高分子电解质分离碱性蛋白的方法及碱性多肽SDS-PAGE条件的优化
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摘要
等电点沉淀(isoelectric precipitation)和聚合高分子电解质沉淀(polyelectrolyte precipitation)是蛋白质分离纯化的基本技术和常用方法，用于蛋白质分离纯化的前期处理，具有设备简单，处理量大，廉价和蛋白质不变性等特点。本试验研究了聚丙烯酸(Polyacrylic Acid，PAA)分离纯化苦瓜种仁碱性蛋白和蜜蜂毒素碱性多肽的方法及其影响因素。同时，研究了丙酮对苦瓜种仁和蜜蜂毒素脂类物质的清除作用，以及碱性多肽的SDS-PAGE分离和分析方法。
丙酮与苦瓜种仁或蜜蜂毒素研磨匀浆，过滤并用丙酮洗涤，室温干燥形成蛋白-丙酮粉，与水抽提法比较，蛋白-丙酮粉的水溶性高、脂类含量少，利于蛋白质的进一步分离纯化。
等电点沉淀试验表明，在pH7.0～3.0范围内，苦瓜种仁的中、酸性蛋白沉淀析出，上清液中等电点pI值大于8.65的碱性蛋白种类和含量与粗提液相似，而等电点pI值为8.65的碱性蛋白含量随溶液pH值的降低而减少。等电点沉淀与酸介质密切相关。以柠檬酸(citric acid)调节苦瓜种仁粗提液的酸碱度，在pH7.0～6.0之间的等电点沉淀作用明显，其中pH6.0时14.62％的苦瓜种仁蛋白被沉淀；以盐酸(hydrochloric acid)调节粗提液酸碱度，在pH7.0～4.0之间的等电点沉淀作用明显，其中pH4.0时32.49％的苦瓜种仁蛋白被沉淀。醋酸(acetic acid)的等电点沉淀作用呈现阶段性特点，在pH7.0～6.0和pH5.0～4.0之间的等电点沉淀作用明显，其中pH6.0和4.0时分别有26.17％和38.72％的苦瓜种仁蛋白被沉淀。
PAA选择性沉淀苦瓜种仁碱性蛋白，与酸介质、PAA浓度和pH值的密切相关。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液，PAA(1％)选择性沉淀碱性蛋白(等电点pI为8.65～9.30)的最佳用量分别为100μl、120μl和100μl。SDS-PAGE结果表明PAA沉淀醋酸处理的苦瓜种仁粗提液碱性蛋白的效率高于沉淀盐酸和柠檬酸处理的苦瓜种仁粗提液的碱性蛋白的效率。以醋酸分别调节苦瓜种仁粗提液pH至5.0、4.0和3.0，等电点沉淀后的上清液用PAA沉淀碱性蛋白，当PAA用量为160μl／mL提取液时，pH5.0和3.0样液分别有33.77％和43.56％蛋白质被沉淀；当在PAA用量为120μl／mL提取液时，pH4.0样液中30.83％蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH＞9.0)，溶解性与NaCl浓度的有关，当溶液NaCl浓度为3.0％时，溶液蛋白质浓度最高。
PAA选择性沉淀的苦瓜种仁碱性蛋白经Sephadex G-75柱层析分离，分别在175min和300min出现2个主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kDa，pI值约为9.3，主峰Ⅱ的分子量约为10kDa，pI值约为9.5。
丙酮处理形成蜂毒-丙酮粉水溶液，PAA选择性沉淀的碱性多肽经Sephadex G-25凝胶层析分离，形成二个独立吸收峰Ⅰ和峰Ⅱ，SDS-PAGE显示其分子量分别约为2800Da和1000Da，IEF显示峰Ⅰ含有等电点pI为9.0的碱性多肽。
高交联度(C≥6.0％)的尿素／SDS-PAGE是多肽分离纯化和分析检测的主要技术，因碱性多肽的分子量和电荷特性，常用酸性电泳分离纯化和分析检测。本试验初步研究了低交联度的SDS-PAGE分析蜂毒碱性多肽的方法及有关条件的优化。
SDS-PAGE结果表明，凝胶交联度C为0.67％时，样品和Marker中低分子成份堆积在凝胶前沿，不能分离；凝胶交联度为1.20％时，样品中的低分子量成分不能分离；Marker中低分子量多肽(分子量≤16950Da)基本分离；凝胶交联度C为1.55％时，样品和Marker中的低分子量成分分离良好；凝胶交联度C为2.00％时，样品和Marker中低分子量多肽不能分离；当凝胶交联度≥3.00％时，样品和Marker中低分子量多肽分离不理想。
Both isoelectric and polyelectrolyte precipitation have been shown to be effective and surprisingly selective in separating proteins from the extracts of various hosts. Because it is a simple unit operation, relatively inexpensive and straightforward to be scaled-up, precipitation is often used in the early stages of protein purification processes for protein recovery and purification. In this study, Polyacrylic Acid (PAA) was used to isolate and purify alkalescence protein from Momordica charantia Zseeds and alkalescence peptides from bee venom.Besides, the effect of acetone on fat and analysis of alkalescence peptides by SDS-PAGE were also evaluated in this paper.
