[1]李辉,翟颖超,窦晓,等.重组猪抑制素蛋白质中毒素残留检测及其理化稳定性[J].江苏农业学报,2015,(02):362-368.[doi:10.3969/j.issn.1000-4440.2015.02.022]
　LI Hui,ZHAI Ying-chao,DOU Xiao,et al.Determination of toxin residue in recombinant pig inhibin protein and its physicochemical stability[J].,2015,(02):362-368.[doi:10.3969/j.issn.1000-4440.2015.02.022]
点击复制

重组猪抑制素蛋白质中毒素残留检测及其理化稳定性()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年02期
页码:
362-368
栏目:
畜牧兽医·水产养殖
出版日期:
2015-04-30

文章信息/Info

Title:
Determination of toxin residue in recombinant pig inhibin protein and its physicochemical stability
作者:
李辉; 翟颖超; 窦晓; 张甜; 果双双; 施振旦
(江苏省农业科学院畜牧研究所，江苏南京210014)
Author(s):
LI Hui; ZHAI Ying-chao; DOU Xiao; ZHANG Tian; GUO Shuang-shuang; SHI Zhen-dan
(Institute of Animal Science，Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
重组猪抑制素蛋白质; 内毒素; β-巯基乙醇; 残留; 理化稳定性
Keywords:
recombinant pig inhibin protein; endotoxin; β-mercaptoethanol; residue; physicochemical stability
分类号:
Q591.2
DOI:
10.3969/j.issn.1000-4440.2015.02.022
文献标志码:
A
摘要:
为探索纯化的重组猪抑制素蛋白质毒素残留情况以及重组猪抑制素蛋白质稳定性，本研究利用鲎试剂、气相色谱和SDS-PAGE方法对重组猪抑制素蛋白质中的细菌内毒素、β-巯基乙醇的残留量及其蛋白质在不同温度、极端pH值和反复冻融条件下的稳定性进行检测。结果显示，经过3次等电点沉淀洗涤后，重组猪抑制素蛋白质中细菌内毒素和β-巯基乙醇的残留量大幅减少。通过在不同温度下的处理发现，重组猪抑制素蛋白质在4 ℃环境下较稳定，可以长期保存；但在37 ℃时，稳定性快速下降，随着温度上升而降解加速。通过极端pH值和反复冻融试验发现，重组猪抑制素蛋白质对极端pH值和反复冻融具有较好的耐受性。
Abstract:
Tachypleus amebocyte lysate, gas chromatography and SDS-polyacrylamide gelelectrophoresis were employed for the determination of residue of bacterial endotoxin and β-mercaptoethanol in recombinant pig inhibin protein, and the stability of protein affected by different temperatures, extreme pH and multigelation, respectively. Results showed that bacterial endotoxin and β-mercaptoethanol residue in the protein were drastically decreased after isoelectric point precipitation washing for three times. The recombinant pig inhibin protein was quite stable at 4 ℃ for long-term preservation, started to degrade at 37 ℃ and accelerated degradation along with the rise of temperature. The protein possessed a good tolerance to the extreme pH and multigelation.

参考文献/References:


