[1]杨小猛,王俊轶,陈涛,等.尘螨肠道微生物蛋白核苷二磷酸激酶的克隆、表达及纯化[J].江西师范大学学报(自然科学版),2014,(05):485-488.
 YANG Xiao-meng,WANG Jun-yi,CHEN Tao,et al.Cloning,Expression and Purification of Intestinal Microflora Protein Nucleoside Diphosphate Kinase in Dust Mites[J].,2014,(05):485-488.
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尘螨肠道微生物蛋白核苷二磷酸激酶的克隆、表达及纯化()
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《江西师范大学学报》(自然科学版)[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2014年05期
页码:
485-488
栏目:
出版日期:
2014-10-31

文章信息/Info

Title:
Cloning,Expression and Purification of Intestinal Microflora Protein Nucleoside Diphosphate Kinase in Dust Mites
作者:
杨小猛;王俊轶;陈涛;刘志刚;杨平常
深圳大学医学院过敏反应与免疫学研究所,广东 深圳,518060;深圳大学医学院过敏反应与免疫学研究所,广东 深圳 518060; 泸州医学院附属医院呼吸内科,四川 泸州 646000;深圳大学医学院过敏反应与免疫学研究所,广东 深圳 518060; 深圳市过敏反应与免疫学重点实验室,广东 深圳 518060
Author(s):
YANG Xiao-meng;WANG Jun-yi;CHEN Tao;LIU Zhi-gang;YANG Ping-chang
关键词:
尘螨肠道微生物蛋白核苷二磷酸激酶
Keywords:
dust miteintestinal microfloranucleoside diphosphate kinase
分类号:
Q938
文献标志码:
A
摘要:
根据 GenBank 中 NDP kinase 的基因序列,采用生物信息学方法将其中稀有密码子改造为大肠埃希菌常用密码子并进行二级结构优化,合成 NDP kinase 基因,构建原核表达载体 pET28a-NDP kinase 并酶切鉴定其序列,在大肠埃希菌 BL21(DE3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,重组产物采用镍离子金属螯合亲和层析柱纯化.经密码子改造和二级结构优化后 NDP kinase 基因长度为490 bp,其编码蛋白理论分子量为21.5 kDa,重组表达载体经酶切鉴定与理论推测结果相符,在大肠埃希菌 BL21(DE3)中该基因经 IPTG 诱导可高效表达,纯化后的重组蛋白分子量约为21.5 kDa,其单一蛋白纯度达95;以上.本研究成功构建了尘螨肠道微生物蛋白核苷二磷酸酶(NDP kinase)基因的 pET28a 原核重组质粒,为进一步研究 NDP kinase 在尘螨疫苗免疫治疗中的作用机理提供了基础.
Abstract:
To research the cloning,expression and purification of intestinal microflora protein Nucleoside diphos-phate kinase(NDP kinase)in dust mites. The gene order of NDP kinase was obtained from the GenBank;the rare codon in the gene was changed to the commonly used codon of Escherichia coli(E. coli)and the secondary struc-ture was optimized by means of bioinformatics. Then the DNA sequence was synthesized and its prokaryotic expres-sion vector pET28a-NDP kinase was constructed. The vector was guided into E. coli BL21(DE3)and expressed in-duced by IPTG. Finally,the recombine protein was purified by means of Ni-NTA affinitycolumn. The gene length of the reformed and optimized NDP kinase was 490 bp and the theoretic molecular mass of the coding protein was a-bout 21 kDa. The enzyme identification of the recombine vector agreed with the theoretic value. Soluble NDP kinase could be expressed efficiently in E. coli BL21(DE3)by IPTG. The molecular mass of purified recombine protein was about 21 kDa. The prokaryotic expression vector pET28a-NDP kinase was constructed successfully in our re-search. Efficient expression and soluble NDP kinase could be used to research the role of NDP kinase in dust mite vaccine immunotherapy.

参考文献/References:

[1] Celano M P,Holsey C N,Kobrynski L J.Home-based family intervention for low-income children with asthma:a randomized controlled pilot study[J].J Fam Psychol,2012,26(2):171-178.
[2] Macchiaverni P,Rekima A,Turfkruyer M,et al.Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma [J].Allergy,2014,69(3):395-398.
[3] Kim J H,Cheong H S,Park J S,et al.A genome-wide association study of total serum and mite-specific IgEs in asthma patients [J].PLoS One,2013,8(8):e71958.
[4] Moed H,Gerth van Wijk R,Hendriks R W,et al.Evaluation of clinical and immunological responses:a 2-year follow-up study in children with allergic rhinitis due to house dust mite [J].Mediators Inflamm,2013,2013:345217.
[5] Moingeon P,Batard T,Nony E,et al.Quality control of house dust mite extracts for allergen immunotherapy [J].Int Arch Allergy Immunol,2013,161(3):285-286.
[6] Jacquet A.Innate immune responses in house dust mite allergy [J].ISRN Allergy,2013,2013:735031.
[7] 万 倩,陈献雄,孙新,等.粉尘螨Der f4基因的克隆和蛋白分子特征分析 [J].江西师范大学学报:自然科学版,2014,38(3):319-323.
[8] 刘晓宇,张强,吉坤美,等.粉尘螨疫苗免疫治疗过敏性鼻炎小鼠鼻黏膜的电镜观察 [J].江西师范大学学报:自然科学版,2011,35(5):532-536.
[9] Aarif O Eifan,Moises A Calderon,Stephen R Durham.Allergen immunotherapy for house dust mite:clinical efficacy and immunological mechanisms in allergic rhinitis and asthma [J].Expert Opin Biol Ther,2013,13(11):1543-1556.
[10] 陈艳丽,徐小玲,张婧,等.不同地区变应性鼻炎患者吸入性变应原的分布研究 [J].河北医药,2012,34(5):754-755.
[11] 马忠校,刘晓宇,杨小猛,等.空气净化器降低室内尘螨过敏原含量及其免疫反应性的实验研究 [J].中国人兽共患病学报,2013,29(2):133-137.
[12] Eifan A O,Akkoc T,Yildiz A,et al.Clinical efficacy and immunological mechanisms of sublingual and subcutaneous immunotherapy in asthmatic/rhinitis children sensitized to house dust mite:an open randomized controlled trial [J].Clin Exp Allergy,2010,40(6):922-932.

备注/Memo

备注/Memo:
国家自然科学基金(31328014,81300028);广东省高等学校国际暨港澳台科技合作创新平台(2012gjhz0009);深圳市科技计划基础研究(JCYJ20120613100657482)
更新日期/Last Update: 1900-01-01