本文選題：sIL-1RI + 蛋白表達(dá)　； 參考：《四川農(nóng)業(yè)大學(xué)》2005年碩士論文

【摘要】：白介素-1(Interleukin-1, IL-1)是類風(fēng)濕性關(guān)節(jié)炎(Rheumatoid arthritis, RA)發(fā)病與病程演變過程中起重要作用的細(xì)胞因子之一,臨床和實(shí)驗(yàn)研究均證明阻斷IL-1可以有效減輕RA癥狀。Ⅰ型白介素-1受體(IL-1RI)與IL-1有很高的親和力,機(jī)體內(nèi)可溶性的IL-1RI可以與IL-1結(jié)合,阻斷IL-1與膜上IL-1RI結(jié)合以消弱IL-1所產(chǎn)生的生物效應(yīng)。本研究通過對IL-1RI細(xì)胞外基因的克隆及體外表達(dá)純化得到具有一定生物活性的IL-1RI細(xì)胞外區(qū)部分即可溶性IL-1RI(sIL-1RI),為人的IL-1RI功能性研究以及通過阻斷IL-1在臨床上治療RA奠定理論基礎(chǔ)。主要結(jié)果如下: 1、從HepG2細(xì)胞中獲取總的RNA,通過RT-PCR及nest PCR得到含有957bp人的Ⅰ型白介素-1受體的細(xì)胞外部分的基因,通過與Gene Bank中的sIL-1RI cDNA序列進(jìn)行比對,所得IL-1RI胞外區(qū)部分的cDNA除有三個(gè)堿基發(fā)生同義突變外其它完全一致。 2、將目的基因片段插入到表達(dá)載體pTIG-Trx,在大腸桿菌BL21(DE3)中進(jìn)行表達(dá)。實(shí)驗(yàn)分析確定最佳表達(dá)條件:誘導(dǎo)時(shí)菌液濃度在OD_(600)=0.8～1.0,IPTG濃度1.0mM,20℃下誘導(dǎo)時(shí)間大約20h。經(jīng)過Western Blot和ELISA檢測證明所表達(dá)蛋白為目的蛋白sIL-1RI;經(jīng)過SDS-PAGE分析,目的蛋白分別以包涵體和可溶形式存在。對包涵體采取SKL溶解然后再經(jīng)過梯度透析復(fù)性,最后超濾離心濃縮得到了較純的sIL-1RI目的蛋白;針對可溶形式存在的目的蛋白采用了SP-Sepharose FF陽離子柱對其進(jìn)行純化。MTT法細(xì)胞活性檢測實(shí)驗(yàn)證明經(jīng)過純化的兩種形式存在目的蛋白都具有一定的生物活性。 3、將目的基因片段插入到表達(dá)載體pPICZαA,經(jīng)SacI單酶切線性化后,用電轉(zhuǎn)的方法轉(zhuǎn)入畢赤酵母GS115中,當(dāng)酵母菌液OD_(600)達(dá)到1.0左右后,加入100%甲醇至終濃度0.5%進(jìn)行誘導(dǎo),并每隔12小時(shí)誘導(dǎo)一次,30℃培養(yǎng)60～72h左右。由于該表達(dá)系統(tǒng)為分泌表達(dá),所以對上清進(jìn)行SDS-PAGE分析,發(fā)現(xiàn)在43kDa處有明顯的表達(dá)條帶,通過ELISA對酵母上清進(jìn)行抗原性檢測,證明所表達(dá)的蛋白即為目的蛋白sIL-1RI,MTT法細(xì)胞活性檢測實(shí)驗(yàn)證明該表達(dá)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，

本文編號：1780396