[1]DAVIS G S, ANDERSON K E, CARROLL A S. The effects of long-term caging and molt of single comb white leghorn hens on heterophil to lymphocyte ratios, corticosterone and thyroid hormones[J]. Poultry Science， 2000，79 (4):514-518.
[2]KLASING K C, PENG R K. Influence of cell sources, stimulating agents, and incubation conditions on release of interleukin-1 from chicken macrophages[J]. Developmental and Comparative Immunology, 1987,11 (2):385-394.
[3]PARMENTIER H K, WALRAVEN M, NIEUWLAND M G. Antibody responses and body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells. 1. Effect of Escherichia coli lipopolysaccharide[J]. Poultry Science ,1998,77 (2):248-255.
[4]邵英光,李京华,魏桂林,等.内毒素的去除策略[J]. 广州化学, 2003 (2):38-45.
[5]罗莉,李坤,王保成,等. 包涵体变复性技术研究进展[J]. 中国医药生物技术,2012 (4):290-293.
[6]包义风,应莲芳,蒋琳. 包涵体蛋白复性技术研究进展[J]. 微生物学免疫学进展, 2012 (2):84-88.
[7]于德强,何询.重组蛋白复性技术研究进展[J]. 生物技术通讯, 2004 (1):67-69.
[8]JUNGBAUER A, KAAR W. Current status of technical protein refolding[J]. Journal of Biotechnology,2007, 128 (3):587-596.
[9]PATRA A K, MUKHOPADHYAY R, MUKHIJA R,et al. Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli[J]. Protein expression and purification ,2000,18 (2):182-192.
[10]HEIDRICK M L, HENDRICKS L C, COOK D E. Effect of dietary 2-mercaptoethanol on the life span, immune system, tumor incidence and lipid peroxidation damage in spleen lymphocytes of aging BC3F1 mice[J]. Mechanisms of Ageing and Development,1984,27 (3):341-358.
[11]SMITH R T, THOMAS L . The lethal effect of endotoxins on the chick embryo[J]. The Journal of Experimental Medicine,1956,104 (2):217-231.
[12]董小英,唐胜球,李海云. 内毒素对家禽的影响及其防治措施[J]. 饲料工业,2005 (11):24-27.
[13]任勇,丁东平,渠刚,等.蛋白质类药物的稳定性研究进展[J]. 华南国防医学杂志, 2006 (4):31-33.
[14]LIU Y P, MAO X B, WEI Y M, et al. Studies on enhancing embryo quantity and quality by immunization against inhibin in repeatedly superovulated Holstein heifers and the associated endocrine mechanisms[J]. Animal Reproduction Science,2013,142 (1-2):10-18.
[15]MEI C, LI M Y, ZHONG S Q,et al. Enhancing embryo yield in superovulated holstein heifers by immunization against inhibin[J]. Reproduction in Domestic Animals,2009,44 (5):735-739. 
[16]MEDAN M S, AKAGI S, KANEKO H, et al.Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate[J]. Reproduction,2004, 128 (4):475-482.
[17]SASAKI K, MEDAN M S, WATANABE G, et al. Immunization of goats against inhibin increased follicular development and ovulation rate[J]. The Journal of Reproduction and Development,2006,52 (4):543-550.
[18]MEDAN M S, WATANABE G, SASAKI K, et al. Effects of passive immunization of goats against inhibin on follicular development, hormone profile and ovulation rate[J]. Reproduction,2003, 125 (5):751-757.
[19]国家药典委员会.中华人民共和国药典（三部）[M]. 北京:中国医药科技出版社,2010.
[20]朱九群,童荣生. 重组人角质细胞生长因子冻干粉针剂中β-巯基乙醇残留量的考察[J]. 临床合理用药杂志, 2013 (1):12-13.
[21]LEVIN J, BANG F B A. Description of cellular coagulation in the limulus[J]. Bulletin of the Johns Hopkins Hospital, 1964,115:337-345.
[22]黄清泉,夏振民. 药品细菌内毒素检查的实验设计[J]. 中国药学杂志,1997 (2):10-12.
[23]苏建勇,龚承元,芮期义,等. 荷电微孔滤膜去除细菌、病毒效果的初步研究[J]. 军事医学科学院院刊,1998 (2):22-24.
[24]苏建勇，龚承元，朱孟府.荷电微孔滤膜去除细菌内毒素的试验研究[J]. 军事医学科学院院刊,1995 (4):289-292.
[25]CHERKIN A. Destruction of bacterial endotoxin pyrogenicity by hydrogen peroxide[J]. Immunochemistry, 1975,12 (6-7):625-627.
[26]LIU S, TOBIAS R, MCCLURE S, et al . Removal of endotoxin from recombinant protein preparations[J]. Clinical Biochemistry,1997, 30 (6):455-463.
[27]MINOBE S, WATANABE T, SATO T, et al. Characteristics and applications of adsorbents for pyrogen removal[J]. Biotechnology and Applied Biochemistry,1988,10 (2):143-153
[28]商振华,周冬梅,郭为,等. 聚酰胺亲和膜色谱用于去除内毒素的研究Ⅱ. 去除条件最佳化和应用[J]. 分析测试学报,1998, 17(2):10-14.
[29]商振华,周冬梅,郭为,等. 聚酰胺亲和膜用于去除内毒素的研究 Ⅰ. 亲和膜的制备和特征[J]. 分析测试学报, 1998, 17 (1):1-4.
[30]GUO W, SHANG Z, YU Y, et al. Removal of endotoxin from aqueous solutions by affinity membrane[J]. Biomedical Chromatography，1997,11 (3):164-166. 
[31]MATSUMAE H, MINOBE S, KINDAN K, et al. Specific removal of endotoxin from protein solutions by immobilized histidine[J]. Biotechnology and Applied Biochemistry, 1990,12 (2):129-140.
[32]ANSPACH F B. Endotoxin removal by affinity sorbents[J]. Journal of Biochemical and Biophysical Methods,2001, 49 (1-3):665-681.
[33]FOWERS K D, JANATOVA J. Specific inhibition of C3 to facilitate general complement inhibition on endotoxin affinity sorbents for apheresis applications[J]. Biomaterials, 2001,22 (13):1749-1761.
[34]ANSPACH F B, HILBECK O. Removal of endotoxins by affinity sorbents[J]. Journal of Chromatography A, 1995,711 (1):81-92.
[35]钟翔，邵丁丁，王恒. 三种去除人巨噬细胞迁移抑制因子（HuMIF）重组蛋白内毒素方法的比较[J]. 寄生虫与医学昆虫学报, 2009, 16 (4):5.
[36]SHARMA V K, KALONIA D S. Temperature- and pH-induced multiple partially unfolded states of recombinant human interferon-alpha2a: possible implications in protein stability[J]. Pharmaceutical Research, 2003,20 (11):1721-1729.
[37]ISHII M, KUNIMURA J S, JENG H T, et al. Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride[J]. Applied Biochemistry and Biotechnology, 2007, 137-140 (1-12):555-571.
[38]SWIETNICKI W, PETERSEN R, GAMBETTI P, et al. pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231)[J]. The Journal of Biological Chemistry, 1997,272 (44):27517-27520.
[39]APPEL T, WOLFF M, VON RHEINBABEN F, et al. Heat stability of prion rods and recombinant prion protein in water, lipid and lipid-water mixtures[J]. The Journal of General Virology,2001,82 (2):465-473.
[40]朱国萍,滕脉坤,伍传金,等. G138P定点突变对葡萄糖异构酶热稳定性的改善[J]. 生物化学与生物物理学报,1998,30 (6):77-80.〖ZK）〗〖FL）〗

备注/Memo

备注/Memo:
收稿日期：2014-09-02 基金项目：江苏省农业科技自主创新基金项目[CX(13)5039]; 江苏省科技支撑计划项目(BE2012392) 作者简介：李辉（1982-），男，山东惠民人，博士，助理研究员，主要从事动物繁殖方面的研究。（Tel）025-84390772；（E-mail）lhlydk@126.com 通讯作者：施振旦，（E-mail）zdshi@scau.edu.cn
更新日期/Last Update: 2015-04-30