摘要
目的构建携带针对CD46基因的pSUPER retro RNAi逆转录病毒载体,研究糖基磷脂酰肌醇(GPI)锚定蛋白CD59与CD46在介导T细胞信号转导中的相关性。方法将能转录产生靶向CD46小发夹RNA(shRNA)的寡核苷酸序列,克隆入逆转录病毒载体pSUPER retro,转化大肠杆菌JM109并转染Jurkat细胞。将Jurkat细胞分为未转染的Jurkat细胞组(Ⅰ组)、转染空质粒的Jurkat细胞组(Ⅱ组)、转染CD59干扰质粒的Jurkat细胞组(Ⅲ组)及转染CD46干扰质粒的Jurkat细胞组(Ⅳ组)。用RT-PCR、Western blot技术检测各组细胞中的CD59、CD46基因的表达水平。用噻唑蓝(MTT)比色法检测CD46与CD59联合作用对4组Jurkat细胞的增殖效应。结果重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序,结果表明序列正确,构建成功,稳定转染后,Ⅳ组细胞CD46分子的表达被成功抑制,Ⅲ组细胞CD59分子的表达被抑制。Ⅰ组和Ⅱ组细胞CD46与CD59单抗联合作用后,增殖能力明显高于Ⅲ组、Ⅳ组(P<0.05);但Ⅰ组和Ⅱ组,Ⅲ组和Ⅳ组之间无差异。结论 CD59可增强CD46对T细胞信号转导的效应。
In order to study the enhancement effect of glycosylphosphatidylinositol （GP1）-anchored protein CD59 on CD46-mediated T cell signal transduction, pSUPER-siCD46 eukaryotic expression vector was constructed, and then two pairs of single-stranded oligo encoding CD59 shRNA sequence were synthesized and inserted into lined pSUPER expression vectors to construct recombinant vectors. The two recombinant vectors and one negative control vector （siCD59-C） were transfected into Jurkat cells. Meanwhile, human Jurkat cells were divided into 4 groups： Jurkat cells （group Ⅰ ）, Jurkat cells transfected with pSUPER plasmid （group Ⅱ）, Jurkat cells transfected with pSUPER-CD59 siRNA plasmid （group Ⅲ）, and Jurkat ceils transfected with pSUPER-CD46 siRNA plasmid （group Ⅳ）. CD59 and CD46 mRNA levels was detected by RT-PCR. After erosslinking of CD46 and CD59 antibodies, T cell proliferation was measured by MTT assay （lymphocyte transformation test）. The results of PCR, enzyme digestion and sequence demonstrated that the target sequence had been successfully inserted into the vector. The mRNA expressions of CD59 and in group Ⅱ and CD46 in group m were significantly inhibited, as compared with group Ⅰ and Ⅱ （P〈 0.05）. After crosslinking of anti-CD46 mAb and anti--CD59 mAb, the proliferation of lymphocyte in group Ⅰ or Ⅱ was evidently increased compared with groups Ⅲ and Ⅳ（P〈 0.05）, but there was no difference between groups I and Ⅱ, or group m and group Ⅳ （P〉 0.05）. These results showed that CD59 can enhance the effect of CD46-mediated T cell signal transduction.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第7期576-579,共4页
Immunological Journal
基金
国家自然科学基金资助项目(3170893)
