小鼠肺腺癌细胞C-erbB-2基因片段TA克隆载体的构建与鉴定

Construction and identification of TA cloning vecter of murine lung cancer cell C-erbB-2 gene fragments

目的:选择C-erbB-2(neu)基因中编码两个HLA-A2限制性CTL抗原肽表位p106-114,p369-377的基因片段,构建克隆载体pGM-neu1(包含p106-114片段)和pGM-neu2(包含p369-377片段)。方法:用免疫组化方法检测小鼠Lewis肺腺癌细胞中C-erbB-2的表达,然后采用RT-PCR技术,从小鼠Lewis肺腺癌细胞总RNA中扩增编码neu1(包含p106-114片段)、neu2(包含p369-377片段)的基因片段,通过连接反应构建重组克隆载体,测序鉴定。结果:免疫组化染色检测C-erbB-2的表达,发现小鼠Lewis肺腺癌细胞有明显深棕色,表现出较强阳性,阳性信号位于细胞膜。RT-PCR扩增产物neu1、neu2基因片段大小与理论预期相符。重组克隆载体经DNA序列测定,结果与GeneBank中小鼠C-erbB-2的基因序列一致。结论:C-erbB-2在小鼠Lewis肺腺癌细胞中有较高表达,成功构建了克隆载体pGM-neu1和pGM-neu2。

Objective： To construct and identify TA cloning vector of C-erbB-2 gene fragments. Methods： The detection of C-erbB-2 in murine Lewis lung cancer cell was carried out by immunohistochemistry.The 164bp region of neul cDNA and 157bp region of neu2 eDNA were amplified by RT-PCR from murine Lewis lung cancer cell total RNA. The PCR products were cloned into TA cloning vector. Sequence analysis was carried out. Results： C-erbB-2 was positive in all murine Lewis lung cancer cells, and the signal mainly located in the membrane of Lewis cells.Prospective amplified products-neul 164bp and neu2 157bp gene were obtained by RT- PCR method.The TA cloning vectors were identified as neul and neu2 with the sequencing methods. Conclusion： The expression of C-erbB-2 gene in murine Lewis lung cancer cell was high. pGM-neul and pGM-neu2 cloning vector were successfully constructed.