鼻咽癌相关新基因NPCEDRG突变型重组表达载体的构建及生物信息学分析

Construction of Eukaryotic Expression Recombinant of Mutational NPCEDRG and Its Bioinformatics Analysis

目的构建带His标签的突变型及野生型pcDNATM3.1/myc-HisB/NPCEDRG重组表达载体。方法以pcDNA3.1-NPCEDRG重组质粒为模板,采用聚合酶链式反应(PCR)技术扩增NPCEDRG基因编码区,利用PCR过程中的碱基错配,随机突变产生NPCEDRG的突变体,分别将野生型及突变型NPCEDRG基因编码区cDNA插入pcDNATM3.1/myc-HisB真核表达载体,双酶切鉴定,测序验证,生物信息学方法进行蛋白质结构预测。结果双酶切突变型及野生型pcDNATM3.1/myc-HisB/NPCEDRG重组表达载体,均产生519 bp长度的目的片断。测序结果证实,突变型NPCEDRG基因有两个点发生了突变,分别是T260-C260和T287-C287,但没有移码突变,在线生物信息学分析结果示其相应氨基酸序列改变为V73-A73和M82-T82;野生型NPCEDRG基因序列与Genebank已知序列一致。两个重组载体插入片段大小均为513 bp,插入方向及位置正确,能表达预期所要的融合蛋白。结论成功构建了带His标签的突变型及野生型pcDNATM3.1/myc-HisB/NPCEDRG重组表达载体,为深入研究NPCEDRG基因功能和揭示鼻咽癌发病分子机制提供实验手段。

Objective To construct two eukaryotic expression vectors of mutational and wild NPCEDRG by Random Mutation. Methods The ORF of NPCEDRG gene was amplified from the recombinant plasmid of pcDNA3.1-NPCEDRG by polymerase chain reaction （PCR）, and mutational NPCEDRG was procured by Random Mutation. The ORFs＇ cDNAs of the wild NPCEDRG gene and its mutant were recombined respectively into the pcDNA^TM3.1/myc-HisB vector with two tags of myc and 6 × His following the target genes. Then the products were transferred into E. coli DH5α,and the positive clones were screened after being identified with restrictive enzymes and sequence analysis. Results The 519 bp target segments of mutational and wild NPCEDRG were obtained successfully in digestion products. The DNA sequence of wild NPCEDRG was consistent with the eDNA sequence of NPCEDRG from GeneBank. And the sequence of the mutational NPCEDRG re- vealed two altered codons,T260-C^260 and T^287-C^287, which resulted in amino acid exchanges V73-A73 and M^82-T^82. Conclu- sion Two recombinant expression vectors of mutational or wild NPCEDRG gene were constructed successfully.