EB病毒BHRF1基因的克隆及其表达产物抑制细胞凋亡的研究

Cloning of EBV BHRF1 gene and study for the effect of the recombinant protein on apoptosis suppression

目的ＥＢ病毒ＢＨＲＦ１基因克隆，其表达产物抑制ＣＨＯ细胞凋亡功能的研究。方法以Ｂ９５－８细胞株ＥＢＶＤＮＡ为模板设计一对引物，采用ＰＣＲ技术扩增ＢＨＲＦ１基因，用目的基因内的两个单酶切点ＢａｍＨⅠ、ＢｇｌⅠ进行酶切鉴定。用引物上所引入的两个酶切位点ＮｃｏⅠ、ＳｃｌⅠ酶切目的基因后定向连接到ｐＢＶ２２１载体上，转入宿主菌ＤＨ５α经质粒抽提，单、双酶切鉴定，从所获克隆中筛选重组质粒克隆。温控诱导目的基因表达，ＳＤＳ－ＰＡＧＥ及凝胶薄层扫描分析。采用缺血清培养方法建立ＣＨＯ细胞凋亡模型。结果所得扩增产物长５９２ｂｐ与预期大小一致；筛选出的９个重组质粒克隆经ＳＤＳ－ＰＡＧＥ和扫描表明重组蛋白表达量占菌体蛋白总量的２．５％。将含重组蛋白的菌体蛋白加入缺血清培养体系，通过与空白菌体蛋白对照表明，重组蛋白具有明显的抑制ＣＨＯ细胞凋亡的能力。结论ＢＨＲＦ１蛋白具有有效控制血清缺乏所诱导的ＣＨＯ细胞凋亡功能，这为ＢＨＲＦ１蛋白的广泛应用提供了依据。

The BHRF1 gene was amplified from template DNA of B958 cell by polymerase chain reaction.The amplified fragment was about 592bp in length,and was identified with the method of digestion with two unique restriction endonucleases.PCRamplified fragment and plasmid pBV221 were restricted with NcoⅠ puls SalⅠ,and were orientally ligated by T4 DNA ligase. E.coli DH5α was transformed with recombinant plasmid.Nine positive recombinant colonies harbouring the inserted fragment were selected.The recombinant expressing plasmid was induced to expression.SDSPAGE assay showed that a 170kD protein was expressed as expected and the amount of this protein expressed was about 25% of the total bacterial proteins.CHO cells were cultured and induced apoptosis by being cultured with medium deficient in serum.Then,CHO cells were cultured in two kinds of serumdepleted media which were added by positive and negative strain proteins respectively for 72 hours.The results suggest that the recombinant protein has an effect of suppressing apoptosis.