唾液酸化路易斯-X合成关键酶基因的克隆表达

中国生物工程杂志

2011, Vol. 31

Issue (12): 51-56

研究报告

唾液酸化路易斯-X合成关键酶基因的克隆表达

姚晶^1,2, 任婧², 吴正钧², 孙克杰², 郭本恒^1,2

1. 上海海洋大学食品学院上海 201306;
2. 乳业生物技术国家重点实验室光明乳业股份有限公司技术中心上海 200436

Cloning and Expression of the Key Enzyme Gene in Biosynthesis of Sialyl Lewis X

YAO Jing^1,2, REN Jing², WU Zheng-jun², SUN Ke-jie², GUO Ben-heng^1,2

1. College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;
2. State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy Co., Ltd., Shanghai 200436, China

全文: PDF(1071 KB) HTML

摘要：

唾液酸化路易斯-X(sialyl lewis X,Slex) 是选择素家族的一个共同糖配体,通过与选择素竞争性地结合炎性细胞,可以抑制炎症反应。通过克隆表达Slex合成过程中的关键酶,在体外进行Slex的生物合成,用于相关乳腺炎防治药物的研发。β-1,4-半乳糖基转移酶(β-1, 4-galactosyltransferase,GT)就是参与Slex生物合成过程的关键酶之一。利用相关软件对牛的GT基因进行了生物信息学的分析,了解了GT的相关理化性质。通过人工合成的方法获得了GT基因的CDS,构建了重组质粒pMD18-GT,并亚克隆至表达载体pPIC9K。通过电转化将线性化的表达质粒pPIC9K-GT整合到宿主菌P. pastoris GS115基因组上,构建了重组酵母GS115-GT。经诱导表达后,SDS-PAGE检测到了目的蛋白条带,证明了此基因在P. pastoris GS115中能够可溶性表达;并用苯酚红法测定了粗酶液的活性,其比酶活为16.4 U/ml,这为其进一步研究奠定了基础。

关键词： 乳腺炎; 唾液酸化路易斯-X; 生物合成; 牛; β-1,4-半乳糖基转移酶; 克隆表达

Abstract:

Sialyl lewis X (Slex), one of the common glyco ligands of the selectin family, restrains inflammation reaction by combining with the inflammatory cells competitively. Clone and expression of the key enzymes in the biosynthesis process of Slex could make its biosynthesis in vitro, and some related mastitis therapy research possible. Beta 1, 4 galactosyltransferase (GT) was the very one of the key enzymes in the biosynthesis process. In order to understand some related physicochemical property of GT gene, the gene sequence was analyzed by using approaches of bioinformatics online. The synthetic CDS of this gene was inserted into a recombinant plasmid pMD18-T, and subcloned to the expression plasmid pPIC9K later. The linear expression plasmid pPIC9K-GT was integrated to the genome of P. pastoris GS115 by electrotransformation. After inducible expression, the soluble target protein was detected by SDS-PAGE. It was proved that this gene could be expressed successfully in P. pastoris GS115. The phenol red method was used to determine the activity of the unpurified enzyme, and the specific activity was 16.4U/ml. This might be the foundation work for the further study.

Key words: Mastitis Sialyl lewis X Biosynthesis B. taurus β-1, 4-galactosyltransferase GT Clone and expression

收稿日期: 2011-09-20 出版日期: 2011-12-25

ZTFLH:

Q786

基金资助:

国家"973"计划(2010CB735705)、上海市科委课题(09DZ2251400)资助项目

通讯作者: 郭本恒,电子信箱:guobenheng@brightdairy.com E-mail: guobenheng@brightdairy.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

姚晶, 任婧, 吴正钧, 孙克杰, 郭本恒. 唾液酸化路易斯-X合成关键酶基因的克隆表达[J]. 中国生物工程杂志, 2011, 31(12): 51-56.

YAO Jing, REN Jing, WU Zheng-jun, SUN Ke-jie, GUO Ben-heng. Cloning and Expression of the Key Enzyme Gene in Biosynthesis of Sialyl Lewis X. China Biotechnology, 2011, 31(12): 51-56.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I12/51

[1] Jian Z J,Ma S Z,Yuan J L,et al. Identification and isolation of pathogenic bacteria of bovine subclinical mastitis in Kelamayi City of Xinjiang. Progress in Veterinary Medicine,2008,29(2):12-17.

[2] Wang M Y, Ren W J. Advance in the curing strategies for mastitis in dairy cows. China Cattle Science, 2009, 1:63-70.

[3] He N, Yang H J, Wang C F, et al. Progress on Staphylococcus aureus vaccine against mastitis in dairy cows. Progress in Veterinary Medicine, 2009, 30(1):93-96.

[4] Li H S, Yu J, Li X P, et al. Development of inactivated multi-vaccine against dairy mastitis and observation of its clinical efficacy. Veterinary Science in China, 2007,37(4):363-368.

[5] Andrew S M, Moyes K M, Borm A A,et al. Factors associated with the risk of antibiotic residues adintramammary pathogen presence milk from heifers administered prepartum intramammary antibiotic therapy. Veterinary Microbiology, 2009,134:150-156.

