摘要
为构建以人白细胞介素 2 /γ干扰素(hIL 2 /hIFN γ)融合基因为佐剂的治疗型HBVDNA疫苗 ,采用DNA重组技术分别构建含有HBV包膜中蛋白 (preS2 ·S)抗原和hIL 2 /hIFN γ融合蛋白的真核表达质粒即pcDNAS2 ·S和pcDNAIIF ,酶谱和测序分析表明克隆的preS2 ·S和hIL 2 /hIFN γ融合蛋白基因片段的方向、序列与预期相符。用脂质体转染试剂转染COS 7细胞 ,并用ELISA检测转染细胞培养上清中目的基因表达水平。结果显示 ,质粒转染后 4 8h达峰值 ,分别为HBsAg(P/N) =7.6 3、IL 2 =1 0 .35ng/ml、IFN γ =7.90ng/ml。以CTLL 2依赖细胞株/MTT比色法和微量细胞病变抑制法 (WISH VSV)检测pcDNAIIF转染后 4 8h培养上清中IL 2及IFN γ活性 ,结果分别为 998U/ml和 2 4 9U/ml。证明构建的重组质粒pcDNAS2 ·S和pcDNAIIF结构正确 。
To develop an effective therapeutic HBV DNA vaccine, two eukaryotic expressing plasmids, namely pcDNAS 2 ·S and pcDNAIIF , which respectively encoding HBV middle envelope protein preS 2 ·S and human IL 2/IFN γ fusion protein, were constructed by DNA recombinant technique. After identification by restriction analysis and DNA sequencing, the constructed recombinant plasmids were transfected into COS 7 cells in vitro with transfection reagent Lipofectamine. The expressed product in supernatant was quantified by ELISA , and the biologic activities of IL 2 and IFN γ were assayed by CTLL 2 cell line proliferation and cytopathogenic inhibition, respectively. The results showed that secretive expression of preS 2 ·S and hIL 2/hIFN γ fusion protein peaked at 48h after transfection, and the expression levels were HBsAg(P/N)=7 63, IL 2=10 35ng/ml, and IFN γ=7 90ng/ml. The IL 2 and IFN γ activities in the culture supernatant of COS 7 cells transfected with pcDNAIIF were 998U/ml and 249U/ml, respectively. These results suggested that the recombinant plasmids pcDNAS 2 ·S and pcDNAIIF were correctly constructed and the transfected cells could express secretive active protein.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2003年第6期493-497,共5页
Medical Journal of Chinese People's Liberation Army
基金
国家高技术研究发展计划 (863计划 )基金资助课题 (编号2 0 0 1AA2 1 71 4 1 )
