摘要
本研究构建包含人B7.1cDNA的重组真核表达载体pDisplay-hB7.1并鉴定,实现其在白血病细胞株HL-60细胞上的表达,获得HL60-hB7.1。应用分子克隆、PCR法扩增人B7.1基因片段,采用ApaI、SalI分别双酶切PCR产物和表达载体pHook,纯化后的酶切产物由T4DNA连接酶连接,转导大肠杆菌DH5α;应用ApaI、SalI双酶切和DNA序列分析技术进一步鉴定阳性克隆pDisplay-hB7.1,应用脂质体转化技术将B7.1转染HL-60细胞,同一标本分别采用直接免疫荧光法、SABC法及FACS法鉴定hB7.1在HL-60细胞上的表达,阳性细胞命名为HL60-hB7.1。结果表明,PCR法扩增出约620bp的基因片段;连接产物转导感受态细胞后,共筛选出5个阳性克隆,均可经ApaI、SalI双酶切出大小约620bp的基因片段,与人B7.1基因片段大小相符,提示重组载体构建成功。进一步DNA序列分析证明人B7.1基因序列和读码框架正确,与文献报道人B7.1cDNA序列一致,无基因突变。直接免疫荧光法鉴定表明,HL60-hB7.1阳性细胞数为70%,SABC法鉴定为65%,FACS法鉴定为92.7%。经以上鉴定证实hB7.1cDNA已成功转染HL-60细胞株并在其膜上高效表达,获得了HL60-hB7.1。结论:应用分子克隆方法可成功构建人B7.1(CD80)重组真核表达载体,并稳定、高效表达B7.1-于HL-60细胞胞膜上,推测应用这种方案制备的瘤苗可能具有免疫治疗和免疫保护作用。
The aim of study was to investigate the construction , identification and expression of human B7. 1 （CD80） eukaryotic expressing vector on HL-60 cells. B7. 1 gene was subcloned from the cloning vector using PCR. The PCR products and eukaryotic expressing vector （pHook） both were separatly digested with ApaI, SalI and were ligated using T4 DNA ligase. The ligased products were transduced into DH-5et. B7. 1 gene containing clones was selected by digestion with ApaI and SalⅠ, and were further confirmed by sequencing of DNA. HL-60 cells were transfected with B7.1 by using lipofectamine and detected by immunofluorescence, SABC and FACS methods. The results showed that the size of PCR products was about 620 bp. Five clones were ampicillin-resistant and all could be digested by ApaI and SalⅠ to produce 620 bp gene fragment that had the same size of B7.1, which means that the B7.1 recombinant vector has been constructed successfully. Further sequencing confuaned the validity of the construction. No nucleotide mutation was found, B7.1 effectively expressed on HL-60 cells with 70%, 65% and 92. 7% by immunofluorescence, SABC and FACS respectively. It is concluded that the human B7.1 （CDS0） eukaryotic expressing vector can be successfully constructed by molecular cloned methods and can stably effectively express on the membrane of B7.1-negative acute myelocytic leukemia （ AML ） cell line HL-60. It is inferred that the vaccine prepared by using this methool may have immunotherepeutic and immuno-protective effects.
出处
《中国实验血液学杂志》
CAS
CSCD
2009年第2期450-454,共5页
Journal of Experimental Hematology
基金
卫生部部属(管)医疗机构临床学科重点项目基金
编号20012131
