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CTL对肿瘤的特异性杀伤作用受到HLA的限制.在中国人群中,HLA-A2的频率最高,所以,克隆HLA-A*0201基因,构建逆转录病毒载体可以用于研究肿瘤抗原排斥基因及HLA-A*0201限制性的肿瘤抗原肽.为了克隆HLA-A*0201全长基因,我们首先抽提B淋巴母细胞(HLA-A*0201,A19)的总RNA,在HLA-A座位cDNA特异性引物两端分别加上HindⅢ,SalⅠ的酶切序列,作RT-PCR.用HindⅢ,SalⅠ同时酶切PCR产物及测序载体pBlueScript SK(+/-),低熔点琼脂糖凝胶分离,切胶、纯化、连接,用TSS转化法转入XL1-Blue细菌中.接种至涂布X-gal,IPTG的含氯苄青霉素的LB平板上,挑选白色菌落,摇菌,抽
The specific cytotoxicity of CTL is limited by HLA, and HLA-A2 is most frequently expressed in Chinese population. Therefore, cloning HLA-A * 0201 gene and constructing retroviral vector can be used to study tumor antigen rejection gene and HLA -A * 0201-restricted tumor antigen peptide To clone the full-length HLA-A * 0201 gene, we first extracted total RNA from B lymphoblasts (HLA-A * 0201, A19) Hind Ⅲ and Sal Ⅰ digestion sequences were added to both ends of the primers to make RT-PCR. The PCR products were digested with Hind Ⅲ and Sal I and sequenced pBlueScript SK (+/-), low melting point agarose gel, , Purified, ligated, and transformed into XL1-Blue bacteria by TSS transformation onto a LB plate containing Xanthomonas or Xylol coated with X-gal and IPTG, and white colonies,