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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?
Objective To explore the effect of costimulatory molecule B7-l on enhancing the gene immunization of HBsAg eukaryotic expression vector and to eliminate hepatitis B immune tolerance. Methods The high-fidelity PCR method was used to amplify the target gene fragment B7-1 and the IRES-HBs gene fragment with linker and promoter, subcloned into pBluescriptks + vector and sequenced, then cloned into the retroviral vector plxsn. PA317 cells were transfected with liposomes and screened by G418 to obtain resistant clones. The titer of pseudovirions was detected by NIH3T3 and used to infect HepG2,293 and NIH3T3 cell lines. The transcription of B7-1 was detected by RT-PCR, the expression of B7-1 was detected by immunohistochemistry and the expression of HBsAg in supernatant was detected by ELISA . Results The amplified target gene fragment was confirmed by sequencing, and no sequence change was found. B7-1 was inserted into plxsn with the endonuclease EcoRI / XhoI and IRES-HBs was inserted into plxsn-B7-1-HBs with XhoI / BamHI. PA317 transfected 24 h, according to 1:10 passage, a total of 43 clones after 2 weeks, 1: 5 passage of 90 clones, the average titer of pseudovirions 54 × 105cfu / ml. B7-1 is expressed in all the above cells. HBsAg in PA317, NIH3T3, HepG2, 293 cells supernatant content (A value) 24, 48, 72h were 0.15,0.11?