趋化因子BLC表达载体构建及在肿瘤细胞中的表达

目的 建立真核表达重组体pcDNA-BLC的稳定转染细胞株，为研究趋化因子BLC的抗肿瘤免疫作用机制奠定基础。方法 以小鼠脾脏组织总RNA为模板，通过RT-PCR扩增BLC全长cDNA，并将扩增的cDNA片段插入pcDNA3．1( )真核表达质粒，构建重组质粒pcDNA-BLC，重组子经限制性内切酶酶切分析及测序鉴定后，用脂质体转染技术导入Colon26细胞，经G418筛选建立稳定转染细胞株。用免疫印迹法(Western Blot)鉴定转染细胞表达的蛋白质，证实BLC存在。用趋化实验证实转染细胞株表达的BLC有生物学活性。结果 真核表达重组体pcDNA-BLC构建成功，pcDNA-BLC稳定转染细胞株表达有生物学活性的BLC。结论 成功建立了BLC的稳定转染细胞株，为研究BLC的抗肿瘤免疫作用机制继而发展肿瘤疫苗奠定了基础。

Construction of pcDNA-BLC and Expression of BLC in Tumor Cells

Objective To establish a stable transformants of B-lymphocyte chemoattractant(BLC) with the eukaryotic expression vector for investigation of its anti-tumor function further. Methods The BLC full-length cDNA was amplified by RT-PCR from the total RNA isolated from the spleen of mouse. After sequence determination, the BLC full-length cDNA was cloned into the eukaryote expression vector pcDNA3.1 (+), the resulted recombinant was designated as pcDNA-BLC. Then pcDNA-BLC was transfected into murine tumor cell line colon 26 by LIPOFECTIN. The stable transformants were selected by G418 and identified by immunoblot assay. The bioactivity of BLC was confirmed by chemotaxis assay. Results The recombinant pcDNA-BLC was successfully constructed. The stable transformants of BLC can express the bioactive BLC in tumor cell lines. Conclusion The stable transformants can stably express BLC in tumor cell lines. These stable transformants will be useful for further study of the BLC biological effect on tumor and for the development of cancer vaccine.