摘要
为建立能表达人白介素-28A(hIL-28A)基因的稳定转化家蚕卵巢细胞(BmN)系,构建了重组表达载体pIZT/V5-His-hIL-28A并转染BmN细胞,采用终浓度为300～400μg/mL的博莱霉素(zeocin)筛选2个月后,获得了稳定转化BmN细胞系。SDS-PAGE和Western blotting检测显示在稳定转化BmN细胞中表达的重组hIL-28A蛋白分子质量约为26kD;用ELISA试剂盒测定hIL-28A的表达水平约为2.035×10-5ng/个细胞。用噻唑蓝法(MTT法)检测hIL-28A的体外抗肿瘤活性,结果显示其对肺癌细胞A549、急性早幼粒细胞白血病细胞HL60、肝癌细胞BEL-7402和乳腺癌细胞M231的半数抑制浓度(IC50)分别为3.21、2.84、6.29和9.32ng/mL。研究结果表明hIL-28A可在稳定转化BmN细胞中表达,表达产物具有体外抗肿瘤活性。
In order to establish a stably transformed silkworm BmN cell line which can continuously express human interlukin-28A （hIL-28A） gene, a recombinant expression vector pIZT/V5-His-h/L-28A was constructed and used to transfect BmN cells. After screening with 300 -400μg/mL zeocin （the final concentration） for two months, a stably transformed BmN cell line expressing hIL-28A was obtained. SDS-PAGE and Western blotting results indicated that the recombinant hIL-28A protein expressed in the stably transformed cells had a molecular weight of about 26 kD. The expression level of hIL-28A, determined by ELISA, was about 2. 035 ng in 105 cells. In vitro anti-tumor activity of hIL-28A by MTT assay revealed that the 50% inhibitory concentration （ICso） of the recombinant hIL-28A on A549 （ lung cancer cells）, HL60 （acute promyelocytic leukemia cells）, BEL-7402 （liver cancer cells） and M231 cells （breast cancer cells） was approximately 3.21, 2.84, 6.29 and 9.32 ng/mL, respectively. It was concluded that hIL-28A could be stably expressed in the transformed BmN cells, and has certain anti-tumor activity in vitro.
出处
《蚕业科学》
CAS
CSCD
北大核心
2010年第3期452-457,共6页
ACTA SERICOLOGICA SINICA
基金
国家重点基础研究发展计划"973"项目(No.2005CB-121000)
