鸡抗病毒Mx蛋白修饰及其真核表达载体的构建

Mutagenesis Modified and Recombinant Expression Plasmid Construction of Anti-virus Mx Protein from Chicken

利用高保真RT-PCR的方法从Poly(I).Poly(C)诱导的鸡成纤维细胞总RNA中扩增出鸡全长MxcD-NA,扩增产物克隆到pMD19-T Simple载体上,利用PCR突变技术将其第2 032位的碱基由G突变为A,经克隆测序证实突变成功后,将已突变的Mx基因插入真核表达载体,通过PCR、酶切鉴定插入片段正确,提取质粒转染COS-Ⅰ细胞进行RT-PCR鉴定。结果表明:正确克隆了鸡Mx基因,并成功对该基因进行PCR诱变修饰,构建了能够表达鸡Mx基因的重组真核表达载体,为Mx基因体内外表达及生物学活性的进一步研究奠定了基础。

A full-length cDNA of chicken Mx gene was obtained using reverse transcription polymerase chain reaction （RT-PCR） amplification of total RNA extracted from chicken embryo fibroblast （CEF） which was induced with Poly（I）·Poly（C）. The RT-PCR product was subcloned into pMD19-T Simple Vector and mutagenesis was performed in PCR site-directed mutagenesis, sequencing analysis confirmed the successful mutation from G to A in the site 2 032 of chicken Mx cDNA. The fragments amplified by PCR containing the mutation site were subcloned into an eukaryotic expression vector, then transfected into COS-Ⅰ cell. Enzyme, PCR and RT-PCR analysis indicated that the recombinant expression plasmid pcDNA3.0-MMx was successfully constructed, which may provide a basis of expression of Mx protein genes in vivo and in vitro and its specific biological activity research.