优化Ⅰ型胶原蛋白的纯化工艺

使用0.5 mol/L乙酸和胃蛋白酶提取Ⅰ型胶原蛋白后,结合等电点沉淀法和超滤纯化技术对Ⅰ型胶原蛋白进行纯化。通过鞣酸沉淀法和聚丙烯酰胺凝胶电泳(SDS-PAGE)证实等电点沉淀可有效除去胃蛋白酶;经超滤纯化后的胶原蛋白紫外光谱检测在235nm处有最大吸收峰;在SDS-PAGE谱图上出现3条Ⅰ型胶原蛋白特征谱带;采用凯氏定氮法也证实其纯度符合YY/T1511—2017医药行业标准;通过傅里叶变换红外光谱、圆二色谱法(Circular Dichroism, CD)测定证实胶原蛋白三螺旋结构被完整保留下来。采用等电点沉淀法结合超滤纯化技术,可保证Ⅰ型胶原蛋白的高纯度及三螺旋结构的完整性。     

1～100kDalton的胶原水解物的分子量分布测定的研究

采用凝胶板配方和电泳条件优化后的SDS-PAGE技术对酸法明胶水解物、碱法明胶水解物、胃蛋白酶法明胶水解物、胃蛋白酶法猪皮胶原水解物、明胶以及猪皮胶原蛋白透析物等进行表征,获得了将分布于一块凝胶板上的1～100kD的棒状胶原蛋白各组分的色谱图。胃蛋白酶法猪皮胶原蛋白水解物经电泳分离后的色谱图中,各谱带清晰明显,分子量均一,且均匀分布于1～100kD不等,这对棒状胶原蛋白分子的研究具有重要价值。     

鸡软骨Ⅱ型胶原蛋白的提纯与鉴定

目的探讨可溶性Ⅱ型胶原蛋白(CⅡ)的提纯方法。方法以雏鸡胸软骨为原材料,酸性条件下经胃蛋白酶消化,离心后,应用DEAE-Sephadex A-50进行离子交换层析,透析,盐析,得到纯化的CⅡ。结果自提的CⅡ样品与CⅡ标准品采用SDS-PAGE电泳显示条带一致。结论本法提纯CⅡ具有材料丰富,操作简便,重复性好等优点。     

栝楼籽蛋白分离纯化及抗氧化性的研究

采用超声波辅助碱溶法提取栝楼籽蛋白,以等电点沉淀、乙醇沉淀、硫酸铵沉淀等三种方法对栝楼籽蛋白纯化,并对纯化蛋白分别进行羟基自由基清除能力,DPPH自由基清除能力,铁还原能力的测定。结果表明,在三种纯化方法的最优条件下,蛋白质析出率最高的为硫酸铵沉淀法,其次为等电点沉淀法,较小的为乙醇沉淀法;三种方法沉淀蛋白质的抗氧化性均随着蛋白质浓度增大而增强,但它们的抗氧化能力不一,抗氧化能力强弱顺序为:硫酸铵沉淀等电点沉淀乙醇沉淀。     

重组人干扰素β1a的纯化及其理化性质

目的纯化重组人干扰素β1a(recombinant human interferonβ1a,rhIFNβ1a),并分析其理化性质。方法应用15 L生物反应器悬浮微载体培养表达rhIFNβ1a的重组CHO细胞,细胞培养收获液经超滤浓缩、蓝胶亲和层析、脱盐和CM离子交换层析纯化后,采用水泡性口炎病毒抑制法测定rhIFNβ1a的效价;Lowry法测定蛋白含量;等电聚焦电泳法测定其等电点;SDS-PAGE和高效液相色谱法测定其纯度;Western blot法分析其免疫活性;SDS-PAGE和质谱仪测定其相对分子质量;圆二色谱法分析其二级结构。结果共纯化了3批样品,纯化的rhIFNβ1a平均蛋白浓度为0.284 0 mg/ml,平均比活可达1.06×108IU/mg,纯度达95%以上,蛋白质平均回收率为58.36%,蛋白质相对分子质量为20 445.0,等电点约为6.6,免疫活性良好,二级结构与国家标准品基本一致。结论成功纯化了rhIFNβ1a,并检测了其理化性质,为建立大规模制备rhIFNβ1a的生产工艺和制造检定规程,实现产业化奠定了基础。     

