摘要
目的研究多表位串联重组核酸疫苗中IL18与HBsAg(S)抗原、P6(NP6)抗原在质粒中所处位置的差异对抗原表达是否有影响。方法分别将IL18连接在融合蛋白P6-S(NP6-S)的氨基端和羧基端,即构建4种质粒:pc-IL18-P6-S、pc-IL18-NP6-S、pc-S-P6-IL18和pc-S-NP6-IL18。采用DNAstar软件分析4种质粒表达的融合蛋白的抗原性。将重组质粒分别转染CHO细胞进行瞬时表达,通过间接免疫荧光和Western blotting检测S、P6(NP6)抗原的表达情况,以确定最佳的连接方式。结果应用DNAstar软件分析可知IL18在融合蛋白的氨基端的抗原性高于在羧基端。间接免疫荧光检测结果发现IL18位于融合蛋白羧基端的质粒表达的抗原性优于氨基端,即:质粒pc-S-P6-IL18和pc-S-NP6-IL18的S、P6(NP6)抗原的表达情况均优于质粒pc-IL18-P6-S和pc-IL18-NP6-S,故选用质粒pc-S-P6-IL18和pc-S-NP6-IL18作为优势质粒。结论成功构建了4种质粒,并确定了优势质粒,此项技术将为新一代多基因核酸疫苗的研制提供实验依据,从而为构建更加有效的多价DNA疫苗奠定了良好的基础。
To study whether the position change of IL18,HBsAg,and P6（NP6） in vector pcDNA3.1 affect the protein expressing.IL-18 was respectively connected with amino terminal and carboxyl terminal of fusion protein P6-S（NP6-S） to construct 4 plasmids： pc-IL18-P6-S,pc-IL18-NP6-S,pc-S-P6-IL18,and pc-S-NP6-IL18.The recombinant plasmids were analyzed by DNAstar,and then transformed into CHO cells.The expressed proteins were characterized by indirect immunofluorescence and Western blotting.Then we can determine which is the best connection.It was certified that IL18 should be connected with amino terminal of fusion protein by DNAstar.However we drew the opposite conclusion by indirect immunofluorescence.The result indicate that IL18 should be connected with carboxyl terminal of fusion protein,which offers the basic conditions for constructing new type of polygenic DNA vaccine.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第4期337-340,共4页
Immunological Journal
基金
山东省卫生厅资助课题(2007HZ037)
山东省卫生厅资助课题(2011QW029)
山东省自然科学基金(ZR2009CQ003)
