明胶亲和层析介质分离纯化猪血浆纤维结合蛋白

目的：研究猪血浆中纤维结合蛋白的分离纯化方法,得到高纯度、高活性的目标蛋白。方法：以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,考察了不同洗脱方式对纯化结果的影响,对产品进行了纯度、生物活性的测定。结果：通过对纯化工艺条件的摸索,采用含不同浓度尿素缓冲液进行分步洗脱,可以得到纯度为95％以上的产品,回收率达到50．96％;经BHK细胞培养实验证明其具有与人血浆纤维结合蛋白同样的生物活性。结论：采用了较温和的洗脱方式以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,提高了动物资源的可利用性。     

膜技术纯化菊花总黄酮的工艺研究

研究膜分离技术分离纯化菊花黄酮的工艺,以菊花总黄酮纯度和操作过程稳定性为评价指标,采用膜分离技术对菊花提取液进行处理,对膜的规格、溶液温度、操作压力和操作时间进行了优选。结果表明:选择孔径0.5μm无机陶瓷膜,在溶液温度50℃、操作压力0.25 MPa条件下,微滤180 min能达到较好地除杂和澄清的效果;选择截留分子量为8×103的超滤膜,在溶液温度40℃、操作压力1.6 MPa条件下,超滤120 min,总黄酮纯度为19.81%。采用膜技术纯化菊花总黄酮的工艺操作简单,纯化效果高。     

超滤膜分离纯化珊瑚藻溴过氧化物酶

利用超滤膜对珊瑚藻中溴过氧化物酶(EC 1.11.1.18,分子质量740kDa)进行分离纯化,对膜的截留分子质量、操作压力、起始蛋白质浓度、搅拌速率、pH、离子强度等条件进行了优化。超滤分离纯化最优条件为:截留分子质量为100kDa的聚偏氟乙烯(PVDF)膜,操作压力为0.02MPa,搅拌速率为600r/min,起始蛋白质浓度为0.1g/L,pH=6.0。对粗酶液先进行热沉淀纯化,再进行超滤纯化,溴过氧化物酶被纯化了21倍,比活为212U/mg,酶活回收率为96%。     

短梗霉多糖超滤器的计算

超滤浓缩是短梗霉多糖分离纯化过程中的重要工序。本文研究了短梗霉多糖溶液的浓度每超滤速率的关系,推导出新的超滤器计算公式。根据预定的生产规模及工艺条件,可以对超滤器进行设计计算。实验结果表明用这种方法计算的结果是可靠的。     

超滤膜分离纯化加纳籽中5-羟基色氨酸的研究

研究了超滤膜从加纳籽中分离纯化5-羟基色氨酸的相关膜工艺条件及参数,并筛选出适合的聚砜超滤膜。研究结果表明,截留分子量为50 K的超滤膜分离纯化5-羟基色氨酸的效果要优于截留分子量分别为10 K和100 K的超滤膜。超滤膜分离纯化5-羟基色氨酸优化工艺条件:操作压力0.10 MPa、操作温度35℃、料液质量浓度1.141 mg/m L、p H值7.0,在此条件下5-羟基色氨酸转移率为83.5%,纯度可达90.5%。     

超滤膜分离纯化加纳籽中5-羟基色氨酸的研究北大核心CSCD

研究了超滤膜从加纳籽中分离纯化5-羟基色氨酸的相关膜工艺条件及参数,并筛选出适合的聚砜超滤膜。研究结果表明,截留分子量为50 K的超滤膜分离纯化5-羟基色氨酸的效果要优于截留分子量分别为10 K和100 K的超滤膜。超滤膜分离纯化5-羟基色氨酸优化工艺条件:操作压力0.10 MPa、操作温度35℃、料液质量浓度1.141 mg/m L、p H值7.0,在此条件下5-羟基色氨酸转移率为83.5%,纯度可达90.5%。     

重组胰高糖素样肽-1融合蛋白的纯化及其活性测定

主要探讨了发酵液中融合蛋白rh（GLP-1A2G）2-HSA的分离纯化过程。发酵上清液经超滤浓缩、离子交换层析、凝胶过滤分离得高纯度的rh（GLP-1A2G）2-HSA。纯化样品经SDS-PAGE检测为单一条带,HPLC分析纯度达98%,^125I标记后用TCA沉淀法测定放射性纯度达97%,符合药效学和药代动力学研究的需要,整个纯化过程回收率达到48.5%。通过细胞增殖实验表明纯化后的rh（GLP-1A2G）2-HSA具有类似GLP-1的促胰岛β细胞增殖活性,说明该分离过程保护了融合蛋白rh（GLP-1A2G）2-HSA的生物活性。通过融合蛋白rh（GLP-1A2G）2-HSA的纯化方法,可获得高纯度的重组融合蛋白rh（GLP-1A2G）2-HSA,为进一步的药效学和药代动力学研究奠定了基础。     

