家鸡Mx基因多态性及其诱变修饰的研究

Study on the Polymorphism and Mutagenesis Modification of Mx Gene in Chicken

研究采用错配PCR-RFLP的方法对家鸡14个品种的Mx基因变异性分布进行了探索;并利用PCR突变技术将Mx基因全长cDNA第2032位碱基由G突变为A(即631位氨基酸的改变),并将突变的Mx基因插入真核表达载体pcDNA3.0,转染COS-Ⅰ细胞,进行RT-PCR与间接免疫荧光(IFA)鉴定,研究结果显示,14个品种Mx基因的2032位点存在2个等位基因(A、G),3种基因型(AA、AG、GG),其中鹿苑鸡、狼山鸡、斗鸡未发现AA型个体,茶花鸡未发现GG个体;并成功对Mx基因进行PCR诱变修饰和构建了能够表达鸡Mx基因的重组真核表达载体,为其体内外表达及生物学活性的进一步研究奠定了基础。

A RFLP of Mx gene from 14 chicken breeds were analysised with mismatch PCR. Meanwhile,a mutation was induced from G to A at the 2032 site of Mx gene cDNA by PCR site-directed mutagenesis technique. The fragments amplified by PCR containing the mutation site were subcloned into a enteukaryotic expression vector,then this vector was transfected into COS-Ⅰ cell,Mx gene and Mx protein in the transfected COS-Ⅰ cell were detected by RT-PCR and IFA. The results showed that two alleles,three genotype was discoveried in the Mx gene cDNA 2032 site. The AA genotype had not been found in Luyuan chicken,Langshan chicken and Cock fight chicken,while GG genotype was not found in Chahua chicken. RT-PCR and IFA had demonstrated COS-Ⅰ cell transfected the recombinant plasmid could stably express the Mx protein.