鸡Mx蛋白基因诱变修饰及抗病活性

Mutagenesis modified of Mx Gene from chicken and Identification of its antiviral specificity

摘要:

【目的】进一步研究鸡Mx蛋白第631位氨基酸的变异与鸡群抗病性的相关性。【方法】本实验利用PCR突变技术将鸡Mx蛋白基因的全长cDNA第2032位的碱基由G突变为A（既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，重组表达载体转染COS-Ⅰ细胞后，进行RT-PCR与间接免疫荧光（IFA）鉴定。【结果】对鸡Mx蛋白基因的cDNA进行PCR诱变修饰正确，构建了能够正确表达鸡Mx蛋白的重组真核表达载体；诱变修饰重组Mx蛋白对抗新城疫病毒（NDV）感染分析结果显示，重组Mx蛋白具有较强的抗新城疫病毒生物活性。【结论】为下一步研究鸡Mx蛋白的抗病机理与制备抗病转基因鸡奠定了坚实的基础。

Abstract:

[Objective] To study whether the antiviral specificity of chicken Mx protein is determined by an amino acid substitution at position 631. [Methods] We used PCR site-directed mutagenesis technique by which a single amino acid was reciprocally substituted G with A at position 2032bp of chicken Mx cDNA. Sequence analysis confirmed successful mutation from G to A at 2032bp of chicken Mx cDNA. The fragments amplified by PCR containing the mutation site were subcloned into a enteukaryotic expression vector. Then the recombinant vector was transfected into COS-Ⅰcell, Mx gene and Mx protein in the transfected COS-Ⅰcell were detected by RT-PCR and indirect fluorescence assay. [Results] The results showed that COS-Ⅰcell transfected the recombinant plasmid could stably express the Mx protein. The antivirus assay showed that Mx protein had characteristics of resistance to infection of Newcastle Disease Virus. [Conclusion] This study may provide a basis of the virus-resistant mechanism of Mx protein and production of virus-resistant transgenic chicken.