HBsAg及B_(7-2)抗原重组腺病毒载体感染免疫研究

目的　为激发机体对ＨＢｓＡｇ的ＣＴＬ反应,寻求对慢性乙型肝炎更有效的治疗方法。方法　构建了共表达ＨＢＳＡｇ及Ｂ７－２抗原的Ｅ１区插入重组腺病毒载体,用脂质体法转染２９３细胞,经空斑筛选表达目的抗原的重组腺病毒,用ＥＬＩＳＡ法及Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ法分别检测ＨＢｓＡｇ及Ｂ７－２抗原表达。在２９３细胞中扩增,再纯化、浓缩后,体内感染Ｃ５７小鼠,检测体液免疫反应和细胞免疫反应。结果　重组腺病毒在体外培养的２９３细胞及ＨｅｐＧ２细胞中高效表达ＨＢＳＡｇ及Ｂ７－２抗原。感染小鼠体液免疫较弱,但可通过注射乙型肝炎疫苗而加强;重组腺病毒载体能诱导强而有效的细胞免疫反应;未观察到明显毒副作用。结论　ＨＢｓＡｇ及Ｂ７－２抗原重组腺病毒载体体外感染２９３、ＨｅｐＧ２细胞后高效表达目的抗原,感染免疫局诱导有效的细胞免疫反应。初步结果显示腺病毒载体是一种高效、安全的载体系统。

Humoral immunization and cell-mediated immunization evoked by HBsAg and B_(7-2) Ag coexpression recombinant adenovirus vector

Objective: To evoke cytotoxic T lymphocytes (CTL) response and seek for a more effective method to treat chronic hepatitis B. Methods The adenovirus vector was constructed with the foreign genes inserted in the early region 1(E1), which directed coexpression of HBV-S and B7-2 antigens by means of an internal ribosomal entry site placed between the two coding sequences. The vector was transfected into 293 cell lines by liposome and the adenovirus expressing the target antigens was obtained by plaque select. The HBsAg and B7-2 antigen expression in in vitro cell culture was measured by ELISA and Western blotting, respectively. The immune responses were measured by ELISA for antibody response and a LDH release assay for CTL activity after immunization with the recombinant adenovirus vector in C57 mice. Results HBsAg and B7-2 antigens were highly expressed after infecting the 293 and HepG2 cell lines in vitro. The humoral response to hepatitis B surface antigen was mildly induced and could be enhanced by reinjecting a regular dose of HBsAg antigen vaccine. The cell-mediated immune response was highly induced by the recombinant adenovirus infection. No clear side effect was observed after immunization. Conclusions This could be a novel strategy for a development of both preventive and therapeutic vaccines against HBV infection. The recombinant adenovirus vector is an effective and safety vector system suitable to the experiments of gene immunization and gene therapy for incurable diseases.