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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。
Objective To study whether the difference of IL18, HBsAg (S) antigen and P6 (NP6) antigen in the plasmid of multi-epitope tandem recombinant nucleic acid vaccine affects the antigen expression. Methods Four kinds of plasmids, pc-IL18-P6-S, pc-IL18-NP6-S and pc-S-P6 were constructed by ligating IL18 to the amino and carboxyl ends of the fusion protein P6- -IL18 and pc-S-NP6-IL18. DNAstar software was used to analyze the antigenicity of the fusion proteins expressed by the four plasmids. The recombinant plasmids were transiently transfected into CHO cells for transient expression. The expression of S, P6 (NP6) antigen was detected by indirect immunofluorescence and Western blotting to determine the optimal connection. Results The results of DNAstar software showed that the antigenicity of IL18 at the amino terminus of the fusion protein was higher than that at the carboxy terminus. Indirect immunofluorescence assay showed that the antigenicity of IL18 at the carboxyl terminus of the fusion protein was superior to that of the amino terminus, ie, the expression of S, P6 (NP6) antigens on pc-S-P6-IL18 and pc-S-NP6- The results were superior to the plasmids pc-IL18-P6-S and pc-IL18-NP6-S, so the plasmids pc-S-P6-IL18 and pc-S-NP6-IL18 were selected as the dominant plasmids. Conclusion Four plasmids were successfully constructed and the dominant plasmids were identified. This technique will provide an experimental basis for the development of a new generation of multi-gene nucleic acid vaccine, which lays a good foundation for the construction of a more effective multivalent DNA vaccine.