摘要
本文用RT_PCR技术从鸡的肌肉组织中扩增出泛素 (ubiquitin_Ub)编码基因 ,再将其定向克隆到真核表达载体pCMV_Script中多克隆位点的BamHⅠ、HindⅢ之间 ,构建成重组质粒pCMV_Ub。经酶切和测序确定为正确后 ,再将来源于E .teella裂殖子的Etmic_2基因克隆到pCMV_Ub质粒中Ub基因下游的SalⅠ位点上 ,经酶切鉴定 ,获得编码Etimc_2基因的真核表达载体pCMV_Ub_mic_2。该载体中的Etmic_2基因与Ub融合表达 ,并将用于DNA疫苗免疫鸡 ,希望融合了Ub的Etmic_2蛋白能更有效的进入MHC_Ⅰ循环 。
The gene of chicken mono_ubiquitin(Ub) was amplified by RT_PCR from chicken muscle tissue with two mutagenic primers,and then inserted into eukaryotic expression vector pCMV_Script between BamH I and HindⅢ sites to construct the recombinant(named as pCMV_Ub).In order to express ubiquitin in eukaryotic cell and drive fussed ubiquitinated Etmic_2 to the ubiquitin pathway,the upper primer was supplied an ATG start codon with an appropriate Kozak sequence,and the lower primer was changed the C_terminal amino acid from glycine(G 76 to alanine(A 76 ).Endonuclease digestion and DNA sequencing show that modified mono_Ub cDNA gene is correctly inserted into vector pCMV_Script.In order to under control of the ubiquitin gene,Etmic_2 gene was inserted at the Sal I site of pCMV_Ub to construct the recombinat pCMV_Ub_mic2.Thus,DNA vaccine expression vector encoding Etmic_2 fussed with modified chicken mono_ubiquitin was successfully constructed for further DNA vaccine study.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2002年第3期175-177,共3页
Chinese Journal of Preventive Veterinary Medicine
基金
瑞典国际科学基金 (InternationalFoundationforScience
IFS)资助项目 (B/ 2 910_1)
