中文摘要：

蛋白质内部多个位点的翻译后修饰在基因的功能调节过程中发挥重要作用,基因的点突变体在其结构和功能研究中发挥非常关键的作用,因此,高效、快速构建基因的多个点突变体在基因的功能研究中意义重大.本研究在建立了对目的基因进行高效准确的单点突变方法的基础上,以SRrp53点突变体的构建为例,设计了新型的以反向PCR为基础的多个点突变的实验流程,获得的多位点突变体质粒经测序后均与预期相符,将测序正确的多位点突变体质粒转染293T细胞后,均表达了分子质量正确的蛋白质.以上结果表明,该实验设计方案能够高效、方便地用于基因多个点突变体的构建,为进一步研究它们的分子功能打下了基础.

英文摘要：

Protein post-translational modifications play very important roles in the regulation of gene functions and successful site-directed mutagenesis is vital to determine a protein＇s modification sites and hence, to define its structure and function. Yet, researches are always confronted with the problem on how to generate multiple-site mutations of a target gene rapidly. In this study, a novel strategy to generate multiple-site mutations was developed based on accurate single site mutagenesis with SRrp53 as an example. Firstly, the entire SRrp53 coding sequence was amplified from the human breast cDNA library and then cloned into the expression vector pcDNA3-FLAG. Five sets of primers for K196R, K171R, K163R, K146R and K309R mutations were synthesized and were then phosphorylated at their 5＇ terminus. In the next step, the first run of inverse PCR was performed with one specific set of phosphorylated primers. Further, the PCR products were subjected to Dpn I treatment, agarose gel purification, dam methyltransferase treatment, self-ligation and purification with PCR extraction kit. Afterwards, the second run of inverse PCR was performed and the PCR products were treated as above protocol. The second processed PCR products were then used as the template for the third run of inverse PCR. The exact run times of inverse PCR are according to the number of mutation sites that you wanted. The PCR products of the last run were treated with Dpn I , purified with agarose gel extract kit, self-ligated and were transformed into DH5ot. Ten colonies were randomly picked up for plasmids extraction and DNA sequencing. The expression of both the SRrp53 wild type and five-site mutants was analyzed by transfection of these plasmids into 293T cells. The DNA sequencing results showed that 9 of 10 extracted plasmids gained the correct mutations. And after these 9 correct plasmids were transfected into 293T cells, all of the mutants were expressed with the right molecular mass. In a word, our novel strategy can be applied to generate m