electrospun polyamide nanofibers supported modest axonal regeneration in injured adult rat spinal cord. Covalent modification of the nanofibers with a bioactive peptide derived from the neuroregulatory extracellular matrix molecule tenascin‐C enhanced the ability of the nanofibers to facilitate axonal regrowth. However, the random orientation of the nanofibrillar fabric folds was an impediment to the forward movement of axons. CONCLUSIONS: Polyamide nanofibers covalently modified with neuroactive molecules provide a promising material for grafts to promote spinal cord regeneration. However, for the proper guidance of regrowing axons, attention must be paid to the engineering of ordered nanofibrillar structures. Copyright © 2007 Society of Chemical Industry     

Presence of indigestible peptide aggregates of soybean meal in pig ileal digesta residue

With the purpose of analysing the molecular size and composition, proteinaceous material was extracted from the insoluble components of a digesta sample obtained from pigs fed a feed consisting of only soybean meal. Gel permeation chromatography indicated that the alkali‐extractable fraction of the proteinaceous material from the residue was of relatively high apparent molecular weight. However, the combined results from gel electrophoresis, RPLC‐MS, and MALDI‐ToF MS showed that the extracted protein material was in fact, to a high extent, composed of aggregated peptides. To our knowledge this has not previously been described. Aggregates extracted by dilute alkali were fully degraded upon subsequent proteolytic treatment. N‐terminal sequencing of selected protein bands from SDS‐PAGE gels indicated the presence of partly degraded β‐conglycinin alpha subunits in the residue. Copyright © 2007 Society of Chemical Industry     

Exogenous ANP Treatment Ameliorates Myocardial Insulin Resistance and Protects against Ischemia–Reperfusion Injury in Diet-Induced Obesity

Increasing evidence suggests natriuretic peptides (NPs) coordinate interorgan metabolic crosstalk. We recently reported exogenous ANP treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and attenuating hepatic steatosis in diet-induced obesity (DIO). We herein investigated whether ANP treatment also ameliorates myocardial insulin resistance, leading to cardioprotection during ischemia–reperfusion injury (IRI) in DIO. Mice fed a high-fat diet (HFD) or normal-fat diet for 13 weeks were treated with or without ANP infusion subcutaneously for another 3 weeks. Left ventricular BNP expression was substantially reduced in HFD hearts. Intraperitoneal-insulin-administration-induced Akt phosphorylation was impaired in HFD hearts, which was restored by ANP treatment, suggesting that ANP treatment ameliorated myocardial insulin resistance. After ischemia–reperfusion using the Langendorff model, HFD impaired cardiac functional recovery with a corresponding increased infarct size. However, ANP treatment improved functional recovery and reduced injury while restoring impaired IRI-induced Akt phosphorylation in HFD hearts. Myocardial ultrastructural analyses showed increased peri-mitochondrial lipid droplets with concomitantly decreased ATGL and HSL phosphorylation levels in ANP-treated HFD, suggesting that ANP protects mitochondria from lipid overload by trapping lipids. Accordingly, ANP treatment attenuated mitochondria cristae disruption after IRI in HFD hearts. In summary, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with mitochondrial ultrastructure modifications in DIO. Replenishing biologically active NPs substantially affects HFD hearts in which endogenous NP production is impaired.     

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.