胃蛋白酶提取牛皮胶原蛋白的工艺研究

以新鲜牛皮为原料,分析了牛皮的基本组成;采用胃蛋白酶提取牛皮胶原蛋白,研究了提取过程中加酶量、提取温度、提取时间和料液比对胶原蛋白提取率的影响,得到酸酶结合法提取牛皮中胶原蛋白的最佳工艺条件为:以0.5 mol/L乙酸作提取剂,每1 g牛皮加入30 mL浸提液(即料液比1∶30),胃蛋白酶加入量3 000 U/g牛皮,提取温度30℃,提取时间为8 h,在该条件下胶原蛋白的提取率为61.7%。提取的胶原蛋白经过初步纯化后进行紫外扫描,测得其最大紫外吸收波长为232 nm,与Sigma标准Ⅰ型胶原蛋白一致。     

栝楼籽蛋白分离纯化及抗氧化性的研究

采用超声波辅助碱溶法提取栝楼籽蛋白,以等电点沉淀、乙醇沉淀、硫酸铵沉淀等三种方法对栝楼籽蛋白纯化,并对纯化蛋白分别进行羟基自由基清除能力,DPPH自由基清除能力,铁还原能力的测定。结果表明,在三种纯化方法的最优条件下,蛋白质析出率最高的为硫酸铵沉淀法,其次为等电点沉淀法,较小的为乙醇沉淀法;三种方法沉淀蛋白质的抗氧化性均随着蛋白质浓度增大而增强,但它们的抗氧化能力不一,抗氧化能力强弱顺序为:硫酸铵沉淀>等电点沉淀>乙醇沉淀。     

L-硒代蛋氨酸的合成及工艺优化

将蛋氨酸（I）烷基化后再经过溴取代反应生成溴代高丝氨酸溴酸盐（III）；接着将硒分别和三种还原剂反应得到二硒化二钠后与化合物III反应；得到的硒代高胱氨酸（IV）经由三乙酰氧基硼氢化钠还原、碘甲烷甲基化获得硒代蛋氨酸（V）。对其主要步骤进行优化筛选，获得最优的合成工艺条件：采用新型还原剂三乙酰氧基硼氢化钠制备二硒化二钠可以获得的中间产物IV收率较高，而且当氢氧化钠与三乙酰氧基硼氢化钠摩尔比为4.0：1.0时，化合物III的最高产率为62.42%；采用化合物IV、碘甲烷摩尔比1.0：2.0，以及氢氧化钠与三乙酰氧基硼氢化钠的摩尔比2.7：1.0，可获得终产物V的最高产率为75.79%；最终产物V的结构结晶纯化（纯度95%）后结合质谱、氢谱、碳谱进行确证。     

酸酶结合法提取牛皮胶原蛋白的工艺研究

以新鲜牛皮为原料,分析了牛皮的基本组成;采用胃蛋白酶提取牛皮胶原蛋白,研究了提取过程中加酶量、提取温度、提取时间和料液比对胶原蛋白提取率的影响,得到酸酶结合法提取牛皮中胶原蛋白的最佳工艺条件为：以0.5 mol／L乙酸作为提取剂,料液比为1：30,胃蛋白酶加入量3 000 U／g牛皮,提取温度35℃,提取时间为8 h,在此条件下得胶原蛋白的提取率为61.7％。提取的胶原蛋白经过初步纯化后进行紫外扫描,测得其最大紫外吸收波长为232nm,与sigma标准Ⅰ型胶原蛋白一致。     

聚乙二醇修饰重组人干扰素α1b的制备及其性质

目的制备聚乙二醇(PEG)修饰的重组人干扰素α1b(rhIFNα1b),并分析其部分性质。方法采用PEG修饰rhIFNα1b,通过离子交换层析分离纯化修饰混合物,单点修饰产物经超滤浓缩后,经Lowry法检测蛋白的浓度,SDS-PAGE和RP-HPLC检测蛋白的纯度,等电聚焦电泳检测蛋白的等电点,质谱法检测蛋白的相对分子质量,细胞病变抑制法检测蛋白的体外活性,ELISA法检测蛋白的抗原性。结果单点修饰产物的蛋白得率为33%;SDS-PAGE分析纯度为96.1%,RP-HPLC分析纯度为98.1%;等电点为6.22;相对分子质量为39946;比活为1.28×106IU/mg,活性保留率为14.4%;抗原性至少降至原来的1/625。结论制备的单点修饰rhIFNα1b药学性质获得改善,有望开发为安全、长效的新型干扰素。     

Improving collagen extraction through an alternative strategy based on succinic anhydride pretreatment to retain collagen's triple‐helix structure

