狂犬病“靶向性”Th表位DNA疫苗的构建与瞬时表达研究

用RT-PCR扩增BALB/c小鼠P31基因,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;采用定向克隆方法将狂犬病病毒核蛋白、糖蛋白及NS蛋白主要Th抗原表位核苷酸序列替换P31序列中CLIP区域,进行新一轮PCR反应获得带有Kozak调控序列的P31-Th分子,以增加目的基因的表达量,并将含有Kozak序列的P31-Th分子克隆入真核表达载体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Th表位表达载体转染COS-7细胞,Western-blot检测目的基因的瞬时表达。测序分析结果表明,BALB/c小鼠P31基因CDS区长648bp,编码215个氨基酸,与Gen-Bank中登录的Ii分子（NM₀10545）的核苷酸序列、氨基酸序列均存在多个位点差异;定点突变后获得了P31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P31-Th分子的真核表达载体;Western-blot分析证实,各P31-Th分子均在COS-7细胞中获得瞬时表达,且表达的目的产物能够与兔抗P31多克隆抗体发生抗原抗体结合反应。结果表明,作者成功构建了狂犬病病毒主要Th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。

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

The BALB/c mouse invariant chain P31 was obtained by using RT-PCR and a P31 mutant was obtained by site-directed mutagenesis to the Nco Ⅰ restriction endonuclease site at 461 nt with the amino acid coding sequence no changes. Then the CLIP domain was substituted by the Th antigenic epitopes of rabies virus nucleoprotein,glycoprotein and NS protein. PCR was performed to obtain the P31-Th sequence that containing the Kozak regulative sequence to increase the expression efficiency of target genes.The P31-Th genes were then cloned into the multiple cloning sites of the eukaryotic expression vector pCI-neo and the recombinant plasmids were confirmed by PCR and restriction enzyme digestion methods.Then the Th antigenic epitopes expressing plasmids were transfected respectively into COS-7 cells. Western-blotting was used to detect transient expression products of target genes. Sequencing analysis showed that the BALB/c mouse P31 gene CDS was 648bp in length encoding 215 amino acids and there were multiple different nucleotides or amino acid sites compared with the sequence in the GenBank（NM₀10545）.The site-mutant P31 was obtained after site-directed mutagenesis,and the P31-Th expressing plasmids were successfully constructed. The transiently expression products of target genes in COS-7 cells could react with the rabbit anti-P31 polyclonal antibodies.These results indicated that the target DNA vaccines expressing Th antigenic epitopes of rabies virus was successfully constructed and could be used for further immune experiments in mice.