猪细小病毒NS1基因的克隆、表达及密码子优化提高表达水平

Cloning and expression of porcine parvivirus NS1 gene and its codon optimization for enhancing the expression

通过PCR方法从猪细小病毒（PPV）基因组中扩增NS1基因,将其克隆到pcDNA3.1A质粒中,构建成与Myc/His标签融合的NS1真核表达载体。将表达载体转染PK-15细胞使其表达。利用猪偏爱的密码子对NS1全基因进行了密码子优化,通过构建表达载体和转染细胞,检测并比较了密码子优化前后的表达水平。结果显示：扩增的NS1基因与GenBank序列一致,构建的表达载体结构正确;Western blot检测表明,NS1基因能够在PK-15细胞中进行表达,但表达水平很低,有时检测不到,从而严重影响对其的进一步研究;密码子优化后NS1基因的表达水平由优化前的7μg/T25培养瓶增加至32μg/T25培养瓶,是原来的4倍。可见密码子优化可明显提高NS1蛋白在PK-15细胞中的表达,这为下一步研究NS1蛋白的功能提供了有利条件。

To investigate the biological functions of porcine parvovirus （PPV） non-structure protein 1 （NS1）,the NS1 gene was amplified from PPV genome and cloned into pcDNA3.1A plasmid to generate Myc/His-tag-fused NS1 eukaryotic expression vector,which was transfected into PK-15 cells for expression. Results showed that the amplified NS1 gene sequence was consistent with that in GenBank,and the constructed NS1 vector was identified to be correct. Western blot detec- tion indicated that the NS1 gene could be expressed in the PK 15 cells, however, its expression level was too low to be detected. To enhance the NS1 protein expression in PK-15,the NS1 gene was codon-optimized using porcine bias codons. By constructing its expression vector and transfecting PK-15 cells,the expression levels of codon-optimized NS1 gene was compared with its wild type gene. Results showed that after codon optimization, the NS1 expression significantly increased from 7 μg/T25 to 32 μg/T25, indicating that codon optimization can significantly increase NS1 gene expression in PK-15 cells. This study provided a necessary conditions for further investigation on NS1 function.