摘要
根据减蛋综合征病毒(EDSV)AV-127株的全基因序列(GenBank序列号AC000004)设计了1对引物,采用PCR扩增出五邻体(Penton)完整基因,将其连接到克隆载体pMD18-T,经过鉴定后测序。序列分析表明,所获得的DNA片段核苷酸序列和GenBank中AV-l27株的五邻体比较有9个碱基突变,但利用DNAstar分析,两者的氨基酸完全一致。然后酶切胶回收后将目的片段连接到质粒表达载体pET-30a(+),获得的阳性克隆命名为pET-30a-Penton。将pET-30a-Penton转化E.coli Rosetta,经37℃、1.0 mmol·L-1 IPTG诱导表达5 h,SDS-PAGE分析超声裂解后的上清和沉淀,结果显示目的蛋白以包涵体形式存在,蛋白分子质量约为54.0 ku。Western Blot试验结果表明目的蛋白具有较好的抗原活性。
With a pair of primers designed according to the complete gene sequence of egg drop syndrome virus（EDSV） AV-127 strain in GenBank（AC000004）, the complete Penton gene was amplified by PCR, and cloned into pMD18-T vector. The sequence analysis revealed that the target gene has 9 bases mutation from Penton gene of EDSV reported in GenBank. The recombined plasmid was transformed into E. coli Rosetta and induced by 1.0 retool· L^-1 of IPTG, 37 ℃ for 5 h. The SDS-PAGE analysis showed that the expressed fusion protein with a molecular weight of 54.0 ku existed in the the protein had good antigenicity. inclusion body. The Western Blot analysis showed that
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2009年第2期83-87,共5页
Journal of Northeast Agricultural University
关键词
减蛋综合征病毒
五邻体
原核表达
抗原性
egg drop syndrome virus
Penton
prokaryotic expression
antigenicity
