摘要
目的 了解增生性瘢痕Fb中羟基丙酮酸异构酶同系物（HYI）与P311蛋白相互作用的结合区域. 方法（1）以克隆有P311的pMD18-T质粒为模板,对P311进行PCR扩增；Trizol法提取人增生性瘢痕Fb的总RNA,对HYI进行RT-PCR扩增,采用双酶切法分别构建pGA DT7-P311和pGBKT7-HYI重组载体,PCR及测序验证.采用Prot Seale软件和HNN软件对HYI蛋白进行二级结构分析,据此结果扩增HYI-1（1 ～447 bp）、HYI-2（247 ～447 bp）、HYI-3（1 ～279 bp）、HYI-4（247 ～654 bp）片段,双酶切法构建pGBKT7-HYI-1、2、3、4重组载体,经PCR及测序验证.（2）采用醋酸锂法将酵母细胞AH109制备成感受态细胞,粗略等量分为HYI全长与HYI-1、2、3、4杂交组,以及阳性对照组和阴性对照组.采用聚乙二醇-醋酸锂法,前5组分别同时转化pGBKT7-HyI全长、各片段的重组载体与pGADT7-P311重组载体,后2组分别同时转化pGBKT7-53与pGADT7-RecT重组载体、pGBKT7-Lam与pGADT7-RecT重组载体.转化后即刻,取各组部分细胞分别涂布在缺乏亮氨酸、色氨酸、腺嘌呤及组氨酸的培养基（简称四缺培养基）上,另取各组部分细胞分别涂布在缺乏亮氨酸和色氨酸的培养基（简称二缺培养基）上,培养3～6d,观察菌株生长情况,5-溴-4-氯-3-吲哚-β-D-半乳糖苷显色反应检测菌落β半乳糖苷酶表达. 结果（1）克隆出的P311片段与Genbank中P311（序号hsu36189）序列一致.与Genbank中HYI（序号AY775560）序列对比,HYI基因扩增片段在496 bp处的A替换为了G.经验证各重组载体插入片段无误.（2）计算机模拟结果显示,组成HYI蛋白的216个氨基酸中,可能形成α螺旋的氨基酸有101个,可能形成无规则卷曲的氨基酸有90个,可能形成延伸链的氨基酸有25个.（3）HYI-1、2、3、4克隆片段大小与预期相符,经验证各重组载体插入片段无误.（4）除阴性对照组菌株未见在四缺培养基上生长外,其他组菌株均在2种培养基上生长,�
Objective To explore the binding domain of hydroxypyruvate isomerase homologues （HYI）in the interaction with protein P311 in hypertrophic scar fibroblasts（Fb）. Methods （ 1 ）P311 was amplified by PCR using plasmid pMD18-T-P311 as template.The total RNA of hypertrophic scar Fb was extracted by Trizol to amplify HYI with RT-PCR.Recombinant vectors pGADT7-P311 and pGBKT7-HYI were constructed by double-enzyme digestion,and they were verified by PCR and sequencing.The secondary structure of protein HYI was analyzed with software Prot Seale and HNN.Fragments of HYI-1（1-447 bp）,HY1-2（247-447 bp）,HYI-3（1-279 bp）,and HYI-4（247-654 bp）were amplified based on the result of software analysis.And then the recombinant vectors pGBKT7-HYI-1,2,3,and 4 were constructed by double-enzyme digestion and verified by PCR and sequencing.（2）AH109 yeast cells were transformed into competent cells by lithium acetate method and divided into 7 groups roughly in the same amount,including HYI full length,HYI-1,HYI-2,HYI-3,and HYI-4 hybrid groups,positive control group,and negative control group.Cells in the first five groups were respectively transformed with recombinant vector pGBKT7-HYI full length,pGBKT7-HYI-1,pGBKT7-HYI-2,pGBKT7-HYI-3,pGBKT7-HYI-4 and recombinant vector pGADT7-P311,and that in the rest two groups were transformed with recombinant vectors pGBKT7-53 and pGADT7-RecT,pGADT7-RecT and pGBKT7-Lam by polyethyleneglycol/lithium acetate method.Immediately after transformation,a part of the transformed cells in each group was spread onto the medium lacking leucine,tryptophan,adenine,and histidine（briefly called four-factor lacking medium）,and another portion of the cells was spread onto the medium lacking leucine and tryptophan（briefly called two-factor lacking medium）.After 3 to 6 days＇ culture,the growth of yeast was observed,and the expression of β-galactosidase of yeast was detected by color reaction with 5-bromo-4-chloro-indolyl-β-D-galactopyranoside.Results （1）Cloned P311 and the re
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2012年第1期60-64,共5页
Chinese Journal of Burns
基金
国家自然科学基金（30872695）
四川省科技厅应用基础研究项目（2008JY0082）
