人外周血单核细胞B7.2基因的克隆及其可溶剪接体的发现

Cloning of human B7.2 gene in PBMC and discovering of a soluble splice variant

目的研究B7.2基因在肿瘤免疫方面作用,从人外周血单核细胞中克隆人B7.2基因的全长基因,并构建含此基因的重组质粒。方法根据人B7.2基因序列设计特异引物,用RT-PCR、巢式PCR方法扩增B7.2基因的全长基因,并构建至PGEM-T载体中,测序鉴定后进行序列分析。结果成功构建B7.2/PGEM-T重组载体,并发现一种新的B7.2异常的剪接体,保留第1,2,3,5外显子,第四外显子全部缺失,共144 bp,其编码蛋白质由此缺少一段跨膜结构。结论新剪接体在灵长类动物中的存在可能是个普遍现象,其对细胞功能有待进一步的研究,为研究B7.2编码蛋白质的结构和生物学功能及其在肿瘤中的作用奠定基础。

Objective B7 family members include B7.1 and B7.2, which are important costimulatory molecules in immune system. So we clone the full-length CDS of human B7.2 gene from PBMC （peripheral blood mononuclear cell）. Methods It was designed that specific primers of human B7.2 gene and gained its full-length CDS by RT-PCR and nested PCR, then ligated the objective fragment into pGEM- T vector and transformed into E, coli for screening. The positive clones which were identified by Colony PCR were taken for sequence analysis. Results It was constructed that the B7.2/ pGEM-T recombinant plasmid successfully and discovered a new B7.2 splice variant. This variant keeps its exon 1,2,3,5 ,but its exon 4 is absent. This lost exon takes 144 bp,whieh leads to an absence of a transmembrane region in the integrated B7.2 protein. Conclusion The clone of human B7.2 gene lays solid base for the investigation of the structure and function of B7.2 protein and its biological effect in tumor immunology. However, the potential impact of this new splice variant sill needs further investigation.