摘要
目的 研究 ＨＢｓＡｇ真核表达质粒ｐＣＩ—Ｓ和 ｐｃＤＮＡ３．１—Ｓ在真核细胞中的表达和质粒ＤＮＡ的免疫效果。方法 应用基因重组技术构建ＨＢｓＡｇ Ａ核表达质粒ｐＣＩ—Ｓ和ｐｃＤＮＡ３．１—Ｓ；经酶切和测序鉴定无误后，用阳离子脂质体介导的方法将重组质粒转染 ＨｅＰＧ２和 ＣＯＳ—７细胞；４８ｈ后，用 ＥＬＩＳＡ的方法检测重组质粒在细胞中ＨＢｓＡｇ的表达。同时用质粒ＤＮＡ免疫小鼠，用ＥＬＩＳＡ检测免疫小鼠血清抗－ＨＢｓ抗体水平；用乳酸脱氢酶释放法检测小鼠脾细胞 ＨＢｓＡｇ特异性 ＣＴＬ叵应。结果 重组质粒ＤＣＩ—Ｓ和 ｐｃＤＮＡ３．１—Ｓ转染的 ＨｅｐＧ２和 ＣＯＳ—７细胞培养上清液和细胞裂解液中ＨＢｓＡｇ均为阳性；ＤＮＡ免疫小鼠血清可检测到高滴度的抗－ＨＢｓ抗体；免疫小鼠脾细胞可检测到较强的 ＨＢｓＡｇ特异性 ＣＴＬ叵应。结论 ＨＢｓＡｇ 真核表达质粒ｐＣＩ－Ｓ和 ｐｃＤＮＡ３．１—Ｓ可在 ＨｅｐＧ２和 ＣＯＳ－７细胞中高效表达，ＤＮＡ免疫小鼠成功地诱导出抗－ＨＢｓ和ＨＢｓＡｇ特异性ＣＴＬ反应。
Objective To study the HBsAg transient expression in HepG2 or COS-7 cells with eukaryotic expres- sion plasmids inserting HBsAg gene (pCl-S and pcDNA3.1-S) and the efficacy of naked DNA immunization in mice. Methods Firstly, the recombinant plasmids of pCI-S and pcDNA3.1-S were constructed by the cloning technique and the accuracy of these constructs was confirmed by restriction enzyme digestion and DNA sequencing. Secondly, plasmids of pCI-S and pcDNA3.1-S were transferred into HepG2 and COS-7 cells, respectively by means of cationic liposome. HBsAg transient expression was assayed by ELISA in cell culture supernatants and cell lysates. Thirdly, plasmids were injected into quadriceps muscles of BALB/C mice and serum samples were obtained from individual immunized or control mice 4 weeks after injection and boost injection, respectively. Anti-HBs were assayed in mice sera by ELISA. HBsAg-specific CTL re- sponses of spleen cells from immunized mice were tested by the LDH method. Results Plasmids of pCI-S and pcDNA3.1- S allowed HBsAg transient expression in cell culture supernatants and cell lysates of HepG2 or COS-7 cells. Intramuscular immunization of BALB/C mice with plasmids of pCI-S or pcDNA3.1-S elicited the antibody and cytotoxic T Iymphocyte responses to HBsAg. Conclusions The vectors used in this study are effective to induce prime antibody and HBsAg-spe- cific-cytotoxic T Iymphocyte responses to HBsAg in mice after intramuscular immunization.
出处
《中华肝脏病杂志》
CAS
CSCD
2002年第2期106-108,共3页
Chinese Journal of Hepatology
基金
国家自然科学基金重点项目(395-7660)
