医学论文-人IL.docVIP

医学论文-人IL ??????????? 作者：孙杰, 李伯安, 戚扬, 周继文, 朱雷, 迟淑萍, 程云 【关键词】? IL 　　Cloning, expression and identification of human interleukin10 gene? 　　[Abstract]? AIM: To construct a prokaryotic expression vector containing human interleukin10 (IL10) gene, express and identify the protein. METHODS: Human IL10 gene from HepG2 was isolated and identified by DNA sequencing, and then cloned into the expression vector PET28 a (+) to construct prokaryotic expression vector. The recombinant protein was expressed in BL21(DE3) and identified by Western blot and ELISA. RESULTS: The expressed protein? was mainly located in the inclusion body. The relative molecular mass of the expressed product was identical to that of prediction. The expressed protein had binding activity with specific antibody. CONCLUSION: The human IL10 gene was successfully cloned and expressed in E.coli. The expressed product had antigenicity which provides foundation for preparation of monoclonal antibodies against IL10. 　　[Keywords]IL10; cloning; prokaryotic expression 　　[摘 要]? 目的: 构建含人IL10全基因序列的原核表达载体, 并进行表达及产物活性的鉴定。方法: 利用RTPCR方法, 从人肝癌细胞系HepG2的总RNA中扩增IL10的全长编码序列。编码序列经测序验证后, 将其克隆入表达载体PET28 a（+）, 构建IL10基因的重组原核表达载体。在大肠杆菌中诱导表达目的蛋白, 表达产物采用Western blot和ELISA进行鉴定。结果: 表达产物主要位于包涵体内。经Western blot分析和ELISA检测显示, 表达产物的相对分子质量(Mr)同预期的结果相符, 有结合抗体活性。结论: 成功地扩增人IL10全基因序列, 并构建了含该基因序列的原核表达载体, 在大肠杆菌中高效表达的表达产物具有抗原活性, 为下一步制备抗IL10的单克隆抗体(mAb)提供了必要的前提。 　　[关键词]IL10; 克隆; 原核表达 　　IL10也叫细胞因子合成抑制因子, 主要由Th2细胞、 巨噬细胞及活化的B细胞产生, 具有维持机体免疫反应稳定, 抑制病原体引起对机体有害炎症反应的功能[1]。IL10是含有178个氨基酸残基(其中N端的18个氨基酸为信号肽序列)相对分子质量(Mr)约为18000的酸性糖蛋白。其成熟形式含有160个氨基酸, 活性形式通常是以二硫键形成的Mr约为39000的二聚体, 含有两个N糖基化位点。为进一步探讨IL10的生物学活性及其与疾病发生防治的关系, 我们从人肝癌细胞系HepG2中扩增了IL10全长基因编码序列, 成功地构建了IL10基因的原核表达载体, 并对表达产物进行了鉴定。 　　1? 材料和方法 　　1.1? 材料? HepG2细胞及大肠杆菌DH5α、 BL21(DE3)均为本室保存。pGEMT、 PET28质粒购自Promega公司。各种限制性内切酶, 连接酶, Taq DNA聚合酶, dNTP、?? PCR产物及质粒纯化试剂盒, 均为Promega公司产品。总RNA提取试剂Trizol购自Gibco公司。IPTG为Sigma公司产品。检测IL10的试剂盒购自晶美试剂公司。兔抗IL10血清为301医院颜光涛教授惠赠。HRP羊抗兔IgG购自中杉金桥公司。寡聚核苷酸引物由北京三博远志公司合成。扩增IL10基因的引物设计根据GenBank中登