重组人尿激酶原纯化中浓缩方法的比较

对尿激酶原纯化中的浓缩方法进行了探索,先后采用了3种不同的方法,即超滤、CM-阳离子柱、疏水色谱柱浓缩。结果表明,超滤只适合于大量样品的浓缩,样品量较小时损失较大,而且机械作用较强会使部分产品分解;CM-阳离子枉浓缩回收率较高,但得到的产品盐浓度高,还需要经过脱盐处理,增加了操作步骤,而疏水柱浓缩方法较为理想,经它浓缩后的产品用SDS-PAGE分析,非还原条件下,纯度在98％以上,还原条件下单链占90％以上,浓缩16倍,尿激酶原浓度达到3mg/ml以上。     

超滤过结合密度梯度离心纯化鲍曼不动杆菌噬菌体方法的建立

目的 探讨获得低浓度内毒素和高滴度鲍曼不动杆菌噬菌体的方法,为制备安全的噬菌体生物制剂提供参考.方法 用可截留100 kD以上分子量的超滤离心管浓缩噬菌体裂解液并滤出分子量约为10 kD的内毒素,然后用蔗糖密度梯度离心纯化噬菌体浓缩液;分别测定超滤前、超滤后和纯化后的噬菌体滴度,采用鲎试验测定超滤前后内毒素的浓度,通过SDS-PAGE分析超滤前后和纯化后噬菌体蛋白的纯度.结果 经超滤离心法噬菌体滴度从3.9×1010 PFU/mL提高至1.68×1012PFU/mL,并可去除99.2％的内毒素;超滤过结合密度梯度离心后的SDS-PAGE可清晰呈现7种蛋白,分子量为29～100 kD.结论 超滤过结合密度梯度离心是一种简便、快速浓缩和纯化噬菌体的方法,并可有效地去除裂解液中的内毒素.     

右旋糖酐40制备过程中细菌内毒素含量的影响因素

右旋糖酐40生产中分离纯化采用乙醇沉淀和超滤方法,将细菌内毒素作为分离纯化的一项指标,对影响因素进行探究,将细菌内毒素含量引入右旋糖酐40原料药产品标准,并且达到国际标准。随着乙醇浓度的增加,相应沉淀产品的细菌内毒素含量逐渐降低;在乙醇分步沉淀中,随着乙醇浓度逐级提高,分离产品的细菌内毒素含量也在降低。分离纯化用水和环境因素均会影响右旋糖酐40分离纯化的细菌内毒素指标。乙醇沉淀分离纯化的右旋糖酐40产品,细菌内毒素含量均<6.00 EU/g,最低含量<3.00 EU/g,优于国际标准(≤10 EU/g),重均分子量和分子量均符合《中国药典》质量标准。100 kDa+20 kDa二膜组合超滤分离纯化的右旋糖酐40的细菌内毒素含量可以达到10 EU/g以下。100 kDa+20 kDa+50 kDa三膜组合超滤分离纯化,右旋糖酐40的细菌内毒素含量达到3 EU/g以下,重均分子量及分子量分布达到《中国药典》标准。改进右旋糖酐40生产工艺,提高产品品质,细菌内毒素含量引入右旋糖酐40原料药质量指标,使其达到和超过国际最高质量标准。     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.     

Ochratoxin A in porcine blood and in consumed feed samples

The aim of the study was to estimate occurrence of ochratoxin A (OA) in feeds and the metabolite residues in porcine blood serum in Poland. Samples were collected in the period from February to May, 1999, in the southern Wielkopolska region. Altogether 40 and 45 samples of feed and porcine blood serum, respectively, were analyzed for OA. Percentage of samples contaminated with OA, both in case of feeds and blood, collected in the winter season was considerably higher than that for the spring season. The percentages for feeds were as follows: 47.6 and 26.3 %, while for porcine serum: 66.7 and 50.0 %, respectively winter and spring. In 25 % of cases ochratoxin A was present in both types of investigated material (feed, blood), whereas in 27.5 % of samples this metabolite was detected in blood only, or in 7.5 % only in the feed. The presence of OA was found neither in the feed nor in the serum in 40 % of all cases. In subgroups (feed, blood) the concentration in the whole collective of positive samples were in the range 0.3–13.5 ng/g and 0.3–69.5 ng/ml, respectively, while median values were 2.3 ng/g and 6.0 ng/ml. Only one feed and three porcine serum samples, were found to be contaminated at concentration levels higher than 10 ng/g or 10 ng/ml.     

Preparation of lipid-free human hemoglobin by dialysis and ultrafiltration

Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.     

Purification of lysozyme using ultrafiltration

This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.     

Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay

Two hybridoma cell lines secreting antibodies against thromboxane synthase of porcine lung were produced. Clone TS1 secretes IgG2a antibody of lower affinity, while clone TS2 secretes IgG1 antibody of higher affinity. Both antibodies (when bound to rabbit anti-mouse IgG-Staphylococcus aureus complex) can immunoprecipitate thromboxane synthase from crude enzyme preparations in an active form suggesting that binding was not directed at the active site. Each antibody showed a distinctive pattern of cross-reactivity with thromboxane synthase from different porcine tissues. Neither of the antibodies cross-reacted with the enzyme from tissues of other species tested, indicating the heterogeneous nature of the enzyme among species. Competitive binding assay revealed that TS1 and TS2 recognized different determinants on the enzyme. The fact that two antibodies bind to separate epitopes on the same enzyme allows the development of a sensitive tandem immunoradiometric assay. The assay, based on binding of 125I-TS2 to thromboxane synthase immobilized on TS1-S. aureus complex, was linear with 7.5 approximately 75 ng of purified lung thromboxane synthase as standards and applicable to enzyme preparations regardless of their purity. The concentration of immunoreactive thromboxane synthase in porcine tissues as determined by this assay followed the order of platelet greater than colon greater than duodenum greater than lung greater than kidney greater than stomach. The fact that gastrointestinal tract is enriched with thromboxane synthase suggests that thromboxane may have significant physiological roles to be recognized in these organs.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

A continuous-flow high-yield process for preparation of lipid-free hemoglobin

Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.     

Elimination of porcine hemopoietic cells by macrophages in mice.

The difficulty in achieving donor hemopoietic engraftment across highly disparate xenogeneic species barriers poses a major obstacle to exploring xenograft tolerance induction by mixed chimerism. In this study, we observed that macrophages mediate strong rejection of porcine hemopoietic cells in mice. Depletion of macrophages with medronate-encapsulated liposomes (M-liposomes) markedly improved porcine chimerism, and early chimerism in particular, in sublethally irradiated immunodeficient and lethally irradiated immunocompetent mice. Although porcine chimerism in the peripheral blood and spleen of M-liposome-treated mice rapidly declined after macrophages had recovered and became indistinguishable from controls by wk 5 post-transplant, the levels of chimerism in the marrow of these mice remained higher than those in control recipients at 8 wks after transplant. These results suggest that macrophages that developed in the presence of porcine chimerism were not adapted to the porcine donor and that marrow-resident macrophages did not phagocytose porcine cells. Moreover, M-liposome treatment had no effect on the survival of porcine PBMC injected into the recipient peritoneal cavity, but was essential for the migration and relocation of these cells into other tissues/organs, such as spleen, bone marrow, and peripheral blood. Together, our results suggest that murine reticuloendothelial macrophages, but not those in the bone marrow and peritoneal cavity, play a significant role in the clearance of porcine hemopoietic cells in vivo. Because injection of M-liposomes i.v. mainly depletes splenic macrophages and liver Kupffer cells, the spleen and/or liver are likely the primary sites of porcine cell clearance in vivo.     

Somatostatin action on insulin secretion induced by chicken and porcine vasoactive intestinal peptide in the perfused rat pancreas

The isolated perfused rat pancreas with duodenal exclusion was used to study the stimulation of glucose-induced insulin release in response to chicken and porcine vasoactive intestinal peptide (VIP). The insulin response to 5.5 or 16.7 mM glucose was markedly enhanced by 750 pM porcine VIP and a concentration of 250 pM was still effective. At 250 pM, chicken VIp exhibited a slightly higher potency than porcine VIP at both glucose concentrations. The main difference between the two peptides was that the effect of porcine VIP disappeared immediately after the peptide suppression but tha of chicken VIP persisted for an additional period of 8-10 min. Somatostatin (10 ng/ml) blocked the stimulatory effect of both VIP molecules on glucose-induced insulin secretion. After suppression of VIP and somatostatin from the perfusion medium, insulin release increased to levels higher than those with glucose alone in the case of the avian peptide, but not in that porcine VIP. The data are consistent with previous results in the literature on stimulation of exocrine pancreas secretion and interaction with intestinal epithelium.     

Retrograde transfer of testosterone to the porcine ovary in follicular and luteal phase of the estrous cycle in vivo

This study was designed to determine the efficiency and rate of testosterone (T) retrograde transfer during the follicular and luteal phase of the estrous cycle in gilts (n=27). The efficiency and the rate of the retrograde transfer of T from the ovarian effluent into blood supplying the ovary were determined for the first time under in vivo conditions. Ovarian arterial blood concentration of T was higher than that in systemic blood during both, the follicular phase (p<0.01) and luteal phase (p<0.0001). The efficiency of the retrograde transfer of T into ovarian arterial blood was not dependent on concentration of testosterone in the ovarian venous blood. However, the efficiency and the rate of the retrograde transfer of T differed between phases of the estrous cycle. The presented results suggest that local retrograde transfer enriched the porcine ovary with an additional amount of T which may affect the ovarian secretory function.