人信号转导抑制分子突变重组质粒的构建及鉴定

目的 构建人细胞因子信号转导抑制分子(SOCS)3编码区(CDS)和3’非编码区(UTR)突变基因重组真核表达质粒.方法 以pEGFP-C1-SOCS3野生型表达质粒为模板,PCR方法扩增获得野生型SOCS3 CDS和3'UTR基因的序列.根据SOCS3 mRNA与miR-155预测结合靶点,分别设计3个SOCS3突变型:3' UTR-1、3'UTR-2和CDS.通过融合PCR方法将获得的SOCS3突变基因上、下游片段连接,再将融合片段定向克隆到pEGFP-C1表达载体上,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性.结果 成功构建人SOCS3 CDS和3'UTR区突变基因重组表达质粒.结论 构建的突变重组质粒将为进一步探讨人SOCS3和miR-155相互作用的研究提供实验依据.

Construction and identification of mutant recombinant plasmids of human suppressor of eytokine signa- ling

Objective To construct the mutant recombinant plasmids express the CDS and 3＇ UTR gene of human suppressor of cytokine signaling （SOCS） 3. Methods pEGFP-C1-SOCS3 wild type plasmid as the template, the CDS and 3＇ UTR gene of SOCS3 was obtained by PCR amplification. According to the SOCS3 mRNA and miR-155 prediction of binding sites, respectively, to design SOCS3 gene mutation types ： 3＇ UTR-1, 3 ＇ UTR-2 and CDS. By two rounds PCR will get the mutations of the SOCS3 fusion fragments which were cloned into pEGFP-C1 expression vector. The fusion recombinant plasmids were confirmed by PCR and DNA sequen- cing. Results The mutant recombinant plasmids express the CDS and 3＇ UTR gene of human SOCS3 were constructed and identified by DNA sequencing as same as the prediction. Conclusions The successfully con- structed pEGFP-C1-SOCS3 mutant plasmids are useful to study the interaction of SOCS3 and miR-155.