水貂生长激素的原核表达及特异性抗血清制备

doi:10.16431/j.cnki.1671-7236.2018.04.004

摘要/Abstract

摘要：

本研究旨在建立水貂生长激素（mink growth hormone，mGH）的原核高效表达体系，探索mGH蛋白规模化生产方法，利用获得的目的蛋白制备特异抗血清。将已构建的原核表达载体pET28b-mGH转化大肠杆菌Rosetta（DE3）^TM pLysS感受态细胞，经IPTG诱导表达，超声破碎菌体后离心获得包涵体蛋白，利用8 mol/L尿素对其进行溶解和复性，经Ni-NTA树脂亲和层析纯化获得重组蛋白。通过Western blotting检测及MALDI-TOF-MS质谱分析对获得的蛋白进行鉴定。用SDS-PAGE电泳法检测蛋白纯度，用BCA法测定其浓度。复性mGH蛋白与完全弗氏佐剂或不完全弗氏佐剂乳化制备免疫抗原，采用皮下多点注射法免疫3只新西兰大白兔，首次免疫剂量为600 μg/只，二免剂量为600 μg/只，三免剂量为400 μg/只。三免10 d后分离制备血清，Western blotting法检测其特异性，间接ELISA法检测其效价。结果表明，原核表达的包涵体经Ni-NTA柱纯化后所得蛋白鉴定为mGH融合蛋白；SDS-PAGE电泳鉴定复性mGH蛋白条带单一，纯度达90%以上，BCA法计算蛋白浓度为669 μg/mL，均达到了免疫动物的要求；制备的抗血清经Western blotting检测具有良好的结合特异性，间接ELISA法测定其抗体效价达1：256 000以上。上述结果表明，原核表达载体pET28b-mGH可在大肠杆菌Rosetta（DE3）^TM pLysS细胞中高效表达mGH融合蛋白，表达蛋白经变性、复性和纯化后免疫新西兰大白兔，可制备高效价特异性抗血清。

关键词: 水貂生长激素(mGH); 原核表达; Ni-NTA树脂; 质谱分析; 兔抗mGH血清

Abstract:

This study was aimed to establish a prokaryotic expression system of mink growth hormone (mGH),explore a scale production method,and get specific anti-mGH serum from the immune rabbit.In this study,the prokaryotic expression vector pET28b-mGH was transferred into Rosetta(DE3)^TM pLysS competent cells and the inclusion body protein was got successfully which induced by IPTG.The protein was dissolved and refolded with 8 mol/L urea solution,the recombinant protein was obtained by Ni-NTA resin affinity chromatography purification.The recombinant protein was confirmed by Western blotting and MALDI-TOF-MS methods,the purity and concentration of mGH fusion protein were determined by SDS-PAGE and BAC methods,respectively.In the case of refolding mGH protein,the immune antigen was prepared with a complete fluoride adjuvant or incomplete fluoride adjuvant.Three New Zealand White rabbits were immunized by subcutaneous multipoint injection the immune antigen,the first dose was 600 μg per rabbit,the second dose was 600 μg per rabbit,the third dose was 400 μg per rabbit.We took the rabbits' blood sampling on 10 day after the third immunization,and then separated antibody serum,the anti-mGH serum specificity and antibody titer were identified by Western blotting and indirect ELISA,respectively.The result showed that the purified protein was mGH fusion protein,SDS-PAGE results showed that the purity of the mGH fusion protein was more than 90%,and the BCA result showed that the concentration reached to 669 μg/mL.The rabbit anti mGH serum was specificity,the anti-mGH sera with a titer of over 1:256 000.The results showed that the prokaryotic expression vector pET28b-mGH could express efficiently mGH fusion protein in Rosetta (DE3)^TM pLysS cells,the expressed protein was immunized with New Zealand White rabbits after denaturation,renaturation and purification,which could be used to prepare high titer specific antiserum.

