GST-SRY融合蛋白原核表达和纯化

张小玉; 段君君; 王江; 贾霄; 汪小波; 何颖红

GST-SRY融合蛋白原核表达和纯化

张小玉^1,2,
段君君^1,2,
王江^1,2,
贾霄^1,2,
汪小波^1,2,
何颖红^1,2

1. 大理大学基础医学院
2. 云南省高校细胞生物学重点实验室

基金项目:

基金：国家自然科学基金资助项目 (31360288);

计量
- 文章访问数: 2402
- HTML全文浏览量: 1002
- PDF下载量: 198
- 被引次数: 0
出版历程
- 收稿日期: 2017-11-20

Prokaryotic Expression and Purification of GST-SRY Fusion Protein

Zhang Xiao Yu ^1,2,
Duan Jun Jun ^1,2,
Wang Jiang ^1,2,
Jia Xiao ^1,2,
Wang Xiao Bo ^1,2,
He Ying Hong ^1,2

Funds:

基金：国家自然科学基金资助项目 (31360288);

摘要

摘要: 目的构建GST-SRY融合蛋白原核表达载体, 在E.coli中诱导其表达, 并进行纯化.方法 PCR扩增SRY基因, 将SRY基因插入原核表达载体p GEX-6P-1, 经酶切和测序验证后, p GEX-6p-1-SRY转化到原核表达菌株E.coli Rosetta DE3, IPTG诱导表达GST-SRY融合蛋白, 用GST标签纯化树脂对其进行纯化.结果经双酶切和测序证实p GEX-6P-1-SRY构建正确.重组质粒转化到Rosetta DE3经IPTG诱导后表达了分子质量单位约为49 KD的重组蛋白.结论 p GEX-6P-1-SRY原核表达载体构建成功, SRY蛋白成功诱导表达并纯化, 为今后深入研究SRY蛋白的功能奠定了实验基础.
- SRY /
- 基因重组 /
- 基因表达 /
- 蛋白纯化
Abstract: Objective To construct GST-SRY fusion protein expression vector and induce its expression in E.coli, then purificate so as to obtain GST-SRY fusion protein. Me thods SRY gene was amplified by PCR and cloned into prokaryotic expression vector p GEX-6 P-1. p GEX-6 P-1-SRY plasmid was verified by restriction enzyme digestion and DNA sequencing. After that, the recombinant plasmid was transformed into E.coli Rosetta DE3.Expression of GST-SRY fusion protein was induced by IPTG, which was purified by Glutathione Sepharose later on.Re s ults Restriction enzyme digestion and sequencing demonstrated that p GEX-6 P-1-SRY was successfully obtained. The result of SDS-PAGE assay indicated that SRY was successfully expressed in E.coli Rosetta DE3 and expressed a 49 KD SRY protein.Conclus ion The successful construction of SRY gene recombinant prokaryotic expression vector and expression of SRY protein will be helpful for further studies of SRY gene function.
- SRY /
- DNA recombination /
- Gene expression /
- Protein purification

HTML全文

参考文献(15)

