摘要： 猪博卡病毒是近年来新发现的一种细小病毒.本研究根据其部分基因组序列(GenBank登录号GU902967-GU902971)设计了一对引物,采用PCR方法扩增出了猪博卡病毒的NP1基因,将其克隆到表达载体pET32a(+)中,构建了重组质粒pET32a-NP1,经测序鉴定正确后,将其转化感受态细胞BL21(DE3)中,并进行IPTG诱导表达.结果表明,重组菌可表达分子量为6 kDa的融合蛋白,蛋白表达量较高,而且蛋白以可溶性形式存在于菌体中.表达产物经纯化后,目的蛋白纯度较好.Western Blot结果显示,表达的NP1蛋白能与His抗体发生特异性的免疫反应,表明原核表达的NP1蛋白具有良好的反应原性.
关键词:
猪博卡病毒,
NP1基因,
克隆,
原核表达
Abstract: Porcine bocavirus(PBoV) was a recently discovered parvovirus from pigs.One pair of primers was designed according to the published sequences of genome of the porcine bocaviruses(GenBank accession number GU902967-GU902971).The NP1 gene was amplified by PCR and cloned into the prokaryotic expression vector pET32a(+) to construct a recombinant pET32a-NP1.After sequencing,the recombinant was transformed into Escherichia coli BL21(DE3) competent cells.The transformed bacteria were induced by IPTG to produce a recombinant protein of 46 kDa of molecular weight.The result showed that recombinant protein was higher expression and purity by the Ni-NTA resin,and in soluble form in the bacteria.The recombinant protein could react with the antibody of His in a Western blot test,indicating that the expressed protein possessed strong reactinogenicity.
Key words:
Porcine bocavirus,
NP1 gene,
Cloning,
Prokaryotic expression
中图分类号:
李彬, 毛立, 何孔旺, 温立斌, 茅爱华, 张雪寒, 倪艳秀, 郭容利, 马俊杰, 周俊明, 吕立新, 俞正玉. 猪博卡病毒NP1基因的克隆与原核表达[J]. 华北农学报, 2011, 26(3): 28-31. doi: 10.7668/hbnxb.2011.03.006.
LI Bin, MAO Li, HE Kong-wang, WEN Li-bin, MAO Ai-hua, ZHANG Xue-han, NI Yan-xiu, GUO Rong-li, MA Jun-jie, ZHOU Jun-ming, LU Li-xin, YU Zheng-yu. Cloning and Prokaryotic Expression of NP1 Gene of Porcine Bocavirus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(3): 28-31. doi: 10.7668/hbnxb.2011.03.006.