The improvement of the extraction yield of collagen while retaining its triple‐helix structure continues to represent a significant challenge for the high‐value utilization of collagen. In this study, pigskin was pretreated by succinic anhydride via the chemical linking of additional carboxylic groups to epsilon amino groups with a conversion degree of 90.2% to obtain pretreatment acid‐pepsin‐solubilized collagen (PAPC). The pretreatment by succinic anhydride increased the tropocollagen distance from 1.39 to 1.42 nm. This permitted acid and pepsin to more easily enter into the fiber clearance and, thus, improved the collagen extraction yield by 9.6%. Furthermore, X‐ray diffraction, Fourier transform infrared spectroscopy, circular dichroism, ultrasensitive differential scanning calorimetry, and atomic force microscopy analysis demonstrated that the triple‐helix conformation of PAPC was well‐maintained. The equilibrium surface tension and isoelectric point of PAPC were 57.48 mN/m and 4.01, respectively; this suggested that the PAPC had surface activity and better solubility in a neutral pH solution. The novelty of PAPC lay in its facilitating fibroblast proliferation, and no extra cytotoxicity was introduced into the collagen after pretreatment. According to these results, our study revealed that succinic anhydride pretreatment as an alternative strategy retained the triple‐helix structure of collagen and improved its extraction ratio; this might be a feasible, yet promising paradigm for the high‐value utilization of collagen. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45424.     

α_1-抗胰蛋白酶制剂的制备及其病毒灭活

目的 从Ｃｏｈｎ　ＦⅣ中提纯α１－抗胰蛋白酶（α１－Ａｎｔｉｔｒｙｐｓｉｎ　α１－ＡＴ，制备较高纯度α１－ＡＴ制剂。方法 对Ｃｏｈｎ ＦⅣ抽提液经沉淀、超滤、离子交换及凝胶过滤，提纯 α１－ＡＴ用巴氏法（液态，６０℃，１０ｈ加热）作病毒灭活， 并考察其效果。用免疫单扩散、双向交叉免疫电泳、ＳＤＳ－ＰＡＧＥ及合成基质法检测α１－ＡＴ蛋白及生物活性。用区带 扫描法测定α１－ＡＴ制剂的纯度。结果３批试制的α１－ＡＴ制剂的纯度为９１．３±１．５２％，比活０．４９±０．０８μ／ｍｇ，比活性 比抽提液提高了２７．４倍。巴氏法处理可使ＶＳＶ、Ｓｉｎｄｂｉｓ、Ｐｏｌｉｏ　Ｉ型疫苗病毒分别降低１０．０±０．３，１０．０±０．３１及７．１１ ±０．３ｌｇＴＣＩＤ５０／ｍｌ，但仍能检出Ｐｏｌｉｏ型疫苗病毒。结论 可从 Ｃｏｈｎ ＦⅣ中制得较高纯度的 α１－ＡＴ制剂，巴氏法能对 α１－ＡＴ制剂作有效的病毒灭活处理。     

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

A simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A_{545 nm}/A_{280 nm} > 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (V_r) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry     

Facile purification and characterization of recombinant human antithrombin III from transgenic goat milk

BACKGROUND: Antithrombin III (AT III) is a serine protease inhibitor that inhibits thrombin and the activated forms of factors X, VII, IX, XI and XII. Transgenic expression of therapeutic proteins in animal systems has gradually matured from laboratory scale to industrial practice, demanding efficient and scalable purification processes. The purification and characterization of recombinant human antithrombin III (rhAT III) from transgenic goat milk are described here. RESULTS: The rhAT III was purified by isoelectric precipitation, heparin affinity chromatography, and size exclusion chromatography, resulting in a 90.6% yield and > 99% purity. The goat β‐casein secretion peptide introduced to the rhAT III was cut off using enterokinase and removed by size exclusion chromatography using a Superdex 75 column. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT III were measured and found to be the same as those of the plasma‐derived AT III (phAT III). CONCLUSION: A facile process is introduced for the purification of rhAT III from transgenic goat milk. The rhAT III with high purity was achieved after an initial isoelectric precipitation step in which most of the bulk protein impurities are removed, followed by affinity chromatography and size exclusion chromatography. The rhAT III was demonstrated to have the same structure as phAT III. Copyright © 2011 Society of Chemical Industry     

猪皮胶原的降解

采用低温-胃蛋白酶法从新鲜猪皮中提取胶原。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）结合紫外、红外光谱分析证明所提取的胶原为Ⅰ型活性胶原,即胶原分子量约为32万道尔顿,组成为α1）2α2,且具有完整的三股螺旋结构;利用天然胶原的特性降解酶细菌胶原酶进行低温降解,得到分子量在3万到20万道尔顿之间的降解产物,降解产物红外光谱证明大部分仍然保持三股螺旋结构,并与市售的胶原多肽（失活）的红外光谱图比较,充分体现了此降解方案的优越性。     

