人的I型白介素-1受体细胞外区基因的克隆表达及其活性鉴定

发布时间：2018-04-21 01:39

  本文选题：sIL-1RI + 蛋白表达　； 参考：《四川农业大学》2005年硕士论文

【摘要】：白介素-1(Interleukin-1, IL-1)是类风湿性关节炎(Rheumatoid arthritis, RA)发病与病程演变过程中起重要作用的细胞因子之一,临床和实验研究均证明阻断IL-1可以有效减轻RA症状。Ⅰ型白介素-1受体(IL-1RI)与IL-1有很高的亲和力,机体内可溶性的IL-1RI可以与IL-1结合,阻断IL-1与膜上IL-1RI结合以消弱IL-1所产生的生物效应。本研究通过对IL-1RI细胞外基因的克隆及体外表达纯化得到具有一定生物活性的IL-1RI细胞外区部分即可溶性IL-1RI(sIL-1RI),为人的IL-1RI功能性研究以及通过阻断IL-1在临床上治疗RA奠定理论基础。主要结果如下: 1、从HepG2细胞中获取总的RNA,通过RT-PCR及nest PCR得到含有957bp人的Ⅰ型白介素-1受体的细胞外部分的基因,通过与Gene Bank中的sIL-1RI cDNA序列进行比对,所得IL-1RI胞外区部分的cDNA除有三个碱基发生同义突变外其它完全一致。 2、将目的基因片段插入到表达载体pTIG-Trx,在大肠杆菌BL21(DE3)中进行表达。实验分析确定最佳表达条件:诱导时菌液浓度在OD_(600)=0.8～1.0,IPTG浓度1.0mM,20℃下诱导时间大约20h。经过Western Blot和ELISA检测证明所表达蛋白为目的蛋白sIL-1RI;经过SDS-PAGE分析,目的蛋白分别以包涵体和可溶形式存在。对包涵体采取SKL溶解然后再经过梯度透析复性,最后超滤离心浓缩得到了较纯的sIL-1RI目的蛋白;针对可溶形式存在的目的蛋白采用了SP-Sepharose FF阳离子柱对其进行纯化。MTT法细胞活性检测实验证明经过纯化的两种形式存在目的蛋白都具有一定的生物活性。 3、将目的基因片段插入到表达载体pPICZαA,经SacI单酶切线性化后,用电转的方法转入毕赤酵母GS115中,当酵母菌液OD_(600)达到1.0左右后,加入100%甲醇至终浓度0.5%进行诱导,并每隔12小时诱导一次,30℃培养60～72h左右。由于该表达系统为分泌表达,所以对上清进行SDS-PAGE分析,发现在43kDa处有明显的表达条带,通过ELISA对酵母上清进行抗原性检测,证明所表达的蛋白即为目的蛋白sIL-1RI,MTT法细胞活性检测实验证明该表达蛋白具有一定的生物活性。
[Abstract]:Interleukin-1 (IL-1) is one of the important cytokines that play an important role in the pathogenesis and progression of rheumatoid arthritis (RA) in rheumatoid arthritis. Clinical and experimental studies have shown that blocking IL-1 can effectively relieve RA symptoms. Type I interleukin-1 receptor IL-1RI has a high affinity to IL-1, and soluble IL-1RI can bind to IL-1. Blocking the binding of IL-1 to IL-1RI on the membrane attenuates the biological effects of IL-1. In this study, extracellular gene of IL-1RI was cloned and purified in vitro. The extracellular region of IL-1RI with certain biological activity was obtained, which can provide a theoretical basis for the functional study of human IL-1RI and the clinical treatment of RA by blocking IL-1. The main results are as follows: 1. Total RNAs were obtained from HepG2 cells. The extracellular genes containing type I interleukin-1 receptor of 957bp were obtained by RT-PCR and nest PCR. The genes were compared with sIL-1RI cDNA sequences in Gene Bank. The cDNA of the extracellular region of IL-1RI was identical except for three bases with synonymous mutations. 2. The target gene fragment was inserted into the expression vector pTIG-Trx and expressed in E. coli BL21DDE3. The optimal expression conditions were determined by experimental analysis: the concentration of bacteria solution was induced for about 20h at the concentration of 1.0mM-1 IPTG at 0. 8mmM20 鈩，

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