人类免疫共刺激信号B7-2分子逆转录病毒表达载体的构建

The construction of reverse transcription virus expressing vector of human immuno-costimulating signal B7-2

目的 :构建含B7 2基因片段的克隆载体pGEM B7 2和表达载体pLSN B7 2。方法 :应用逆转录聚合酶链反应 (RT PCR)方法从人类慢性淋巴细胞白血病Raji细胞株中扩增到人B7 2cDNA基因片段 ,并经回收纯化酶切后 ,分别连接到质粒pGEM Teasy和pLXSN上。结果 :琼脂糖凝胶电泳及序列测定证实 ,扩增的B7 2cDNA片段的碱基组成与Genebank提供的人B7 2cDNA的基因组成相同。结论 :酶切证实成功构建得了克隆载体pGEM B7 2和表达载体pLSN B7 2 ,为将B7 2基因转导到肿瘤细胞制备肿瘤疫苗以及研究免疫细胞间相互作用。

Objective: To construct reverse transcription virus expressing vector of human immuno-costimulating signal B7-2. Methods: In order to use RT-PCR assay to amplify the fragment of immuno-costimulating signal B7-2cDNA in Raji cells which belong to chronic lymphocytic leukemia cell line, we designed a couple of primers for human B7-2 according to the sequence obtained from genebank. The amplified product was examined by electrophoresis and sequence determination. This fragment was inserted to pGEM-T Easy plasmid and pLXSN expressing vector, then the recombinant plasmid was transformed to E.Coli.JM109 and amplified. Results: ①The base sequence of amplified fragment is in accordance with human B7-2cDNA offered by Genebank.②Recombined cloning vector pGEM-T-B7-2 and pLSN-B7-2 expressing vector were determined by restriction enzyme digestion and PCR assay. Conclusion: pLSN-B7-2 expressing vector can be transinfected into tumor cells by using gene engineering technique. This is useful for researching the tumor immunity and tumor immune-genic treatment.