猪附红细胞体DnaJ基因的克隆及真核表达

Cloning and eukaryotic expression of DnaJ gene of Mycoplasma suis

为研究猪附红细胞体DnaJ基因的功能,根据GenBank中的猪附红细胞体全基因组序列(NC_015153.1),应用DNAStar、DNAMAN和ExPASy网络服务器筛选出1个候选蛋白基因DnaJ,设计合成1对特异性引物,经PCR技术扩增出DnaJ基因片段,克隆至pMD18-T载体上,并亚克隆到真核表达载体pVAX1上,构建了重组真核表达质粒pVAX-DnaJ,脂质体介导转染Vero细胞进行真核表达,RT-PCR和IFAT检测DnaJ基因在Vero细胞中的表达。结果表明,PCR扩增DnaJ基因的片段长度为876bp,编码292个氨基酸,与GenBank中的DnaJ基因同源性为99%;DnaJ基因在Vero细胞中获得瞬时表达。本试验为猪附红细胞体DnaJ基因的生物学特性及核酸疫苗的研究奠定了基础。

In order to study the function of DnaJ gene of Mycoplasma suis,DNAStar,DNAMAN software and ExPASy network server were applied to screen a candidate protein gene DnaJ.A pair of specific primers was designed based on the DnaJ gene in the complete genome sequence of M.suis in GenBank（NC_015153.1）.The DnaJ gene was amplified by PCR,cloned into the pMD18-T vector and then subcloned into the eukaryotic expression vector pVAX1 to construct the recombinant eukaryotic expression plasmid pVAX-DnaJ.pVAX-DnaJ was transfected into Vero cells and the expression of DnaJ gene was detected by using RT-PCR and indirect fluorescent antibody test（IFAT）.The results showed that the length of DnaJ gene is 876 bp,encoding 292 amino acids.The amplified DnaJ gene shared 99% nucleotide identity with that in GenBank.RT-PCR and IFAT results indicated that the DnaJ gene was transiently expressed in Vero cells.This study laid foundations for the research on the biological characteristics of the DnaJ gene and the development of DNA vaccine against M.suis.