减蛋综合征病毒五邻体重组蛋白的原核表达及抗原性鉴定

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。

Cloning and expression of vecombinent of protein Penton gene from egg drop syndrome virus and identification of expressed protein antigenicity

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 mmol·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 inclusion body.The Western Blot analysis showed that the protein had good antigenicity.