摘要
目的:构建白细胞介素-21(interleukin-21,IL-21)和乙型肝炎病毒前S2S抗原(S2S)的融合表达质粒,并研究其在293T细胞中的表达。方法:采用PCR方法扩增IL-21和HBV前S2S基因片段,分别克隆入pcDNA3真核表达质粒,用分子克隆方法构建融合表达质粒,并以脂质体2000转染293T细胞,分别应用ELISA法和Western Blot法检测细胞上清及细胞中IL-21和HBsAg的表达水平。结果:经酶切鉴定及DNA序列证实重组质粒内插入片段序列正确,三种重组质粒分别命名为pcDNA-IL-21、pcDNA-S2S和pcDNA-IL-21-S2S,并且重组质粒能在293T细胞内表达并分泌相关蛋白。结论:成功构建IL-21和乙型肝炎病毒前S2S抗原的融合表达质粒,重组质粒能在真核细胞内表达。
Objective: To construct eukaryotic expression plasmids carried mouse interleukin-21 (IL-21) and hepatitis B virus pre-S2 and S (HBV S2S), and to investigate the expression of the plasmids in 293T cells. Methods: IL-21 and HBV $2S were amplified by polymerase chain reaction (PCR), and then they were cloned to pcDNA3 eukaryotic expression plasmid. Recombinant plasmid containing both IL-21 and HBV $2S were constructed by using molecular cloning. Plasmids were transfected into 293T cells by lipofectamine 2000. Expressions of IL-21 and HBsAg in supematant and cells were tested by ELISA and Western Blot, respectively. Results: The sequences of insertion elements were confirmed by enzyme digestion and DNA sequencing. Three recombinant plasmids were named as pcDNA-IL-21, pcDNA-S2S, and pcDNA-IL-21-S2S, respectively. These plasmids could be expressed and secreted to related proteins in 293T cells. Conclusions: Recombinant eukaryotic expression plasmids containing IL-21 and HBV S2S were successfully constructed and could be expressed in eukaryotic cells.
出处
《现代生物医学进展》
CAS
2012年第23期4401-4404,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金(81072434)
国家"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2008ZX10002-006
2012ZX10002-007)
唐都医院2011年苗子人才资助计划