Acetone and Momordica charantia L. seeds (bitter melon seeds) or bee venom were homogenated, filtrated and washed (with acetone). The acetone powder was obtained by evaporation. Compared to water extraction, protein by acetone extraction was much water-soluble with little fat, which contributed to recovery and purification of protein.
By the isoelectric precipitation, the neutral and acidic protein from Momordica charantia L. seeds was removed at the pH 7.0~3.0. In the upper solution, the kinds and content of the alkalescence protein with pI above 8.65 was the same to the extraction. The alkalescence protein with pI 8.65 decreased with pH falling. The isoelectric precipitation was related to acid medium. When the Momordica charantia L. seeds extraction was adjusted by the citric acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0, and 14.62% protein precipitated at pH6.0. When the extraction was adjusted by the hydrochloric acid, the isoelectric precipitation was obvious during the range of pH7.0～4.0, and 32.49% protein precipitated at pH4.0. When the extraction was adjusted by the acetic acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0 and pH5.0～4.0, and 26.17% and 38.72% protein precipitated at pH6.0 and pH4.0, respectively.
Precipitation of PAA with alkalescence protein from Momordica charantia L. seeds was influenced by acid medium, PAA concentration and pH. In the solution (1 mL) adjusted by acetic acid, hydrochloric acid and citric acid, PAA (1%) was added at 100μl,120μl and 100μl. SDS-PAGE showed that PAA precipitation protein in extraction adjusted by acetic acid was more efficient than hydrochloric acid and citric acid. The extraction was titrated to pH 5.0. pH 4.0, and pH 3.0 by acetic acid. After isoelectric precipitation, the PAA precipitation protein was performed. When concentration of PAA was 160μl/mL, the protein decreased in the supernatant was 33.77% at pH 5.0 and 43.56% at pH 3.0. When concentration of PAA was 120μl/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA -Protein complex counld redissolve in alkaline conditions(pH>9.0)and the solubility was related to NaCl concentration. When the NaCl was 3.0%, the protein most.easilly redissolved.
The bitter melon seeds extraction after PAA purification flowed through the Sephadex G-75 columns. The peaks I and II were obtained after 175min and 300min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks I was 30kDa with pI 9.3, peaks I 10kDa with pI 9.5.
The bee venom extraction after PAA purification flowed through the Sephadex G-25 columns. Two peaks were obtained. Their molecule weights were about 2800Da and 1000Da. IEF showed that peak I included the protein with p/9.0.
SDS-PAGE with urea and high cross lingking (C, C≥6.0%) has been used to separate and examine the alkalescence peptides. Because of its characteristics of molecule weight and charge, the polyacrylamid electrophoresis in acid environment is the method accustomed. This study evaluated the effect of SDS-PAGE with the low C separating alkalescence peptides under the different conditions.
SDS-PAGE showed that, the low molecule weight peptides in samle and marker accumulated in the front of the gel with the C 0.67%. When the value of C was 1.20%, though the low molecule weight peptides in samle were not still separated, the peptides (Mr≤16950Da) in marker appeared clear. When the C was 1.55%, the peptides in both were separated efficiently. When the C was up to 2.00%,the bands were little visually. When the C was above 3.00%, the peptides couldn't be separated.
