【摘要】 目的为探索共刺激分子Ｂ７－ｌ增强ＨＢｓＡｇ真核表达载体基因免疫的效果，并用于消除乙型肝炎免疫耐受。方法用高保真ＰＣＲ法扩增目的基因片段Ｂ７－１及带接头和启动子的ＩＲＥＳ—ＨＢｓ基因片段，亚克隆到ｐＢｌｕｅｓｃｒｉｐｔｋｓ＋载体中测序，再克隆到逆转录病毒载体ｐｌｘｓｎ中。用脂质体转染ＰＡ３１７细胞，经Ｇ４１８筛选获得抗性克隆。用ＮＩＨ３Ｔ３检测假病毒颗粒的滴度，并用于感染ＨｅｐＧ２、２９３、ＮＩＨ３Ｔ３细胞系，以ＲＴ－ＰＣＲ检测Ｂ７－１的转录，免疫组织化学检测Ｂ７－１表达， ＥＬＩＳＡ检测细胞上清液中ＨＢｓＡｇ表达。结果所扩增的目的基因片段经测序证实，未发现序列有改变。用内切酶 ＥｃｏＲＩ／ＸｈｏＩ将 Ｂ７－１插入 ｐｌｘｓｎ中，然后用 ＸｈｏＩ／ＢａｍＨＩ将 ＩＲＥＳ－ＨＢｓ插入构建成 ｐｌｘｓｎ－Ｂ７－１－ＨＢｓ。转染后 ＰＡ３１７ ２４ ｈ，按 １∶１０传代， ２周后共得 ４３个克隆， １∶５传代得 ９０个克隆，假病毒颗粒滴度平均为５４ × １０５ｃｆｕ／ｍｌ。 Ｂ７－１在上述细胞中均有表达。 ＨＢｓＡｇ在ＰＡ３１７、 ＮＩＨ３Ｔ３、 ＨｅｐＧ２、 ２９３细胞上清液中的含量（Ａ值）２４、 ４８、 ７２ｈ分别为０．１５、０．１１?更多还原

【Abstract】 Objective To investigate the enhanced role of B7-l in gene immunization of eukaryotic vector expressing HBsAg, and further to eliminate the immunotolerance to hepatitis B virus. Methods B7-l and IRES-HBs with linker and promoter sequence prior to HBs gene were amplified with high fidelity PCR, then subcloned into plasmid pBluescriptks+, which was used to sequence. The eukaryotic expressing vector was constructed by inserting B7-l and IRESHBs into plxsn. Recombinant vector was transfected into PA317 by means of lipal mixtures. Titre of pseudovirus packaged in PA317 cells was detected by infecting NIH3T3 cells. B7-l and HBsAg expression in PA317, NIH3T3, HepG2, 293, EL4 cell lines was detected by RT-PCR, immunohistochemistry and ELISA, respectively. Results No change was found in the sequences of target genes amplified by high fidelity PCR compared with the known sequences. Plxsn-B7-l-HBs was constructed by first inserting B7-l into plxsn with enzyme EcoRI/XhoI, then IRES-HBs was inserted in the same way with enzyme XhoI/BamHI. 43 and 90 G418-resistant clones were obtained two weeks later after l:10 and l:5 passages at 24 hours after transfection. Titre of the peudovirus was 5.4 × 105 CFU/ml. B7-1 was expressed in all the cell lines above by RT-PCR test. The contents of HBsAg in the supernatant of the PA317, NIH3T3, HepG2, and 293 cells at 24, 48, 72 hours intervals were 0.15, 0.1l, 0.20, 0.13; 0.63, 0.47, 0.76, 0.58; 0.77, 0.61, 0.89, 0.69, respectively. Conclusions The retroviral vector expressing B7-l and HBsAg simultaneously is constructed successfully. The target antigens are expressed in above cell lines in vitro after transfection with lipids mixtures. It may be used to conduct gene immunization and to investigate the role of B7-1 on HBsAg immune response, which may be further used to the prevention and therapy of hepatitis B.更多还原