pcDNA3.1/NT-GFP小凹蛋白1及突变体表达载体的构建及功能分析

目的 构建pcDNA3．1/NT-GFP小凹蛋白1及对突变体表达载体及表达蛋白生物活性进行分析。方法采用硫化修饰引物与Pfu酶结合的高度保真性聚合酶链反应体系，自行设计多对引物，分别扩增带His标签的小凹蛋白1全长目的片段、小凹蛋白1（缺失81～101位氨基酸）突变体1片段及小凹蛋白1（缺失143～156位氨基酸）突变体2片段；分别亚克隆入peDNA3．1／NT-GFP-TOPO真核表达栽体，转化TOP10E．coil大肠杆菌，在含氨苄的固体LB培养基上随机挑取6个克隆，分别提取质粒后，用PCR法筛选含正确插入阅读框的阳性克隆子并测序鉴定。用脂质体介导法瞬时转染入HepG2细胞，用MTT法、Western blot法初步鉴定GFP-His小凹蛋白1及突变体重组融合蛋白的生物学活性。结果筛选得到正确peDNA3．1／NT-GFP-His小凹蛋白1及突变体表达我体，测序结果无碱基突变及阅读框移码，GFP-His小凹蛋白1及突变体重组融合蛋白具有天然表达蛋白相似的生物学活性。结论成功构建具有生物学活性的pcDNA3．1／NT-GFP小凹蛋白1及突变体表达栽体，为小凹蛋白1的功能研究奠定基础。

Construction, Identification and Primary Functional Analysis of pcDNA3.1/NT-GFP-Caveolin-1 and Mutants Plasmids

Aim To construct and identified the eukaryotic expression recombinant plasmids containing caveolin-1 gene and mutants for the potential functional analysis. Methods Using a novel high fidelity gene expression profiling strategy mediated by Pfu proofreading polymerases combining with phesphorothioate modified primers, PCR-amplified ORFs of caveolin-1 and its mutants were inserted into a N-GFP tagging expression vector with the help of topoisomerase 1-mediated ligation. After transformation into chemically competent One Shot TOP10 E. coli, the respective destination clones were selected for by plating on ampicillin selective agar plates. The correct orientation and reading frames of the constructs were identified through the PCR approach with the respective sense primer and the BGH antisense primer. The corrected reconstructs would add about 100 bp more than the RT-PCR fragements. The integrity of ORFs was verified by sequencing to exclude errors introduced in the amplification step. MTT and western blot approaches were carried out to determine whether the overexpressed tagged proteins present the same protein activities as endogenous proteins. Results We obtained 3 recombined eaveolin-1 mutant plasmids. In addition, we demonstrated that overexpression of two caveolin-1 mutants in HepG2 cells could result in apoptosis of HepG2. However the other caveolin-1 mutant could promote the growth of HepG2 cells. The overexpressed tagged proteins present the same protein activities as endogenous caveohn-1 protein. Conclusion The eukaryotic expression recombinant plasmids containing caveolin-1 germ and mutants were successfully constructed.