狂犬病“靶向性”中和表位DNA疫苗的构建与瞬时表达  

Construction and transient expression study of target DNA vaccine expressing neutralization epitopes of rabies virus

作　　者：肖跃强[1,2] 谢金文[1,2] 李书光[1,2] 魏凤[1,2] 丁壮[3] 刘吉山[1,2] 沈志强[1,2] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600 [3]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2013年第7期1021-1026,共6页Chinese Journal of Veterinary Science

基　　金：山东省青年自然科学基金资助项目(Q2008D04)

摘　　要：采用RT-PCR获得BALB/c小鼠P41基因,并在不影响氨基酸序列前提下,对序列中461nt NcoⅠ酶切位点进行定点突变;然后采用定向克隆方法将狂犬病病毒糖蛋白主要中和抗原表位核苷酸序列替换P41序列中CLIP区域,进行新一轮PCR反应以获得带有Kozak调控序列的P41-N分子,以增加目的基因的表达量,并将该含有Kozak序列的P41-N分子克隆插入到真核表达载体pCI-neo多克隆位点,通过PCR与限制性内切酶酶切方法进行鉴定;最后将各中和表位表达载体转染COS-7细胞,Western blotting检测目的基因的瞬时表达。测序分析结果显示,BALB/c小鼠P41基因CDS区为840bp,编码279个氨基酸,与GenBank已登录(NM_010545)核苷酸序列、氨基酸序列均存在多个位点差异;定点突变后获得了P41分子突变体,经PCR及限制性内切酶酶切方法证明成功构建了携带P41-N分子的真核表达载体;Western blotting证实各P41-N分子均在COS-7细胞获得瞬时表达,且表达的目的产物能够与兔抗P41多克隆抗体发生抗原抗体结合反应。结果表明,成功构建了狂犬病病毒主要中和抗原表位的"靶向性"核酸疫苗,为开展在小鼠的免疫学试验奠定了基础。The BALB/c mouse invariant chain P41 was obtained by RT-PCR and a P41 mutant was also obtained by site-directed mutagenesis of the Nco I restriction endonuclease site at 461nt that didn＇t affect the amino acid coding sequences. Then the CLIP domain was substituted by the neu- tralization epitopes of rabies virus glycoprotein. Another round PCR was performed to obtain the P41-N sequences that contain the Kozak regulative sequence for increasing expression efficiency of target genes. The P41-N genes were cloned into the multiple cloning sites of the eukaryotic ex- pression vector pCI-neo and the recombinant plasmids were identified by PCR and restriction en- zyme digestion methods. Then the neutralization epitopes expressing plasmids were transfected in- to COS-7 cells respectively,Western blotting was used to detect transient expression products of target genes. Sequencing analysis showed that BALB/c mouse P41 gene CDS was 840 bp in length encoding 279 amino acids and there were multiple different nucleotide or amino acid sites when compared with the sequences published in GenBank （NM_010545）. The site-mutant P41 was ob- tained after site-directed mutagenesis, the P41-N expressing plasmids were constructed successfully and the transiently expression products of target genes in COS-7 ceils could react to the rabbit anti-P41 polyclonal antibodies. These results indicated that the target DNA vaccines expressing neutralization epitopes of rabies virus were successfully constructed and could be used for further immune experiments in mice.

关 键 词：狂犬病病毒中和表位 BALB c小鼠恒定链 定点突变 定向克隆 瞬时表达 

分 类 号：S852.655[农业科学—基础兽医学]