中文摘要：

目的：构建PIAS3（活化型STAT3抑制蛋白）的Myc融合蛋白真核表达重组质粒，并表达融合蛋白Myc-PIAS3。方法：采用PCR方法，扩增鼠PIAS3基因全长cDNA编码区序列。将该1851bp的特异性片段连接到T载体上，将其亚克隆至pCMV-Myc真核表达载体的Sal I和Not I位点之间。将pCMV-Myo-PIAS3重组质粒转染前列腺癌PC3细胞，利用Myc抗体通过Western blotting法检测融合蛋白Myc-PIAS3的表达。结果：重组pCMV-Myc-PIAS3质粒通过酶切和测序鉴定了其正确性。EcoR I酶切鉴定时有4357和1318bp2条带，Xba I酶切鉴定时有3291bp和2384bp 2条带，与预期结果一致。测序结果显示，13个氨基酸Myc在N端，然后是阅读框架正确的PIAS3的基因序列。pCMV-Myc-PIAS3重组质粒转染PC3细胞，利用Myc抗体通过Western blotting法检测融合蛋白Myo-PIAS3的表达，在相对分子质量68000处检测到特异的蛋白表达条带。结论：成功构建pCMV-Myc-PIAS3重组质粒，并表达融合蛋白Myc-PIAS3。

英文摘要：

Objective To construct the eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein and express the fusion protein Myc-PIAS3. Methods The full length PIAS3 fragment of 1851bp was amplified by PCR and ligated into pMD18-T vector. The full length PIAS3 fragment was suhcloned into eukaryotic pCMV-Myc vector between Sal I and Not I sites. The recombinant pCMV-Myc-PIAS3 plasmid was trandfected into PC3 cells. The eukaryotic expression Myc-PIAS3 protein was checked by Western blotting with Myc antibody. Results The recombinant plasmid showed right sequence by the full length sequencing. The recombinant plasmid of pCMV-Myc-PIAS3 was identified by enzyme digestion. As expected, by EcoR I digestion, it showed two bands of 4357bp and 1318bp. By Xba I digestion, it showed two bands of 3291 bp and 2384 bp. The sequencing result showed a N-terminal Myc of 13 amino acids followed with PIAS3 gene sequence in right reading frame. The pCMV-Myc-PIAS3 plasmid was transfected into prostate cancer PC3 cells. A specific protein expression band at relative molecular mass 68000 was obtained by using Myc-antibody with Western blotting method. Conclusion The recombinant plasmid of pCMV-Myc-PIAS3 is sucssesefully constructed, and Myc-PIAS3 fusion protein is sucssesefully expressed.