巴什拜羊SPLUNC1基因的克隆与真核表达

doi:10.16431/j.cnki.1671-7236.2018.03.009

Abstract

Abstract:

In order to clone Bashibay sheep SPLUNC1 gene and express its protein,total RNA was extracted from lung of Bashibay sheep,RT-PCR was used to amplify SPLUNC1 gene open reading frame sequence,the gene fragment was inserted into eukaryotic expression plasmid pPIC9K to construct plasmid pPIC9K-SPLUNC1,then the plasmid pPIC9K-SPLUNC1 was linearized and electroporated into Pichia pastoris GS115,the protein was induced by methanol,and the target protein was identified by SDS-PAGE and Western blotting.The results showed that the 748 bp SPLUNC1 gene was successfully amplified by RT-PCR;The plasmid pPIC9K-SPLUNC1 was consistent with expected results after PCR,enzyme digestion and sequencing identification;Detection results of SDS-PAGE and Western blotting showed that the SPLUNC1 protein was successfully expressed with 25.53 ku.This study laid a foundation for further research of SPLUNC1 protein biological activity of Bashibay sheep.

Key words: Bashibay sheep; SPLUNC1 gene; cloning; eukaryotic expression

CLC Number:

S826

WANG Jixue, SHEN Wen, SUN Yanming. Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep[J]. , 2018, 45(3): 628-634.

References

[1] 决肯·阿尼瓦尔,韩业东,李齐发,等.巴什拜羊微卫星标记多态性及其与生长指标关联性分析[J].中国农业科学,2010,43(16):3425-3432. ANIWASHI J K,HAN Y D,LI Q F,et al.Analysis of microsatellite polymorphism and growth index association of Bashibai sheep[J].Scientia Agricultura Sinica,2010,43(16):3425-3432.(in Chinese)
[2] DI Y P.Functional roles of SPLUNC1 in the innate immune response against gram-negative bacteria[J].Biochemical Society Transactions,2011,39(4):1051-1055.
[3] ZHOU H D,LI G Y,YANG Y X,et al.Intracellular co-localization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain[J].Molecular Immunology,2006,43(11):1864-1871.
[4] ZHOU H D,LI X L,LI G Y,et al.Effect of SPLUNC1 protein on the Pseudomonas aeruginosa and Epstein-Barr virus[J].Molecular and Cellular Biochemistry,2008,309(1-2):191-197.
[5] ZHOU H D,FAN S Q,ZHAO J,et al.Tissue distribution of the secretory protein SPLUNC1 in the human fetus[J].Histochemistry and Cell Biology,2006,125(3):315-324.
[6] GALLY F,DI Y P,SMITH S K,et al.SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice[J].The American Journal of Pathology,2011,178(5):2159-2167.
[7] CHU H W,THAIKOOTTATHIL J,RINO J G,et al.Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation[J].The Journal of Immunology,2007,179(6):3995-4002.
[8] GARCIA-CABALLERO A,RASMUSSEN J E,GAILLARD E,et al.SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage[J].Proceeding of the National Academy of Sciences of the United States of America,2009,106(27):11412-11417.
[9] DI Y P,BIRRU R.Epithelial SPLUNC1 mediated antimicrobial activity against Klebsiella pneumoniae[J].The Journal of Immunology,2012,188(1):112-114.
[10] RIFFAULT S,CARRAT C,VAN REETH K,et al.Interferon-α-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets[J].Veterinary Research,2001,32(1):71-79.
[11] 傅小蒙,孔令聪,裴志花,等.毕赤酵母表达系统优化策略概述[J].中国生物工程杂志,2015,35(10):86-90. FU X M,KONG L C,PEI Z H,et al.Summary of optimization strategy for Pichia pastoris expression system[J].Chinese Journal of Biotechnology,2015,35(10):86-90.(in Chinese)
[12] 谭博敏,余希尧,钟泽民,等.猪流行性腹泻病毒S1基因在毕赤酵母中的表达[J].中国畜牧兽医,2014,41(6):64-67. TAN B M,YU X Y,ZHONG Z M,et al.Expression of porcine epidemic diarrhea virus S1 gene in Pichia pastoris[J].China Animal Husbandry & Veterinary Medicine,2014,41(6):64-67.(in Chinese)
[13] 孙亚范,王海宽,向微,等.甲醇毕赤酵母的高效电转化方法[J].食品与发酵工业,2004,30(5):35-39. SUN Y F,WANG H K,XIANG W,et al.Efficient electroporation method for Pichia pastoris[J].Food and Fermentation Industries,2004,30(5):35-39.(in Chinese)
[14] 江成英,冮洁,曹畅.甲醇毕赤酵母电转化条件的研究[J].齐齐哈尔大学学报,2005,21(1):8-11. JIANG C Y,JIANG J,CAO C.Study on the conditions of electroporation of Pichia pastoris[J].Journal of Qiqihar University,2005,21(1):8-11.(in Chinese)
[15] 李涛,朱玉楠,韩延杰,等.蛋白印迹技术的优化[J].河南大学学报(医学版),2014,33(2):141-144. LI T,ZHU Y N,HAN Y J,et al.Optimization of protein imprinting technique[J].Journal of Henan University (Medical Edition),2014,33(2):141-144.(in Chinese)
[16] 赵师师,高旭红,常君,等.石榴皮多糖的三氯乙酸法脱蛋白工艺研究[J].广州化工,2015,43(17):64-66. ZHAO S S,GAO X H,CHANG J,et al.Study on protein removal of pomegranate peel polysaccharide by three chloroacetic acid method[J].Guangzhou Chemical,2015,43(17):64-66.(in Chinese)
[17] 杨震,王爱霞,姬元忠,等.盐酸快速水解酪蛋白工艺研究[J].食品研究与开发,2011,32(7):44-47. YANG Z,WANG A X,JI Y Z,et al.Study on rapid hydrolysis of casein by hydrochloric acid[J].Food Research and Development,2011,32(7):44-47.(in Chinese)
[18] 纪晓方,李丽英,常娜.高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究[J].首都医科大学学报,2016,37(5):616-620. JI X F,LI L Y,CHANG N.High content analysis and Western blotting method and its application in the study of the distribution and comparison of nuclear protein[J].Journal of Capital Medical University,2016,37(5):616-620.(in Chinese)
[19] 张华荣,陈正明,文海燕,等.免疫印迹法(WB)对隐性梅毒抗体确证检测的价值探讨[J].口岸卫生控制,2013,18(2):20-23. ZHANG H R,CHEN Z M,WEN H Y,et al.Value of Western blotting in the detection of antibodies against latent syphilis[J].Port Health Control,2013,18(2):20-23.(in Chinese)
[20] 刘志云.中国莱姆病阿弗西尼基因型感染蛋白免疫印迹法诊断方法的研究[D].衡阳:南华大学,2011. LIU Z Y.Study on the diagnosis method of Chinese lyme disease avicnienniki by immuno-imprinting method[D].Hengyang:University of South China,2011.(in Chinese)

Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep

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