论文标题:HMCD59糖基化前后的抗补体活性研究
Study of Anti-complement Activity of Mutant Human CD59 Before and After Glycation
论文作者 张艳丽
论文导师 高美华,论文学位 硕士,论文专业 病原生物学
论文单位 青岛大学,点击次数 513,论文页数 68页File Size3699k
2004-03-12论文网 http://www.lw23.com/lunwen_236942/ 人突变CD59,真核表达,中国仓鼠卵巢细胞,抗补体活性
human mutant CD59,eukaryotic expression,CHO anti-complement activity
目的：构建表达人突变CD59(HMCD59)蛋白的重组真核表达载体，建立HMCD59真核表达系统，获得表达HMCD59蛋白的中国仓鼠卵巢细胞(CHO)细胞株，深入研究HMCD59在糖尿病血管增殖症中的作用。为阐明糖尿病血管并发症的发病机理奠定基础。 方法：分别构建两种含有HMCD59全长CDNA序列的重组pALTER质粒，并与pCDNA3质粒，按3：1比例混合后，运用脂质体介导法共转染CHO细胞(编号为HM5-CHO及HM6-CHO)，用新霉素类似物G418初步筛选出阳性克隆。应用荧光抗体技术、流式细胞术、免疫酶标技术、western-blot技术进一步检测HMCD59蛋白在真核细胞膜表面的表达；收集CHO细胞作为抗原，免疫家兔以获得多克隆抗体，并通过BCECF染料释放试验检测HMCD59蛋白糖化前后的抗补体活性。 结果：1、成功构建了表达HMCD59真核细胞(CHO)表达系统。运用脂质体介导法将重组突变CD59-pALTER及pcDNA3共转染CHO细胞，用含有400ug／mlG418的1640培养基培养2周，筛选出阳性克隆。筛选出的阳性克隆细胞以荧光抗体技术、免疫酶标技术检测，表明在细胞膜表面有HMCD59分子表达。流式细胞术检测证明转化CHO细胞的两种突变人CD59的表达率分别为44.06％(HM5)和64.67％(HM6)。将细胞裂解物进行免疫印迹分析，证实在20KD有一条与CD59分子量相等的蛋白条带。2、成功获得了高效价的抗CHO细胞的多克隆抗体，经抗体效价滴定，测定其效价在1：16万以上。3、BCECF染料释放试验提示两种突变质粒的表达产物均具有抗补体活性，糖化后其抗补体活性减弱。 结论：建立了两个表达HMCD59的真核表达系统，获得表达HMCD59的细胞株。获得了抗CHO细胞的高效价多克隆抗体。初步研究了HMCD59的活性，发现HMCD59仍具有抗补体活性，但糖化后其抗补体活性减弱． 意义：成功建立了两种HMCD59的真核表达系统并获得了CHO细胞的多克隆抗体，为深入研究HMCD59在糖尿病患者血管并发症中的作用英文摘要及进一步研究其生物学功能及其作用机制奠定了基础。为临床糖尿病血管增殖症的诊断、防治开辟一条新途径。
Objective: To establish a eukaryotic expression vector and a expression cell line of mutant human CD59(HMCD59).To investigate the effect of HMCD59 on diabetic vascular complications.To laid a foundation for explaining the mechanism of diabetic vascular complication. Methods: Two recombinant palter plasmid containing whole CDNA sequences of HMCD59 transfected CHO cells by lipofectamine with PCDNA respectively. Then G418 was used to select positive clone. A series of methods such as fluorescent antibody technique, immunoenzymatic histochemistry, western blot and flow cytometrey were used to make sure that HMCD59 protein expressed on cell membrane ;CHO cells was harvested to immunize rabbit for muticlonol antibody; it"s activity of anti-complement were studied through the method of BCECF release. Results:1.Two stable cell lines expressing HMCD59 were successfully established, using lipofectamine mediating method. Two recombinant mutant CD59-palter transfected into CHO cells with PCDNA respectively. After two weeks cultivation with RPMI- 1640 medium containing 400ug/ml G418, positive clones was identified. G418-resistant clones was confirmed that HMCD59 molecules expressed on the surface of CHO cells by the method of fluorescent antibody technique and immunoenzymatic histochemistry .The rate of expression of HM5-CHO is 44. 06%;HM6-CHO,64.67%. The cell lysates were identified by western blot that there are a protein band at the position of the relative molecule mass of 20 000 which molecular weight is the same to CD59 molecule. 2, Muticlonol antibody to CHO cells was successfully acquired , and its titer was determined beyond 1 :160, 000. 3, Two expression product possesthe character of resisting complement, and the character weakened if the expression product was glycated. Conclusion: Successfully established two cells expressing HMCD59, acquired high-titered muticlonal antibody to CHO cells. Made a preliminary study of the resisting-complement character of HMCD59, and found that both of the two HMCD59s bore the character of resisting complement, and the character of them weakened after glycation.
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