本文選題：環(huán)核苷酸磷酸二酯酶4B2 + 重組表達��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：人磷酸二酯酶同工酶4(phosphodiesterase,PDE4)選擇性水解cAMP,并分為PDE4A、4B、4C、4D四種亞型。參與炎癥反應(yīng)的單核細胞和中性粒細胞以表達PDE4B2為主。因此,相應(yīng)的PDE4抑制劑可用于治療哮喘、慢性阻塞性肺病等炎癥相關(guān)疾病。磁珠固定化靶蛋白可快速篩選潛在抑制劑混合物,但需要低成本大量表達高活性靶蛋白。前期研究發(fā)現(xiàn),PDE4B2在原核細胞可溶表達豐度低且純化過程不穩(wěn)定。有報道顯示,現(xiàn)有PDE4B2抑制劑的毒副作用與人全長PDE4B2以咯利普蘭(R-rolipram)表征的高親和力結(jié)合構(gòu)象相關(guān),而治療作用與以R-rolipram表征的活性位點低親和力結(jié)合構(gòu)象相關(guān)。因此,本論文通過不同融合表達方式對PDE4B2全長及152-528aa截短突變體進行克隆表達純化及表征,以期獲得大量可溶表達的高表觀比活且處于以R-rolipram表征的低親和力結(jié)合狀態(tài)的PDE4B2融合蛋白。將人磷酸二酯酶4B2(hPDE4B2)全長和152-528aa截短突變體融合6His-SUMO在大腸桿菌重組表達,經(jīng)Ni2+-NTA純化獲得hPDE4B2全長SF-h PDE4B2和截短突變體ST-hPDE4B2重組蛋白。用Western blot檢測多肽成分;用偶聯(lián)堿性磷酸酶的孔雀綠定磷法測定酶活性,表征其km和對模型化合物的抑制常數(shù)。結(jié)果顯示,純化后的st-hpde4b2純度較sf-hpde4b2高,st-hpde4b2(0.03u·mg-1)表觀比活性接近sf-hpde4b2(1.31u·mg-1)的40倍,活性收率超過100倍;此表達純化方案總活性收率不高,且westernblot顯示均有較多降解片段;測定(r)-rolipram和罌粟堿的抑制常數(shù)表明,sf-hpde4b2對(r)-rolipram主要為高親和力結(jié)合構(gòu)象,而st-hpde4b2主要為低親和力結(jié)合構(gòu)象。對代表化合物的抑制常數(shù)進行數(shù)學(xué)分析,發(fā)現(xiàn)其雙對數(shù)模型具有單調(diào)相關(guān)性。因此,與sf-hpde4b2相比,st-hpde4b2用于初步篩選抗炎活性hpde4b2抑制劑可能更有優(yōu)勢。用同源重組的方法將hpde4b2全長和152-528aa截短突變體基因克隆到帶有g(shù)st標(biāo)簽的載體pgex-6p-1上,表達后經(jīng)gst親和層析柱純化分別獲得n-端融合gst的人全長hpde4b2(gf-hpde4b2)和截短突變體(gt-hpde4b2)。然而,純化后的gf-hpde4b2最大表觀比活為0.01u·mg-1,gf-hpde4b2最大表觀比活為0.05u·mg-1。gf-hpde4b2和gt-hpde4b2酶活性收率均較低。進一步探索雙標(biāo)簽純化方法,將hpde4b2的n-端和c端與mbp和6-his標(biāo)簽三種形式融合:n-mbp/6his-c、n-6his/mbp-c和n-6his-mbp/6his-c,分別獲得mcf-hpde4b2/mct-hpde4b2(帶n-mbp/6his-c)、mnf-hpde4b2/mnt-hpde4b2(帶n-6his/mbp-c)、mf-hpde4b2/mt-hpde4b2(帶n-6his-mbp/6his-c)六種融合蛋白基因質(zhì)粒,表達后經(jīng)ni2+-nta和mbp親和層析純化獲得的全長hpde4b2表觀比活性依次是:mcf-hpde4b2(1.11u·mg-1)mf-hpde4b2(0.1U·mg-1)MNF-hPDE4B2(0.01 U·mg-1);截短突變體表觀比活性依次是:MCT-hPDE4B2(1.25 U·mg-1)MNF-hPDE4B2(0.6 U·mg-1)MF-hPDE4B2(0.3 U·mg-1);全長hPDE4B2和截短突變體重組蛋白的活性收率也顯著高于前兩種方案。鑒于測定PDE4活性篩選抑制劑對靶蛋白活性的要求,以MCF-h PDE4B2和MCT-hPDE4B2為靶蛋白在成本上顯得更有優(yōu)勢。綜上所述,本研究成功獲得hPDE4B2全長和截短突變體與三種不同標(biāo)簽的融合表達重組蛋白:融合6His-SUMO標(biāo)簽的hPDE4B2截短突變體的活性收率效果較hPDE4B2全長好;融合GST標(biāo)簽活性收率效果均不理想;N端融合MBP標(biāo)簽C端融合6his標(biāo)簽的hPDE4B2全長和截短突變體融合表達及純化方案的活性收率最好。因此,推薦高通量初篩潛在抗炎活性的PDE4抑制劑用MCT-hPDE4B2,確認潛在抗炎活性的PDE4抑制劑用MCF-hPDE4B2。
[Abstract]:The human phosphodiesterase isoenzyme 4 (phosphodiesterase, PDE4) selectively hydrolyzed cAMP and divided into four subtypes of PDE4A, 4B, 4C, 4D. The monocytes and neutrophils involved in the inflammatory response expressed PDE4B2. Therefore, the corresponding PDE4 inhibitors can be used in the treatment of asthma, chronic obstructive pulmonary disease and other inflammatory related diseases. Magnetic beads immobilized target protein The potential inhibitor mixture can be quickly screened, but high activity target proteins are needed at low cost. Previous studies have found that PDE4B2 has low soluble expression in prokaryotic cells and is unstable in purification process. It is reported that the toxic side effects of existing PDE4B2 inhibitors are associated with the high affinity of human full-length PDE4B2 with the high affinity