Key words: mink growth hormone (mGH); prokaryotic expression; Ni-NTA resin; mass spectrometry; rabbit anti-mGH serum

中图分类号:

Q786

苏俊, 刘曼, 王吉贵, 孙燕, 刘维全. 水貂生长激素的原核表达及特异性抗血清制备[J]. 《中国畜牧兽医》, 2018, 45(4): 866-872.

SU Jun, LIU Man, WANG Jigui, SUN Yan, LIU Weiquan. Prokaryotic Expression and Specific Antiserum Preparation of Mink Growth Hormone[J]. , 2018, 45(4): 866-872.

参考文献

[1] FORSYTH I A,WALLIS M.Growth hormone and prolactin——Molecular and functional evolution[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(3):291-312.
[2] 顾志良,王慧娟,郁建锋.动物GH/IGF-Ⅰ轴调控机制的研究进展[J].中国畜牧兽医,2011,38(10):50-54. GU Z L,WANG H J,YU J F.Advances on the regulation mechanism of growth hormone/IGF-Ⅰ axis[J].China Animal Husbandry & Veterinary Medicine,2011,38(10):50-54.(in Chinese)
[3] 李雨萌,洪盼,郑鑫,等.生长激素促进细胞增殖机理的研究进展[J].中国畜牧兽医,2015,42(1):147-152. LI Y M,HONG P,ZHENG X,et al.Research progress on mechanism of growth hormone promoting cell growth[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):147-152.(in Chinese)
[4] BASKARI S,GOVATATI S,MADHURI V,et al.Influence of autocrine growth hormone on NF-κB activation leading to epithelial-mesenchymal transition of mammary carcinoma[J].Tumour Biology,2017,39(10):1-6.
[5] OLSON K C,FENNO J,LIN N,et al.Purified human growth hormone from E.coli is biologically active[J].Nature,1981,293(5831):408-411.
[6] GEORGE H J,PILACINSKI W P,GLASSMAN D L,et al.High-level expression in Escherichia coli of biologically active bovine growth hormone[J].DNA,1985,4:273-281.
[7] WANG H,MCCONNELL D J,QI S Z,et al.Optimization of the synthesis of porcine somatotropin in Escherichia coli[J].Applied Microbiology and Biotechnology,1993,39(3):324-328.
[8] 龙英娜,马凤龙,乔彦良,等.重组水貂生长激素真核表达载体的构建及在巴斯德毕赤酵母中的表达[J].中国农学通报,2008,3(24):30-34. LONG Y N,MA F L,QIAO Y L,et al.Construction of eukaryotic expression vector of mustela vision growth hormone and expression in yeast P.pastoris[J].Chinese Agricultural Science Bulletin,2008,3(24):30-34.(in Chinese)
[9] 张立垚,周学红,王惠,等.哥本哈根、赫尔辛基部分品种水貂皮十年拍卖数据分析[J].野生动物学报,2017,38(1):110-114. ZHANG L Y,ZHOU X H,WANG H,et al.Furauction data analysis of some mink breeds in Copenhagen and Helsinki in the past decade[J].Chinese Journal of Wildlife,2017,38(1):110-114.(in Chinese)
[10] 李玉梅,丁雪梅,史旭东,等.水貂GH基因SNPs与皮张长度的相关性研究[J].生物技术通报,2010,6(40):167-171. LI Y M,DING X M,SHI X D,et al.Corelation of GH gene polymorphism and dermat-length in mink[J].Biotechnology Bulletin,2010,6(40):167-171.(in Chinese)
[11] HARADA Y,TATSUMI H,NAKANO E,et al.Cloning and sequence analysis of mink growth hormone cDNA[J].Biochemical and Biophysical Research Communications,1990,173(3):1200-1204.
[12] SHOJI K,OHARA E,WATAHIKI M,et al.Cloning and nucleotide sequence of a cDNA encoding the mink growth hormone[J].Nucleic Acids Research,1990,18(21):6424.
