陕西某猪场猪德尔塔冠状病毒N基因的序列分析与原核表达

Sequence Analysis and Prokaryotic Expression of N Gene of Procine Deltacoronavirus in a Pig Farm of Shaanxi

为克隆猪德尔塔冠状病毒N基因并进行序列分析及原核表达,参考GenBank上登录的PDCoV(HKU15-44)N基因序列,合成1对(PDCoV)游引物,PCR扩增N基因,并进行序列分析和原核表达。结果表明,成功克隆了1026 bp的N基因,核苷酸序列分析表明,PDCoV N基因与国内分离株核苷酸同源性最高为98.76%,氨基酸分析表明N蛋白不含信号肽和跨膜区。经IPTG诱导后成功表达了分子质量约47 ku的N蛋白,该蛋白能与PDCoV阳性血清结合,具备良好的抗原性。研究结果为进一步建立PDCoV快速检测方法和研究PDCoV亚单位疫苗奠定了基础。

The cloning,sequence analysis and prokaryotic expression of the procine deltacoronavirus N gene were performed.A pair of upstream and downstream primers were synthesized by referring to the sequence of PDCV(HKU15-44)N gene registered in GenBank.The N gene was amplified by PCR and analyzed by sequence analysis and prokaryotic expression.1026 bp N gene of PDCoV was successfully cloned.The nucleotide sequence analysis showed that the nucleotide homology of PDCoV N gene and domestic isolate N gene was 98.76%.Amino acid analysis indicated that N protein contained no signal peptide and transmembrane region.After induction by IPTG,the N protein with a molecular weight of 47 ku was successfully expressed.The N protein could react with PDCoV positive serum and had good antigenicity.In this study,N gene of PDCov was successfully cloned and expressed in prokaryotes,laying a foundation for the further establishment of a rapid diagnostic method for PDCov and the development of PDCov subunit vaccine.