摘要
通过PCR方法扩增全长P32基因和截去跨膜区的P32基因(MP32),将其分别克隆到真核表达载体pcDNA3·1(+)和已插入CpG序列的pcDNA3·1-CpG中,构建pcDNA3·1-P32、pcDNA3·1-CpG-P32和pcDNA3·1-CpG-MP32质粒;用脂质体法转染BHK-21细胞,通过间接免疫荧光(IFA)试验验证其表达效果;经肌肉免疫注射健康BALB/c小鼠,用间接ELISA法检测抗体;在免疫后的第3、5周取免疫小鼠的脾细胞,用流式细胞仪检测CD4+和CD8+T细胞亚群。结果所构建的真核表达载体在BHK-21细胞中都能表达P32蛋白;免疫小鼠血清在免疫第2周后均能检测到羊痘特异性IgG抗体;免疫组小鼠脾脏CD4+T细胞数目和CD4+/CD8+T细胞比值明显高于对照组。结果提示,所构建的真核载体可诱导小鼠产生特异性体液免疫应答,并能刺激小鼠产生较强的细胞免疫应答。
The full-length P32 gene and the truncated P32 gene（MP-32） were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction （PCR） and cloned into pcDNA3.1 （＋） and pcDNA3.1-CpG respectively. The recombinant plasmids （pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3.1-CpG-MP32） were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay （IFA）. The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4^＋ and CD8^＋ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post immunization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4^＋/CD8^＋T cells was significantly higher than that of the control. In conclusion,these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.
出处
《病毒学报》
CAS
CSCD
北大核心
2008年第2期133-137,共5页
Chinese Journal of Virology
基金
甘肃省重大科技专项(ZGS063-A43-013)
关键词
羊痘病毒
P32基因
真核表达载体
表达
免疫原性
goat pox virus
P32 gene
eukaryotic expression vector
expression
immunogenicity