丙酮与苦瓜种仁或蜜蜂毒素研磨匀浆，过滤并用丙酮洗涤，室温干燥形成蛋白-丙酮粉，与水抽提法比较，蛋白-丙酮粉的水溶性高、脂类含量少，利于蛋白质的进一步分离纯化。
等电点沉淀试验表明，在pH7.0～3.0范围内，苦瓜种仁的中、酸性蛋白沉淀析出，上清液中等电点pI值大于8.65的碱性蛋白种类和含量与粗提液相似，而等电点pI值为8.65的碱性蛋白含量随溶液pH值的降低而减少。等电点沉淀与酸介质密切相关。以柠檬酸(citric acid)调节苦瓜种仁粗提液的酸碱度，在pH7.0～6.0之间的等电点沉淀作用明显，其中pH6.0时14.62％的苦瓜种仁蛋白被沉淀；以盐酸(hydrochloric acid)调节粗提液酸碱度，在pH7.0～4.0之间的等电点沉淀作用明显，其中pH4.0时32.49％的苦瓜种仁蛋白被沉淀。醋酸(acetic acid)的等电点沉淀作用呈现阶段性特点，在pH7.0～6.0和pH5.0～4.0之间的等电点沉淀作用明显，其中pH6.0和4.0时分别有26.17％和38.72％的苦瓜种仁蛋白被沉淀。
PAA选择性沉淀苦瓜种仁碱性蛋白，与酸介质、PAA浓度和pH值的密切相关。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液，PAA(1％)选择性沉淀碱性蛋白(等电点pI为8.65～9.30)的最佳用量分别为100μl、120μl和100μl。SDS-PAGE结果表明PAA沉淀醋酸处理的苦瓜种仁粗提液碱性蛋白的效率高于沉淀盐酸和柠檬酸处理的苦瓜种仁粗提液的碱性蛋白的效率。以醋酸分别调节苦瓜种仁粗提液pH至5.0、4.0和3.0，等电点沉淀后的上清液用PAA沉淀碱性蛋白，当PAA用量为160μl／mL提取液时，pH5.0和3.0样液分别有33.77％和43.56％蛋白质被沉淀；当在PAA用量为120μl／mL提取液时，pH4.0样液中30.83％蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH＞9.0)，溶解性与NaCl浓度的有关，当溶液NaCl浓度为3.0％时，溶液蛋白质浓度最高。
PAA选择性沉淀的苦瓜种仁碱性蛋白经Sephadex G-75柱层析分离，分别在175min和300min出现2个主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kDa，pI值约为9.3，主峰Ⅱ的分子量约为10kDa，pI值约为9.5。
丙酮处理形成蜂毒-丙酮粉水溶液，PAA选择性沉淀的碱性多肽经Sephadex G-25凝胶层析分离，形成二个独立吸收峰Ⅰ和峰Ⅱ，SDS-PAGE显示其分子量分别约为2800Da和1000Da，IEF显示峰Ⅰ含有等电点pI为9.0的碱性多肽。
高交联度(C≥6.0％)的尿素／SDS-PAGE是多肽分离纯化和分析检测的主要技术，因碱性多肽的分子量和电荷特性，常用酸性电泳分离纯化和分析检测。本试验初步研究了低交联度的SDS-PAGE分析蜂毒碱性多肽的方法及有关条件的优化。
SDS-PAGE结果表明，凝胶交联度C为0.67％时，样品和Marker中低分子成份堆积在凝胶前沿，不能分离；凝胶交联度为1.20％时，样品中的低分子量成分不能分离；Marker中低分子量多肽(分子量≤16950Da)基本分离；凝胶交联度C为1.55％时，样品和Marker中的低分子量成分分离良好；凝胶交联度C为2.00％时，样品和Marker中低分子量多肽不能分离；当凝胶交联度≥3.00％时，样品和Marker中低分子量多肽分离不理想。
Both isoelectric and polyelectrolyte precipitation have been shown to be effective and surprisingly selective in separating proteins from the extracts of various hosts. Because it is a simple unit operation, relatively inexpensive and straightforward to be scaled-up, precipitation is often used in the early stages of protein purification processes for protein recovery and purification. In this study, Polyacrylic Acid (PAA) was used to isolate and purify alkalescence protein from Momordica charantia Zseeds and alkalescence peptides from bee venom.Besides, the effect of acetone on fat and analysis of alkalescence peptides by SDS-PAGE were also evaluated in this paper.
