塞内加谷病毒3C蛋白生物信息学分析及真核质粒构建表达

3C蛋白是塞内加谷病毒重要的非结构蛋白之一,在病毒入侵机制及影响宿主抗病毒天然免疫方面发挥重要作用。试验应用Oligo 7.0和Primer 6.0软件,用BLAST对比SVV世界参考株,设计一对特异性引物。以SVV-SN株病毒液抽提RNA为模板,将序列连接pMD19-T载体后测序,对正确片段作生物信息学分析可知SVV 3C可编码一个较稳定非分泌型蛋白,具有一个保守"催化三联体"。预测发现该蛋白具有16个磷酸化位点,证明其较高活性,预测其二级结构后得到再次证实。将pMD19-SVV-3C和真核表达载体pcDNA3.1(+)双酶切后连接,重组质粒经鉴定后命名为pcDNA3.1(+)-SVV-3C。将pcDNA3.1(+)-SVV-3C转染到BHK-21细胞24 h后制样作Western blotting,成功验证该重组质粒可在BHK-21中真核表达。相比其他同科病毒,当前SVV 3C蛋白研究存在空白。文章旨在为进一步研究SVV 3C蛋白结构提供理论依据,为后续探索SVV 3C蛋白对于宿主作用机制奠定基础。

Eukaryotic expression and bioinformatics analysis of 3C protein from Seneca Valley virus

3C protein is one of the important non-structural proteins of Seneca Valley virus(SVV),and plays an important role in the mechanism of viral invasion and influencing host antiviral natural immunity. By using Oligo 7.0 and Primer 6.0 to analysis. A pair of specific primers were designed by use BLAST to compare the SVV strains in the world. The RNA extracted from the SVV-SN strain virus-liquid was used as a template, and the sequence was ligated into the pMD19-T cloning vector. After sequencing, the correct fragment was subjected to bioinformatics analysis. It could be seen that SVV 3C could encode a relatively stable non-secreted protein with a conservative "catalytic triplet". And it was predicted that the protein had 16 phosphorylation sites, which proved that it had higher activity, and then it could be supported after predicting its secondary structure. Then pMD19-SVV-3C and eukaryotic expression vector pcDNA3.1(+) were digested and ligated. The recombinant plasmid which was identified to be the positive sample would be named as pcDNA3.1(+)-SVV-3C. pcDNA3.1(+)-SVV-3C was transformed into BHK-21 cells for 24 h, then collected the sample for Western blotting. The predicted size band appeared, which proved that the recombinant plasmid could be expressed in BHK-21. Compared to other homologous viruses, there were a lot of gaps in the current research on SVV 3C protein. The study aimed to provide a theoretical basis for further study of the structure of SVV 3C protein, and to lay the foundation for the subsequent study of the mechanism of action of SVV 3C protein on the host.