摘要
目的:克隆并构建含人白介素12(hIL-12)基因p35、p40不同亚基的真核表达载体,瞬时转染真核细胞并诱导IL-12的表达。方法:人单核细胞白血病细胞株(THP-1)和人白血病细胞株(HL-60),经DMSO、IFN-γ和LPS诱导后,用RT-PCR及SOEPCR扩增IL-12p40、p35及p40-p35融合基因;并构建pcDNA-p35、pcDNA-p40及pcDNA-IL-12真核表达载体。以3种重组质粒分别瞬时转染COS-7细胞后,通过RT-PCR及ELISA法检测目的基因的表达。结果:经诱导后,从HL-60细胞中扩增到IL-12p40和p35基因片段;但从THP-1细胞中只扩增到p35基因片段,未能扩增到p40基因片段;经酶切鉴定、PCR扩增及序列测定表明pcDNA-p35、pcDNA-p40及pcDNA-IL-12真核表达质粒构建成功;并在COS-7细胞中可检测到IL-12的表达。结论:含hIL-12基因不同亚基的真核表达质粒的成功构建,对进一步研究IL-12在免疫应答中的调节作用以及作为免疫佐剂改善BCG免疫保护效果的研究奠定了基础。
AIM： To clone and construct the eukaryotic expression piasmids containing different subunits of human IL-12 and to investigate its expression following transient transfection into COS-7 cells. METHODS： Human leukaemia cells （HL-60 and THP-1 ） were stimulated by DMSO, IFN-γ, and LPS, and the target genes including IL-12 p40 and p35 subunits were amplified by RT-PCR. P40 and p35 fragments were then connected and the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 were constructed by double restriction enzymes digestion and connection. Positive plasmids were transiently transfected into COS-7 cells with siPORT XP-1 reagent. The expression of target gene was detected by RT-PCR and ELISA. RE- SULTS： The recombinant plasmids were confirmed by PCR amplification, double restriction enzymes digestion and DNA sequencing. Target gene expression was detected by RT- PCR and ELISA in the transfected COS-7 cells. CONCLUSION： The successful construction of the recombinant plasmids pcDNA-p40, pcDNA-p35 and pcDNA-p40-p35 provides a useful tool for further research on the immune regulatory role of IL-12.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第6期698-702,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30271172)