Acetone and Momordica charantia L. seeds (bitter melon seeds) or bee venom were homogenated, filtrated and washed (with acetone). The acetone powder was obtained by evaporation. Compared to water extraction, protein by acetone extraction was much water-soluble with little fat, which contributed to recovery and purification of protein.
By the isoelectric precipitation, the neutral and acidic protein from Momordica charantia L. seeds was removed at the pH 7.0~3.0. In the upper solution, the kinds and content of the alkalescence protein with pI above 8.65 was the same to the extraction. The alkalescence protein with pI 8.65 decreased with pH falling. The isoelectric precipitation was related to acid medium. When the Momordica charantia L. seeds extraction was adjusted by the citric acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0, and 14.62% protein precipitated at pH6.0. When the extraction was adjusted by the hydrochloric acid, the isoelectric precipitation was obvious during the range of pH7.0～4.0, and 32.49% protein precipitated at pH4.0. When the extraction was adjusted by the acetic acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0 and pH5.0～4.0, and 26.17% and 38.72% protein precipitated at pH6.0 and pH4.0, respectively.
Precipitation of PAA with alkalescence protein from Momordica charantia L. seeds was influenced by acid medium, PAA concentration and pH. In the solution (1 mL) adjusted by acetic acid, hydrochloric acid and citric acid, PAA (1%) was added at 100μl,120μl and 100μl. SDS-PAGE showed that PAA precipitation protein in extraction adjusted by acetic acid was more efficient than hydrochloric acid and citric acid. The extraction was titrated to pH 5.0. pH 4.0, and pH 3.0 by acetic acid. After isoelectric precipitation, the PAA precipitation protein was performed. When concentration of PAA was 160μl/mL, the protein decreased in the supernatant was 33.77% at pH 5.0 and 43.56% at pH 3.0. When concentration of PAA was 120μl/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA -Protein complex counld redissolve in alkaline conditions(pH>9.0)and the solubility was related to NaCl concentration. When the NaCl was 3.0%, the protein most.easilly redissolved.
The bitter melon seeds extraction after PAA purification flowed through the Sephadex G-75 columns. The peaks I and II were obtained after 175min and 300min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks I was 30kDa with pI 9.3, peaks I 10kDa with pI 9.5.
The bee venom extraction after PAA purification flowed through the Sephadex G-25 columns. Two peaks were obtained. Their molecule weights were about 2800Da and 1000Da. IEF showed that peak I included the protein with p/9.0.
SDS-PAGE with urea and high cross lingking (C, C≥6.0%) has been used to separate and examine the alkalescence peptides. Because of its characteristics of molecule weight and charge, the polyacrylamid electrophoresis in acid environment is the method accustomed. This study evaluated the effect of SDS-PAGE with the low C separating alkalescence peptides under the different conditions.
SDS-PAGE showed that, the low molecule weight peptides in samle and marker accumulated in the front of the gel with the C 0.67%. When the value of C was 1.20%, though the low molecule weight peptides in samle were not still separated, the peptides (Mr≤16950Da) in marker appeared clear. When the C was 1.55%, the peptides in both were separated efficiently. When the C was up to 2.00%,the bands were little visually. When the C was above 3.00%, the peptides couldn't be separated.
引文
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Yu J, Liu H, Chen J. Modified flocculation of protein with a combination of anionic copolymer and polyacrylamide. Chemical Engineering Science, 1999, 54: 5839-5843.
云南动物研究所四室，生物化学与生物物理学报，1976，8(2)：151．
周淑平等，生物化学与生物物理进展，1986，(4)：33．
钟立人，吕民主，张合文等．鱼精蛋白的抑菌机理．水产学报，2001，(2)：171-175．
Zhang C, Glatz C E. Process engineering strategy for recombinant protein recovery from canola by cation exchange chromatography. Biotechnol Prog 1999.15:12-18.
Zhang C, Love R T, Jilka J M, et al. Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: An example of b-glucuronidase. Biotechnol. Prog, 2001, 17: 161-167.
Zaman F, Kusnadi A R, Glatz C E. Strategies for recombinant protein recovery from canola by precipitation. Biotech. Prog. 1999, 15(3): 488-492.
