HBV微环DNA重组质粒的构建及初步药效学实验

目的 构建HBV的治疗性微环质粒并进行体内外实验研究. 方法 采用聚合酶链式反应扩增HBV包膜蛋白preS2.S抗原基因和hIL-2-IFNγ融合基因,将2个基因分别克隆于ZY781载体,得到亲本质粒preS2.S/ZY781和hIL-2-IFNγ/ZY781,测序正确后,用阿拉伯糖诱导生成微环质粒pMCS2.S和pMCIIF. 转染微环质粒于COS-7细胞株,用酶联免疫吸附法定量法检测24、48、72 h时目的蛋白的表达情况.双质粒联合在体电脉冲注射BALB/c小鼠,4周后处死小鼠,检测特异性的体液和细胞免疫应答. 结果 微环质粒pMCS2.S和pMCIIF转染COS-7细胞后,目的蛋白HBsAg、白细胞介素-2、干扰素 γ在不同时间均有表达,在48 h达峰值,分别为(60.3±7.8)、(17.7±1.9)、(10.3±0.9) ng/ml. 微环质粒pMCS2.S能诱导小鼠产生HBsAb的抗体保护,佐剂pMCIIF可显著增强其免疫效果,在低剂量5μg/只就能诱导较强特异性细胞和体液免疫. 结论 成功构建了HBV微环质粒pMCS2.S及其佐剂微环质粒pMCIIF,其有较好的体内外活性.

Construction,identification and pharmacodynamics research on a minicircle DNA recombinant plasmid of HBV

Objective To develop a therapeutic minicircle DNA plasmid of HBV and investigate their immune activity in vitro and in vivo. Methods Two genes were amplified, which separately encoded HBV middle envelope protein (preS2.S) as a vaccine and human interleukin (IL)-2/interferon (IFN)γfusion protein as an adjuvant by PCR from plasmids pcNDA3.1/preS2.S and pcDNA3.1/hIL-2-IFNγ. Then two genes were cloned into eukaryotic vector ZY781 and the parent plasmid preS2.S/ZY781and hIL-2-IFN γ/ZY781 were obtained respectively. The new plasmids were analyzed by restriction endonuclease and DNA sequencing. The parent plasmids were induced to produce minicircle pMCS2.S and pMCIIF by arabinose. The minicircle plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000. At 24, 48 and 72 h after transfection, the supernatants were quantified by ELISA. The two minicircle plasmids were injected into BALB/c mice by electroporation in situ together. When the mice were killed 4 weeks later, specific humoral and cellular immune responses were examined. Results The gene segments and inserting direction of pMCS2.S and pMCIIF were correct by genetic analysis. At 48 h after transfection, the concentration of HBsAg, IL-2 and IFNγwere 60.3±7.8 ng/ml, 17.7±1.9 ng/ml and 10.3 ±0.9 ng/ml, respectively, which were the peak values. Protective antibodies of HBsAb were produced, and co-injection of minicircle pMCS2.S together with adjuvant pMCIIF resulted in more evident immune responses. There was strongly specific humoral and cell immunity by only 5μg plasmid per mice. Conclusions The minicircle pMCS2.S and pMCIIF are constructed successfully, and in-duce strongly specific immune activity in vitro and in vivo.