[13] SEREIKAITE J,STATKUTE A,MORKUNAS M,et al.Production of recombinant mink growth hormone in E.coli,biotechnological products and process engineering[J].Applied Microbiology and Biotechnology,2007,74:316-323.
[14] 尚丹,刘维全,孙绍光,等.水貂生长激素cDNA的克隆与序列分析[J].特产研究,2005,2(1):1-4. SHANG D,LIU W Q,SUN S G,et al.Cloning and sequence analysis of mink growth hormone cDNA[J]. Special Wild Economic Animal and Plant Research,2005,2(1):1-4.(in Chinese)
[15] HINTZ R L.Untoward events in patients treated with growth hormone in the USA[J].Hormone Research,1992,38(1):44-49.
[16] 于瑞嵩,沈世缘,董世娟,等.基因工程牛生长激素高密度发酵表达的研究[J].上海农业学报,2009,25(3):10-13. YU R C,SHEN S Y,DONG S J,et al.Expression of genetically engineered bovine growth hormone by high density fermentation of Escherichia coli[J].Acta Agriculturae Shanghai,2009,25(3):10-13.(in Chinese)
[17] 张永红,唐芬芬,邵榆岚,等.重组猪生长激素表达研究进展[J].生物技术进展,2014,4(3):165-170. ZHANG Y H,TANG F F,SHAO Y L,et al.Progress on expression of recombinant porcine growth hormone[J].Current Biotechnology,2014,4(3):165-170.(in Chinese)
[18] 王庆华,高丽丽,朱平,等.影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展[J].药学学报, 2014,49(12):1644-1649. WANG Q H,GAO L L,ZHU P,et al.Research advances of the influence factors of high level expression of recombinant protein in Pichia pastoris[J].Acta Pharmaceutica Sinica,2014,49(12):1644-1649.(in Chinese)
[19] 宋凯.水貂生长激素基因重组腺病毒的构建[D].长春:吉林农业大学,2008. SONG K.Construction of recombinant adenovirus of mink growth hormone gene[D].Changchun:Jilin Agricultural University,2008.(in Chinese)
[20] 王友东,张乐萃.水貂生长激素基因重组腺病毒的构建[J].青岛农业大学学报(自然科学版),2010,27(2):144-149. WANG Y D,ZHANG L C.Construction of recombinant adenovirus vector carrying mink growth hormone gene[J].Journal of Qingdao Agricultural University(Natural Science),2010,27(2):144-149.(in Chinese)
[21] 朱涛.重组腺病毒载体埃博拉疫苗的工艺特点和技术创新[J].生物技术通讯,2017,28(1):12-15. ZHU T.The process features of recombinant adenovirus vector Ebola vaccine and technical innovation[J].Letters in Biotechnology,2017,28(1):12-15.(in Chinese)
[22] 闫立论.重组人生长激素(rhGH)的毕赤酵母工程菌构建、表达、分离纯化及其生物学作用研究[D].新乡:河南师范大学,2015. YAN L L.Construction,expression and purification of the recombinant human growth hormone in P.pastoris and its biological activity[D].Xinxiang:Henan Normal University,2015.(in Chinese)
[23] MCNULTY D E,CLAFFEE B A,HUDDLE-STON M J,et al.Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli[J].Protein Expression and Purification,2003,27(2):365-374.
[24] 杨伟,周华,赵沙沙,等.稀有密码子对人源Id2基因表达的影响[J].重庆医科大学学报,2013,38(4):365-368. YANG W,ZHOU H,ZHAO S S,et al.Effects of rare codon on functional expression of Id2 gene in Escherichia coli[J].Journal of Chongqing Medical University,2013,38(4):365-368.(in Chinese)
[25] 兰泓,李慎涛,张玉祥,等.稀有密码子影响人RNaseH-Apaf1融合蛋白原核表达[J].氨基酸和生物资源,2015,37(4):72-78. LAN H,LI SH T,ZHANG Y X,et al.Effect of rare codon on prokaryotic expression of human RNaseH-Apaf1 fusion protein[J].Amino Acids & Biotic Resources,2015,37(4):72-78.(in Chinese)