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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 expression of eukaryotic cells pCI-S and pcDNA3.1-S of HBsAg and the immunogenicity of plasmid DNA. Methods Recombinant plasmid pCI-S and pcDNA3.1-S of HBsAg A were constructed by gene recombination technique. After identified by restriction enzyme digestion and sequencing, the recombinant plasmids were transfected into HepG2 and COS-7 cells by cationic liposome After 48h, the expression of HBsAg in the cells was detected by ELISA. At the same time, the mice were immunized with the plasmid DNA, the level of anti-HBs antibody in the serum was detected by ELISA, and the HBsAg-specific CTL was detected by lactate dehydrogenase release assay. Results HBsAg was positive in culture supernatant and cell lysate of recombinant plasmid DCI-S and pcDNA3.1-S transfected HepG2 and COS-7 cells; high titer anti-HBs Antibody; Immunostaining mouse spleen cells can detect strong HBsAg-specific CTL 叵 should. Conclusion HBsAg eukaryotic expression plasmids pCI-S and pcDNA3.1-S can be efficiently expressed in HepG2 and COS-7 cells. The DNA-immunized mice successfully induced anti-HBs and HBsAg-specific CTL responses.