Au T K, Collins R A, Lam T L, et al. The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Letters, 2000, 471: 169-172.
Chan W Y, Ng T B, Rong S H, et al. Antiproliferative and teratogenic activities of the bishemisuccinates of α-hydroxycholesterol and β-hydroxycholesterol, General Pharmacology: The Vascular System Ge Pharmacol, 1994, 25(4): 767-772.
Chenming Z, Raymond L, Jackie C, et al. Lysozyme purification from tobacco extract by polyelectrolyte precipitation, Journal of Chromatography A, 2005, 1069: 107-112.
郭尧君．蛋白质电泳技术．北京，科学出版，2005．
Glatz C E. Precipitation. In Downstream Processing Biotechnology; Asenjo, J., Ed.; Marcel Dekker: New York, 1990.
Fong W P, Poon YT, Wong T M, et al. A highly efficient procedure for purifying the ribosome-inactivating proteins α- and β-momorcharins from momordica charantia seeds, n-terminal sequence comparison and establishment of their n-glycosidase activity. Life Sciences, 1996, 59(11): 901-909.
傅明辉 田洁，苦瓜籽核糖体失活蛋白的分离纯化及抗氧化活性的研究中国生化药物杂志．2002，23(3)，134-136．
Lee-Huang S, Huang P L, Nara P L, et al. MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS Lett, 1990, 272: 12-18.
孟延发，杜毅峰，张雪梅．苦瓜籽核糖体失活蛋白的理化性质及生物活性．中国生物化学与分子生物学报，1999，15(6)：920．
Munoz R, Arias Y, Ferreras J.M., et al. Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein Nigrinb, Cancer Lett. 2001, 167: 163-169.
Niederauer M Q, Glatz C E. Selective precipitation. Adv. Biochem. Engr./Biotechnol. 1992, 47:159-188.
Paul M F T, Ng T B, Fong W P, et al. The International Journal of Biochemistry & Cell Biology, 1999, 31(9): 895.
Smith P K, Krohn R I, Hermanson G T, et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150:76-85.
Sternberg M, Hershberger D. Separation of protein with polyacrylic.acids, Biochirn. Biophys. 1974,Acta 342:195.
Wang Jianhua, Nie Huiling, Tam Siucheung, et al. Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity, FEBS Letters, 2002, 531 (2): 295-298.
Valbonesi P, Barbieri L, Bolognesi A, et al. Preparation of highly purified momordin Ⅱ without ribonuclease activity, Life Sciences, 1999, 65(14): 1 485-1491.
王宾香，时祥柱，郭春腾，叶秀云，王中来，饶平凡．阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白．色谱2004，22(5)．543-546．
郭尧君．蛋白质电泳技术．北京，科学出版，2005．
Habermann E. Bee and wasp venom. Science, 1972, 177: 314-322.
Iwadate M, Asakara T, Williamson MP. The structure of the melittin tetramer at different temperatures. Eur J Biochem, 1998, 257: 479-487.
刘岭，凌昌奎，胡晋缸，黄雪强，蜜蜂毒中蜂毒素的分离纯化方法及含量测定，第二军医大学学报，2003，22(7)：609-611．
Niederauer M Q, Glatz C E. Selective precipitation. Adv. Biochem. Engr./Biotechnol. 1992, 47:159-188.
Munoz R, Arias Y, Ferreras J. M., et al. Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein Nigrinb, Cancer Lett. 2001, 167:163-169.
Paul M F T, Ng T B, Fong W P, et al. The International Journal of Biochemistry & Cell Biology, 1999, 31(9): 895.
Smith P K, Krohn R I, Hermanson G T, et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150:76-85.
徐彭，欧阳永伟，黄敬耀．从蜂毒中分离纯化蜂毒肽的实验研究，中草药，2000，12：892-895．
Shin S Y, Lee M K, Kim K L, et al. Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin A-ma-gainin 2 and cecropin A-melittin hybrid peptides. J Pept Res, 1997, 50 (4): 279-285.
Anderson BL, Berry RW, Telser A. A sodium dodecyl sulfate-polyac-rylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 9000. Anal Biochem, 1983, 132, 365-375.
Cleveland DW, Fischer SG, Kirschner MW et al. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by ge lectmphoresis. J Bio Che.1977, 252(3): 1102-1106.
