摘要
目的 克隆和表达可溶型白细胞相关抗原 (HLA) B2 7分子的基因。方法 通过三轮聚合酶链反应 (PCR)方法选择性删除编码跨膜单位的第 5外显子而克隆得到可溶型HLA B2 7基因 ,并以电转法将其转入人B淋巴细胞系 ,使其高效表达。结果 人T、B及单核细胞系及小鼠L细胞系在正常培养条件下均存在选择性剪切现象 ,而产生可溶型HLA I类分子。经PCR方法克隆得到的可溶型HLA B2 7基因 ,经酶切图谱鉴定及序列分析证实确为删除了第 5外显子的HLA B2 7分子的基因 ,将此克隆产物转入不含HLA B2 7基因的细胞系C1R中 ,在其培养上清中检测到高水平的可溶型HLA B2 7分子 ,其产量受培养温度、培养条件等因素影响。结论 HLA B2 7分子前mRNA发生选择性剪切而导致跨膜单位丢失可产生可溶型HLA B2 7分子 ,该细胞系为将来研究环境因素、细胞因子刺激对可溶型HLA B2 7分子表达的影响提供了必需的实验基础。为探讨HLA B2 7分子如何参与脊柱关节病的发病提供理论基础。
Objective To clone and express the soluble HLA-B27 (sB27) gene in human B lymphoid cell line. Methods Soluble B27 cDNA was constructed by 3 consecutive rounds of PCR and then cloned into the RSV5neo vector. The construct was transfected into C1R cells. Results Restriction enzyme digestion and DNA sequencing confirmed that the construct was the HLA-B27 gene lacking exon 5 which encodes the transmembrane domain of HLA molecule. The expression of sHLA-B27 was verified successfully in the culture supernatant of C1R cells. The level of sHLA-B27 changed with variable culture conditions, such as temperature and concentration of fetal calf serum. Conclusion The sHLA-B27 molecules can be generated by alternative splicing of HLA-B27 pre-mRNA. Our construct provide a useful model for studying the effect of environmental factors such as bacterial infections and cytokine stimulation on the expression of soluble HLA-B27 molecule. It will also provide clues for the role of HLA-B27 molecule in the pathogenesis of spondyloarthropathies.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2000年第12期924-927,共4页
National Medical Journal of China
