PRRSV陕西分离株ORF3基因的克隆、序列分析及原核表达载体的构建  

Cloning,Sequence Analysis and Construction of Prokaryotic Expression Vector of ORF3 Gene of PRRSV Shaanxi Strain

作　　者：张琦 王菡 黎径 梁靓 许信刚[2] 

机构地区：[1]陕西出入境检验检疫局检验检疫技术中心,西安710068 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农业学报》2010年第10期21-26,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省自然科学基金项目(2006C111);陕西省科技攻关项目(2009K02-01)

摘　　要：根据GenBank公布的PRRSVORF3基因的核苷酸序列,设计并合成一对特异性引物,用RT-PCR方法扩增PRRSV陕西分离株ORF3基因,将其克隆入pGEM-T载体中,测序并进行序列分析。再将ORF3基因亚克隆入pET-32a中,构建原核表达载体。结果扩增到765 bp的PRRSV全长ORF3基因,序列分析结果表明,分离株与SY0608亲缘关系较近,而与CH-2、HB-2关系较远。重组原核表达质粒经酶切鉴定正确后命名为pET-GP3,为进一步研究GP3蛋白的原核表达、免疫特性、结构与功能奠定了基础。According to the published complete nucleotide sequence of PRRSV（Porcine reproductive and respiratory syndrome virus） in GenBank,a pair of primers specific to the full-length of ORF3 gene encoding the envelope glycoprotein GP3 were designed.The ORF3 gene fragment amplified by RT-PCR was cloned into pGEM-T vector.The recombinant plasmid was sequenced and ORF3 gene was compared with GenBank＇s other PRRSV strains.Then the gene was cloned into vector pET-32a for prokaryotic expression in E.coli.The result showed the ORF3 gene of Shaanxi strain is 765 bp in full-length and encodes 254 amino acids.The sequence analysis indicated that the identity of nucleotide sequence and amino acid sequence is high between strains isolated both in our country and others.The Shaanxi strain has high homology with SY0608 in nucleotide level,whereas it has relatively low homology with CH-1 and HB-2.After the correct identification by enzyme digestion,ORF3 gene was cloned into efficient expression vector pET-32α,the correct clone was named pET-ORF3 by enzyme digestion.The results established fundament to further expressing GP3 protein of PRRSV of efficient expression vector in E.coli BL21 cells and studies of its immunological characteristics,the structure and function.

关 键 词：猪繁殖与呼吸综合征病毒 陕西分离株 ORF3基因 序列分析 原核表达载体 

分 类 号：S852.65[农业科学—基础兽医学]