郭燕捷，陶陵，姚志建．测定多肽分子量的SDS聚丙烯酰胺电泳．生物化学与生物物理进展，1987，2：66-69．
郭尧君，蛋白质电泳技术，北京，科学出版，2005．
金冬雁，黎盂枫译．分子克隆实验指南，第2版．北京；科学出版杜，1992：880-887．
Kyte J, Rodriguez H. A discontinuous electro-phoretic system for separating peptides on polyaerylamide gels. Anal Biochem, 1983, 133: 515-522.
陈亚飞，孙宇，高丰衣等．SDS．聚丙烯酰胺凝胶电泳法测定蛇毒抗瘤蛋白的相对分子质量．中国生化药物杂志，2004，25(5)：300-303．
Schagger H, Jagow G. Tricine-sodium dodecyl sulfate-polyacry]amide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal Biochem, 1987; 166(2): 368-397.
张晓楠，张延凤，曹云新，等．尿素-SDS-PAGE快速测定多肽的相对分子质量[J]．细胞与分子免疫学杂志，2001，17(1)：87-97．
石继红，赵永同，王俊楼，韩苇，廉真，张英起SDS-聚丙烯酰胺凝胶电泳分析小分子多肽，第四军医大学学报，2000，21(6)：761-763．
杨联萍，孔样平，易学瑞．SDS-PAGE电泳对小分子多肽的分析．生物工程进展，1998，18(6)：49-51．
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王燕平，旦欣然．东亚钳蝎蝎毒分离纯化及药理作用的研究进展。中草药，2000，31(1)：59-61．
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谢俊杰，李华栋．鲢鱼(Aristichthys nobilis)鱼精蛋白的纯化和鉴定．江西科学，2001，19(4)：214-217．
叶晓峰，吴乔．类泛素蛋白质-SUMO。细胞生物学杂志，2004，26(1)：10-14．
Yu J, Liu H, Chen J. Modified flocculation of protein with a combination of anionic copolymer and polyacrylamide. Chemical Engineering Science, 1999, 54: 5839-5843.
云南动物研究所四室，生物化学与生物物理学报，1976，8(2)：151．
周淑平等，生物化学与生物物理进展，1986，(4)：33．
钟立人，吕民主，张合文等．鱼精蛋白的抑菌机理．水产学报，2001，(2)：171-175．
Zhang C, Glatz C E. Process engineering strategy for recombinant protein recovery from canola by cation exchange chromatography. Biotechnol Prog 1999.15:12-18.
Zhang C, Love R T, Jilka J M, et al. Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: An example of b-glucuronidase. Biotechnol. Prog, 2001, 17: 161-167.
Zaman F, Kusnadi A R, Glatz C E. Strategies for recombinant protein recovery from canola by precipitation. Biotech. Prog. 1999, 15(3): 488-492.
Au T K, Collins R A, Lam T L, et al. The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Letters, 2000, 471: 169-172.
Chan W Y, Ng T B, Rong S H, et al. Antiproliferative and teratogenic activities of the bishemisuccinates of α-hydroxycholesterol and β-hydroxycholesterol, General Pharmacology: The Vascular System Ge Pharmacol, 1994, 25(4): 767-772.
Chenming Z, Raymond L, Jackie C, et al. Lysozyme purification from tobacco extract by polyelectrolyte precipitation, Journal of Chromatography A, 2005, 1069: 107-112.
郭尧君．蛋白质电泳技术．北京，科学出版，2005．
Glatz C E. Precipitation. In Downstream Processing Biotechnology; Asenjo, J., Ed.; Marcel Dekker: New York, 1990.
Fong W P, Poon YT, Wong T M, et al. A highly efficient procedure for purifying the ribosome-inactivating proteins α- and β-momorcharins from momordica charantia seeds, n-terminal sequence comparison and establishment of their n-glycosidase activity. Life Sciences, 1996, 59(11): 901-909.
傅明辉 田洁，苦瓜籽核糖体失活蛋白的分离纯化及抗氧化活性的研究中国生化药物杂志．2002，23(3)，134-136．
Lee-Huang S, Huang P L, Nara P L, et al. MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS Lett, 1990, 272: 12-18.
