摘要
目的建立并鉴定表达人乳头瘤病毒16型（HPV 16）E7蛋白的实体瘤模型。方法携带有野生型HPV 16 E7基因的重组质粒pcDNA3．1-E7用限制性内切酶及序列测定进行鉴定。采用脂质体将重组质粒pcDNA3．1-E7转染小鼠淋巴瘤细胞RMA，经G418筛选获得稳定转染细胞单克隆，并用RT—PCR方法检测稳定转染细胞HPV 16 E7 mRNA。将转染细胞接种于C57BL／6小鼠，肿瘤形成后，用Western blot分析检测E7蛋白在小鼠肿瘤组织中的表达。结果酶切及测序证实目的基因DNA序列、插入位点及方向均正确。RT—PCR检测显示HPV 16 E7能在RMA转染细胞中稳定表达。RMA转染细胞可在小鼠体内成瘤，且Western blot分析表明E7蛋白在小鼠肿瘤组织中的表达较体外高。结论2．5×10^4个RMA—E7细胞可在小鼠体内成瘤，并且体内E7蛋白表达比体外高．可能导致免疫耐受。
Objective To construct and identify solid tumor model expressing human papilloma virus type 16 E7. Methods The recombination vector pcDNA3.1-E7 carrying wild type HPV 16 E7 was identified by restriction and sequencing. The recombination vector pcDNA3.1-E7 was transfected into mouse lymphadenoma cell RMA by liposome and the monoclone cells transfected stably were obtained by antibiotics G418 sieving. RT-PCR in vitro was used to detect the HPV 16 E7 mRNA of RMA-E7. RMA-E7 cells were subcutaneously inoculated to syngeneic mice, and the expression of E7 protein in tumor tissue of mice was detected by Western blot after tumor formation. Results Identification of recombination vector by restriction and sequencing showed the length, inserted location and direction of the target gene which was inserted into the recombinant were correct, and the expression of HPV 16 E7 was detected. RMA -E7 could form tumor in syngeneic mice, and E7 protein was expressed higher in vivo than in vitro. Conclusion 2.5 × 10^4 RMA-E7 cells could form tumor in syngeneic mice. E7 protein level was much higher in in vivo than in in vitro cultured RMA-E7 cells,which might cause immune tolerance.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第4期513-515,共3页
Chinese Journal of Experimental Surgery
关键词
实体瘤
肿瘤特异性抗原
乳头瘤病毒
基因转染
Solid tumor
Tumor specific antigen
Papilloma virus
Gene transfection