[6] Middleton J R. Staphylococcus aureus antigens and challenges in vaccine development. Expert Review of Vaccines, 2008, 7(6) :805-815.

[7] Li Y J, Zang L, Zhang N S, et al. Localization of expression and distribution of L-selectin in neutrophil of cows. Veterinary Science in China, 2006,36(9):738-742.

[8] Munro J M. Expression of sialyl-lewis X an E-selectin ligand in flammation, immune processes and lymphoid tissues.American Journal of Pathology,1992,141:1397-1408.

[9] Deng C H, Chen R R. A pH-sensitive assay for galactosyltransferase. Analytical Biochemistry,2004,330:219-226.

[10] Xiong A S, Yao Q H, Peng R H, et al. High level expression of a synthetic gene encoding Peniophoralycii phytase in methy lotrophic yeast Pichia pastoris. Applied Microbiology and Biotechnology, 2006, 72(10) : 39-47.

[11] Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling.Bioinformatics, 2006,22:195-201.

[12] Schwede T, Kopp J, Guex N, et al.SWISS-MODEL: an automated protein homology-modeling serve. Nucleic Acids Research,2003,31: 3381-3385.

[13] Joseph S, David W R. Molecular Cloning: a Laboratory Manual, 3^rd ed. New York: Cold Spring Harbor Laboratory Press,2002.

[14] Chang H Y, Zhang Y P. α-1, 3 Galactosyltransferase and related glycosyltransferases: research advances. Bulletin of the Academy of Military Medical Sciences, 2008, 32(5):478-481.

[15] Tadashi I, Mayumi O K, Hiroshi O. Identification of elongating β-1,4-galactosyltransferase activity in mung bean (Vigna radiata) hypocotyls using 2-aminobenzaminated 1,4-linked β-D-galactooligosaccharides as acceptor substrates.Planta, 2004, 219: 310-318.

[16] Inka B, John G R, Meileen J, et al. Acceptor substrate specificity of UDP-Gal: GlcNAc-R β 1,3-galactosyltransferase (WbbD) from Escherichia coli O7:K1. Glycoconjugate Journal,2008,25:663-673.

[17] Jobron L, Sujino K, Hummel G, et a1.Glycosyltransferase assays utilizing N-acetyllactosamine acceptor immobilized on a cellulose membrane. Analytical Biochemistry, 2003, 323(1): 1-6.

[18] Zhong W T. Purification and activity assay of β-1,4-galactosyltransferase. Hangzhou: Zhejiang University, 2005.

[1]	张恒,刘慧燕,潘琳,王红燕,李晓芳,王彤,方海田. 生物法合成γ-氨基丁酸的研究策略^*[J]. 中国生物工程杂志, 2021, 41(8): 110-119.
[2]	李冰,张传波,宋凯,卢文玉. 生物合成稀有人参皂苷的研究进展^*[J]. 中国生物工程杂志, 2021, 41(6): 71-88.
[3]	苗轶男,李敬知,王帅,李春,王颖. 萜烯生物合成中关键酶的研究进展^*[J]. 中国生物工程杂志, 2021, 41(6): 60-70.
[4]	翟君叶,成旭,孙泽敏,李春,吕波. 毛蕊花糖苷的生物合成研究进展[J]. 中国生物工程杂志, 2021, 41(5): 94-104.
[5]	王光路, 王梦园, 周忆菲, 马科, 张帆, 杨雪鹏. 吡咯喹啉醌生物合成研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 103-113.
[6]	郭二鹏, 张建志, 司同. 羊毛硫肽的高通量工程改造方法新进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 30-41.
[7]	刘啸尘, 范代娣, 杨帆, 武占省. 人参皂苷化合物生物合成进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 80-93.
[8]	饶海密,梁冬梅,李伟国,乔建军,财音青格乐. 真菌芳香聚酮化合物的合成生物学研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 52-61.
[9]	段海荣,魏赛金,黎循航. 铜绿假单胞菌中鼠李糖脂生物合成的研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 43-51.
[10]	邓廷山,武国干,孙宇,唐雪明. 苯乳酸生物合成的研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 62-68.
[11]	闫伟欢,黄统,洪解放,马媛媛. 丁醇在大肠杆菌中的生物合成研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 69-76.
[12]	贾晓,邱瑾,舒娟,李华,习书斌,曾溢滔,曾凡一. 血清孕酮水平检测在克隆胚胎移植受体牛的筛选及妊娠诊断中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(7): 1-8.
[13]	刘金丛,刘雪,於洪建,赵广荣. 微生物合成根皮素及其糖苷研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(10): 76-84.
[14]	欧梦莹,王晓政,林双君,关统伟,林宜锦. 链黑菌素研究进展 ^*[J]. 中国生物工程杂志, 2019, 39(7): 100-107.
[15]	马雅婷,刘珍宁,刘雪,於洪建,赵广荣. 微生物异源合成植物异喹啉生物碱的新进展 ^*[J]. 中国生物工程杂志, 2019, 39(11): 123-131.

Viewed

Full text

Abstract

Cited

Shared

Discussed