孟延发，杜毅峰，张雪梅．苦瓜籽核糖体失活蛋白的理化性质及生物活性．中国生物化学与分子生物学报，1999，15(6)：920．
Munoz R, Arias Y, Ferreras J.M., et al. Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein Nigrinb, Cancer Lett. 2001, 167: 163-169.
Niederauer M Q, Glatz C E. Selective precipitation. Adv. Biochem. Engr./Biotechnol. 1992, 47:159-188.
Paul M F T, Ng T B, Fong W P, et al. The International Journal of Biochemistry & Cell Biology, 1999, 31(9): 895.
Smith P K, Krohn R I, Hermanson G T, et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150:76-85.
Sternberg M, Hershberger D. Separation of protein with polyacrylic.acids, Biochirn. Biophys. 1974,Acta 342:195.
Wang Jianhua, Nie Huiling, Tam Siucheung, et al. Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity, FEBS Letters, 2002, 531 (2): 295-298.
Valbonesi P, Barbieri L, Bolognesi A, et al. Preparation of highly purified momordin Ⅱ without ribonuclease activity, Life Sciences, 1999, 65(14): 1 485-1491.
王宾香，时祥柱，郭春腾，叶秀云，王中来，饶平凡．阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白．色谱2004，22(5)．543-546．
郭尧君．蛋白质电泳技术．北京，科学出版，2005．
Habermann E. Bee and wasp venom. Science, 1972, 177: 314-322.
Iwadate M, Asakara T, Williamson MP. The structure of the melittin tetramer at different temperatures. Eur J Biochem, 1998, 257: 479-487.
刘岭，凌昌奎，胡晋缸，黄雪强，蜜蜂毒中蜂毒素的分离纯化方法及含量测定，第二军医大学学报，2003，22(7)：609-611．
Niederauer M Q, Glatz C E. Selective precipitation. Adv. Biochem. Engr./Biotechnol. 1992, 47:159-188.
Munoz R, Arias Y, Ferreras J. M., et al. Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein Nigrinb, Cancer Lett. 2001, 167:163-169.
Paul M F T, Ng T B, Fong W P, et al. The International Journal of Biochemistry & Cell Biology, 1999, 31(9): 895.
Smith P K, Krohn R I, Hermanson G T, et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150:76-85.
徐彭，欧阳永伟，黄敬耀．从蜂毒中分离纯化蜂毒肽的实验研究，中草药，2000，12：892-895．
Shin S Y, Lee M K, Kim K L, et al. Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin A-ma-gainin 2 and cecropin A-melittin hybrid peptides. J Pept Res, 1997, 50 (4): 279-285.
Anderson BL, Berry RW, Telser A. A sodium dodecyl sulfate-polyac-rylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 9000. Anal Biochem, 1983, 132, 365-375.
Cleveland DW, Fischer SG, Kirschner MW et al. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by ge lectmphoresis. J Bio Che.1977, 252(3): 1102-1106.
郭燕捷，陶陵，姚志建．测定多肽分子量的SDS聚丙烯酰胺电泳．生物化学与生物物理进展，1987，2：66-69．
郭尧君，蛋白质电泳技术，北京，科学出版，2005．
金冬雁，黎盂枫译．分子克隆实验指南，第2版．北京；科学出版杜，1992：880-887．
Kyte J, Rodriguez H. A discontinuous electro-phoretic system for separating peptides on polyaerylamide gels. Anal Biochem, 1983, 133: 515-522.
陈亚飞，孙宇，高丰衣等．SDS．聚丙烯酰胺凝胶电泳法测定蛇毒抗瘤蛋白的相对分子质量．中国生化药物杂志，2004，25(5)：300-303．
Schagger H, Jagow G. Tricine-sodium dodecyl sulfate-polyacry]amide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal Biochem, 1987; 166(2): 368-397.
张晓楠，张延凤，曹云新，等．尿素-SDS-PAGE快速测定多肽的相对分子质量[J]．细胞与分子免疫学杂志，2001，17(1)：87-97．
石继红，赵永同，王俊楼，韩苇，廉真，张英起SDS-聚丙烯酰胺凝胶电泳分析小分子多肽，第四军医大学学报，2000，21(6)：761-763．
杨联萍，孔样平，易学瑞．SDS-PAGE电泳对小分子多肽的分析．生物工程进展，1998，18(6)：49-51．