摘要
目的:构建肝细胞癌(hepatocellular carcinoma,HCC)特异性镇痛抗肿瘤肽(analgesic-antitumor peptide,AGAP)基因真核表达载体,利用计算机辅助分析和预测所表达蛋白质的理化和结构特征,为探索生物毒素靶向抗肝癌作用奠定基础。方法:提取东亚钳蝎总RNA,用RT-PCR法扩增出AGAP基因,与载体pGL3/AFP连接构建重组质粒,酶切及测序鉴定,并利用蛋白质分析软件对其表达的蛋白二级结构水平及一些生物学特性进行预测,并转染HepG2细胞检测其是否表达。结果:重组质粒所表达的目的多肽经软件分析,发现在二级结构上没有变化,亲水性、抗原性等生物学活性也未受影响,转染HepG2细胞后能成功表达。结论:人AFP基因顺式作用元件调控AGAP真核表达载体构建成功。
Objective： To construct a gene-modified hepatocellular carcinoma （HCC） specific AGAP expression vector regulated by the cis-acting element of AFP.The physicochemical property and structural characteristics of the protein were analyzed and predicted by computer-aided analysis to establish a basis for exploring biotoxin thepapy for tumor.Method： The AGAP DNA fragment was amplified through RT-PCR from the total RNA of Chinese Buthus Martensii Karsch.It was then cloned into the plasmid pGL3/AFP.The recombinant plasmid pAFP-AGAP was identified by restriction endonucleases and nucleotide sequence.The protein analysis software were utilized to predict the flexibility,hydrophilicity,and antigenicity of the new AGAP protein.Then the expression levels of AGAP mRNA were detected by reverse transcriptase polymerase chain reaction （RT-PCR） after the recombinant plasmid being transfected into HepG2 cell line.Results： Through the analysis of the software,it could be found that the secondary structure of the new AGAP didn’t almost changed and remain their biologic activity.The recombinant plasmid was successfully to express AGAP mRNA in HepG2 cell line.Conclusion： The recombinant vector is constructed successfully.
出处
《温州医学院学报》
CAS
2010年第2期141-144,共4页
Journal of Wenzhou Medical College
关键词
甲胎蛋白
镇痛抗肿瘤肽
真核表达载体
肝肿瘤
alpha fetoprotein
analgesic-antitumor peptide
eukaryotic expression vector
hepatocellular carcinoma
