HPV16L1抗原表达肿瘤模型的建立

CONSTRUCTION OF HPV TYPE 16 L1 ANTIGEN EXPRESSING TUMOR MODEL

应用基因重组技术,构建pcDNA-L1真核表达载体,经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCR法检测HPV16L1 mRNA 的生成,并将转染细胞接种小鼠皮下,观察成瘤情况及RT-PCR法检测HPv16L1 mRNA的生成。结果,酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入位点均正确,在转染的 B16细胞中可见绿色荧光并检测到HPV16L1 mRNA的生成,接种的转染细胞在小鼠皮下可成瘤, 接种后1月在肿瘤组织中可检测到HPV16L1 mRNA的生成,B16细胞转染L1后其致瘤性与转染空载体组和野生型B16细胞组无明显差异。

Recombinant expression vector was constructed by techniques of gene recombination,and identified by restriction endonuclease and sequence analysis. Then the recombinant was transfected into B16 cell by techniques of gene transfection and expressions were detected by RT-PCR and IFA. After that,transfected cells were inoculated into subcutaneous of mouse and the forming tumor and expression of HPV16L1 protein after tumor was formed was observed. Identification of pcDNA- HPV16L1 by enzyme digestion showed that the length,inserted location and direction of the target gene which was inserted into the recombinant was correct and the ex- pression of L1 in transfected cell was observed by IFA. The inoculated cells could form tumor in vivo obviously and HPV16 L1 protein could express in the cells stably.