發(fā)布時間：2018-06-16 02:23

  本文選題：人視黃醇結(jié)合蛋白4 + pET-28a(+)　； 參考：《安徽醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的制備抗人視黃醇結(jié)合蛋白4(Retinal binding protein4, RBP4)的多克隆抗血清,初步應(yīng)用于2型糖尿病(T2DM)患者的血清RBP4檢測。 方法從成人正常肝臟組織中提取總RNA,RT-PCR法擴(kuò)增人RBP4 cDNA全長,與pMD-18T Vector相連,構(gòu)建克隆載體pMD-hRBP4。設(shè)計分別帶BamHⅠ和HindⅢ酶切位點(diǎn)的上下游引物,PCR后酶切,構(gòu)建原核表達(dá)載體pET28a(+)-hRBP4,氯化鈣法轉(zhuǎn)化入BL21(DE3),提取質(zhì)粒雙酶切鑒定并測序,將IPTG誘導(dǎo)表達(dá)的蛋白質(zhì)產(chǎn)物用western blotting鑒定。對誘導(dǎo)表達(dá)的條件如IPTG的濃度、時間和溫度等進(jìn)行優(yōu)化,鎳親和層析法純化rhRBP4,免疫新西蘭大白兔,制備多克隆抗血清,Western blotting檢測其特異性,ELISA法測定抗體效價。用多克隆抗血清檢測T2DM患者血清RBP4水平。 結(jié)果重組質(zhì)粒pET-28a(+)-hRBP4的雙酶切結(jié)果與預(yù)期大小完全一致,測序結(jié)果證實(shí)所得的cDNA序列與GenBank中hRBP4的序列基本一致,Western blotting結(jié)果證實(shí)重組蛋白能夠與hRBP4單克隆抗體特異性結(jié)合。IPTG誘導(dǎo)表達(dá)的最適濃度是0.04 mmol/L,最適時間為6 h,最適溫度為37℃,表達(dá)的重組蛋白最多可達(dá)菌體總蛋白的44%,且以包涵體形式存在。SDS-PAGE電泳的結(jié)果表明親和層析法得到的重組蛋白純度為96%。ELISA法測定抗血清的效價為1:512000。Western blotting結(jié)果顯示抗血清能夠與T2DM患者血清中分子量21kDa的蛋白結(jié)合,與天然RBP4的大小一致。 結(jié)論成功構(gòu)建和獲得了hRBP4 cDNA全長的原核表達(dá)載體及rhRBP4蛋白,制備的多克隆抗血清可初步用于T2DM患者血清中RBP4的檢測,為進(jìn)一步的RBP4功能研究及臨床大規(guī)模血清樣本RBP4的檢測奠定基礎(chǔ)。
[Abstract]:Objective to prepare a polyclonal antiserum against human retinol binding protein 4 (RBP4) and apply it to the detection of serum RBP4 in patients with type 2 diabetes mellitus (T2DM). Methods the full length of human RBP4 cDNA was amplified by RT-PCR from adult normal liver tissue and linked to pMD-18T vector. The cloned vector pMD-hRBP4 was constructed. A prokaryotic expression vector pET28a (pET-hRBP4) was constructed and transformed into BL21DE-DE3 by calcium chloride method. The plasmid was digested and sequenced. The protein products induced by IPTG-induced expression were identified by western blotting. The induced expression conditions such as the concentration, time and temperature of IPTG were optimized. RhRBP4 was purified by nickel affinity chromatography and immunized with New Zealand white rabbits. The polyclonal antiserum was prepared by Western blotting to detect the titer of antibodies by Elisa. The level of serum RBP4 in patients with type 2 DM was detected by polyclonal antiserum. Results the double digestion results of the recombinant plasmid pET-28a (hRBP4) were in good agreement with the expected size. The result of sequencing confirmed that the sequence of hRBP4 was basically consistent with the sequence of hRBP4 in GenBank. The results of Western blotting showed that the optimal concentration of recombinant protein binding to hRBP4 monoclonal antibody was 0.04 mmol / L, the optimum time was 6 h and the optimum temperature was 37 鈩，

本文編號：2024791