聚乙二醇化重组人干扰素α2b的质量检测

目的　建立聚乙二醇化重组人干扰素α2b(PEG rhIFNα2b)的质量检测方法。方法　采用Wish细胞 VSV病毒系统 ,以CPE法测定干扰素的生物学活性 ,基质辅助激光解吸附飞行时间质谱法测定相对分子质量 ,反向高效液相法 (RP HPLC)测定纯度 ,溴化氰裂解 SDS PAGE法测定肽图 ,等电聚焦电泳法测定等电点 ,紫外分光光度法测定紫外吸收光谱。结果　聚乙二醇化重组人干扰素α2b的比活为 6 . 0× 10 6～ 10 . 0× 10 6IU/mg蛋白 ,相对分子质量约为 6 2 6 0 0 ,等电点在 5 . 2 0～ 6. 5 5之间 ,紫外最大吸收峰约在 2 78nm波长处。结论　所建立的检测方法可控制聚乙二醇化重组人干扰素α2b质量。     

幽门螺杆菌重组VacA蛋黄抗体的制备

目的研制高效价、高特异性的抗细胞空泡毒素VacA的鸡蛋黄抗体(IgY),为研制幽门螺杆菌(H.pylori)治疗性抗体奠定基础。方法用纯化的重组细胞空泡毒素抗原免疫22周龄洛曼产蛋母鸡,取免后所产鸡蛋,将蛋黄稀释、离心,收集上清,加硫酸铵沉淀。以ELISA间接法检测抗体效价,Bradford法检测蛋白含量,SDS-PAGE法检测相对分子质量及纯度。结果母鸡初次免疫后76d,抗体效价达到1∶6400,最高可达1∶12800。蛋黄抗体经纯化浓缩后,用SDS-PAGE分析,出现2条蛋白带,纯度可达80%以上,其含量为25·66mg/ml。结论已成功制备了抗重组细胞空泡毒素的特异性蛋黄抗体。     

葡萄球菌蛋白A的纯化新工艺

目的建立一种高效、简便、实用的分离纯化葡萄球菌蛋白A(SPA)的工艺。方法采用超声波破菌和IgG-Sepharose4B亲和层析分离纯化SPA;以Lowry法检测蛋白含量;双向琼脂免疫扩散法测定效价和特异性;用还原和非还原SDS-PAGE法检测纯度和相对分子质量。结果此法制得的3批SPA的蛋白含量分别为4·7、4·4和5·4mg/ml,收率分别为2·70、2·64和3·78g/100g湿菌。对人IgG的免疫双扩散效价均为1:64;与正常人、豚鼠、家兔和小鼠的血清反应均出现一条沉淀线,而与鸡和羊不出现沉淀线。经非还原SDS-PAGE检测只呈现一条蛋白带,相对分子质量约为160000～180000;经还原SDS-PAGE检测呈现两条蛋白带,相对分子质量分别约为67000和34000。结论已成功建立了分离纯化SPA的新工艺。     

Computational modeling of type I collagen fibers to determine the extracellular matrix structure of connective tissues

A method is presented for generating computer models of biological tissues. The method uses properties of extracellular matrix proteins to predict the structure and physical chemistry of the elements that make up the tissue. The method begins with Protein Data Bank coordinate positions of amino acids as input into TissueLab software. From the amino acid sequence, a type I collagen-like triple helix backbone was computationally constructed and boundary spheres were added based on known chemical and physical properties of the amino acids. Boundary spheres determined the contact surface characteristics of the collagen molecules and intermolecular interactions were then determined by considering the relationships of the contact surfaces and by resolving the energy-minimum state using feasible sequential quadratic programming. From this, the software created fibrils that corresponded exactly to known collagen parameters and were further confirmed by finite element modeling. Computationally derived fibrils were then used to create collagen fibers and three-dimensional collagen matrices. By resolving the energy-minimum state, large complex components of the extracellular space as well as other structures can be determined to provide three-dimensional structure of molecules, molecular interactions and the tissues that they form.     

A combined coagulation–ultrafiltration method for enhanced separation of proteins and polyphenols

This work aimed to separate proteins and polyphenols from rapeseed stem extracts. The results showed that coagulation with 40% w/w ethanol and ultra-filtration with 10 kDa membrane were two efficient methods for polyphenols purification. The purity of polyphenols increased from 80.3% to 97.0% after separation. The combination of ethanol coagulation with ultrafiltration permitted decreased the ethanol consumption from 40% to 10% w/w while maintaining high polyphenols purity. Significant protein loss at room temperature was observed during the stability test, which emphasizes the importance of elaborating a fast and effective separation method for rapeseed stem extracts.