[1]李萌, 贺竹梅.遗传学教学中性别决定关键基因的阐述[J].遗传, 2014, 36 (6) :611-617.
[2]	[2]SHE Z Y, YANG W X, YANG W X.Sry and Sox E genes:How they participate in mammalian sex determination and gonadal development[J].Seminars in Cell&Developmental Biology, 2016, 63 (2) :13.
[3]	[3]VETRO A, DEHGHANI M R, KRAOUA L, et al.Testis development in the absence of SRY:Chromosomal rearrangements at SOX9 and SOX3[J].European Journal of Human Genetics, 2015, 23 (8) :1025-1032.
[4]	[4]LARNEYC, BAILEY T L, KOOPMANP.Switching on sex:transcriptional regulation of the testis-determining gene Sry[J].Development, 2014, 141 (11) :2195-2205.
[5]	[5]BERTA P, HAWKINSJR, SINCLAIR A H, et al.Genetic evidence equating SRY and the testis-determining factor[J].Nature, 1990, 348 (6300) :448-450.
[6]	[6]THOMASJO.HMG1AND2:architectural DNA-binding proteins.[J].Biochem Soc Trans, 2001, 29 (4) :395-401.
[7]	[7]FERRARIS, HARLEY V R, PONTIGGIA A, et al.SRY, like HMG1, recognizes sharp angles in DNA[J].Embo Journal, 1992, 11 (12) :4497-4506.
[8]	[8]CONNORF, CARYPD, READ C M, et al.DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.[J].Nucleic Acids Res, 1994, 22 (16) :3339-3346.
[9]	[9]PONTIGGIA A, RIMINI R, HARLEY V R, et al.Sex reversing mutations affect the architecture of SRY DNA complexes[J].The EMBO Journal, 1994, 13 (24) :6115-6124.
[10]	[10]HARLEY V R, Cl ARKSONMA.The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 (SRY-related high-mobility group (HMG) box 9) [J].Endocrine Reviews, 2003, 24 (4) :466-487.
[11]	[11]CHABOISSIER M C, KOBAYASHI A, VIDALVI, et al.Functional analysis of Sox8 and Sox9 during sex determination in the mouse[J].Development (Cambridge, England) , 2004, 131 (9) :1891-1901.
[12]	[12]SEKIDO R, LOVELLBADGE R.Sex determination involves synergistic action of SRY and SF1 on a specific Sox9enhancer[J].Nature, 2008, 453 (7197) :930-934.
[13]	[13]XU Z, GAO X, HE Y, et al.Synergistic Effect of SRY and Its Direct Target, WDR5, on Sox9 Expression[J].PLo S ONE, 2012, 7 (4) :e34327.
[14]SAMBROOK J, RUSSELL D W.分子克隆实验指南[M].第3版.北京:科技出版社, 2002:1228-1232.
[15]赵瑜, 胡月新, 卢敏南, 等.人血管内皮生长因子A (VEGFA) 的原核载体构建、蛋白表达及鉴定[J].昆明医科大学学报, 2015, 36 (06) :31-34.

相关文章(20)

[1]	李静玲, 柯坤彬, 王振丞, 秦德强, 李颢. CASR/VDR/PTH1R信号通路在含钙肾结石发生机制中的初步研究, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220609
[2]	唐健, 何建萍, 罗胜军, 吕梦欣, 党峰博, 罗兰. 6例异常血红蛋白K(Hb K)的基因型分析, 昆明医科大学学报.
[3]	阎婷婷, 田丹, 李新, 俎云芬, 李崇阳, 尚学琴. MicroRNA Let-7a与p53基因在肺癌中表达的相关性, 昆明医科大学学报.
[4]	侯峰强, 董山潮, 雷喜锋, 张伟. 紫杉醇对人胆管癌QBC939细胞wnt/β-catenin信号通路基因和蛋白表达的影响, 昆明医科大学学报.
[5]	芮丹云. 辛伐他汀对体外培养鼠骨形成蛋白BMP-2基因的调控, 昆明医科大学学报.
[6]	方峰. ZAK-β蛋白激酶基因真核表达载体的构建, 昆明医科大学学报.
[7]	任晓丽. 蛋氨酸酶基因重组腺病毒载体构建, 昆明医科大学学报.
[8]	刘建军. 人肿瘤抗原基因Delta-like4(DLL4）可溶性表达、纯化及鉴定, 昆明医科大学学报.
[9]	焦扬. 羊水干细胞体外培养早期和后期基因表达谱分析, 昆明医科大学学报.
[10]	袁聪. 甲泼尼龙对受损脊髓组织中抗菌肽相关基因表达的调节, 昆明医科大学学报.
[11]	胡振良. 重组蛋氨酸裂解酶原核体系的表达及纯化, 昆明医科大学学报.
[12]	徐本峰. XAGE-1 基因在胃癌组织中的表达及其临床意义分析, 昆明医科大学学报.
[13]	李昆仑. RNAi抑制Survivin基因的表达对乳腺癌SKBr-3细胞的影响, 昆明医科大学学报.
[14]	. XAGE-1基因在胃癌组织中的表达及其临床意义分析, 昆明医科大学学报.
[15]	. 腭裂相关基因TTF-2转基因小鼠载体的构建及体外表达, 昆明医科大学学报.
[16]	. 流式细胞术辅助筛选和纯化人缺氧诱导因子-1α基因沉默肝癌细胞株, 昆明医科大学学报.
[17]	李玉. 体外培养神经干细胞NGF、BDNF和NT-3及其受体trkA、trkB和trkC 的表达, 昆明医科大学学报.
[18]	毕城伟. Kringle5基因的克隆、原核表达载体构建和蛋白表达, 昆明医科大学学报.
[19]	詹辉. 改良型TAT -VP3融合蛋白的基因合成、原核表达载体构建及表达的实验研究, 昆明医科大学学报.
[20]	刘健刚. 骨桥蛋白基因在两种颅咽管瘤中的表达差异及意义, 昆明医科大学学报.