of R-rolipram. Conformation is related, and the therapeutic effect is related to the conformation of low affinity binding to the active site characterized by R-rolipram. Therefore, the whole length of PDE4B2 and the truncated 152-528aa mutants were cloned and characterized by different fusion expressions, in order to obtain a large amount of soluble expressed high apparent specific activity and be characterized by R-rolipram. PDE4B2 fusion protein of low affinity binding state. Recombinant human phosphodiesterase 4B2 (hPDE4B2) full length and 152-528aa truncated mutants were fused 6His-SUMO in Escherichia coli, hPDE4B2 full SF-h PDE4B2 and truncated mutant ST-hPDE4B2 recombinant protein was purified by Ni2+-NTA. The polypeptide components were detected by Western blot. The enzyme activity of malachite green and phosphorus was determined to characterize its km and the inhibition constant to the model compound. The results showed that the purity of purified st-hpde4b2 was higher than that of sf-hpde4b2, and the apparent specific activity of st-hpde4b2 (0.03u. Mg-1) was 40 times that of sf-hpde4b2 (1.31u. Mg-1), and the yield was over 100 times, and the total yield of the expression and purification scheme was not high, and Wester was not high, and Wester was not high. Nblot showed that there were more degradation fragments, and the inhibition constants of (R) -rolipram and papaverine showed that sf-hpde4b2 to (R) -rolipram was mainly a high affinity binding conformation, while st-hpde4b2 was mainly a low affinity binding conformation. The mathematical analysis of the inhibition constants for the representative compounds showed that the double logarithmic model had a monotone correlation. Therefore, Compared with sf-hpde4b2, st-hpde4b2 may be more advantageous for the preliminary screening of anti inflammatory hpde4b2 inhibitors. The whole length of hpde4b2 and the 152-528aa truncated mutant gene were cloned to the carrier pgex-6p-1 with the GST label using the homologous recombination method, and the whole length hpde4b2 (gf-) of the n- terminal fusion GST after the expression of the GST affinity chromatography column was expressed after the expression of the GST affinity chromatography column. Hpde4b2) and truncated mutants (gt-hpde4b2). However, the maximum apparent ratio of the purified gf-hpde4b2 is 0.01u. Mg-1, and the maximum apparent ratio of gf-hpde4b2 to the activity of 0.05u. Mg-1.gf-hpde4b2 and gt-hpde4b2 enzyme is lower. Further explore the double label purification method, which combines the n- and C ends of hpde4b2 with the three forms: 6his-c, n-6his/mbp-c and n-6his-mbp/6his-c, respectively obtained mcf-hpde4b2/mct-hpde4b2 (n-mbp/6his-c), mnf-hpde4b2/mnt-hpde4b2 (n-6his/mbp-c), mf-hpde4b2/mt-hpde4b2 (n-6his-mbp/6his-c) six fusion protein gene plasmids, after expression by ni2+-nta and MBP affinity chromatography, the full length hpde4b2 apparent specific activity is in turn: Mcf-hpde4b2 (1.11u. Mg-1) mf-hpde4b2 (0.1U. Mg-1) MNF-hPDE4B2 (0.01 U. Mg-1); the apparent specific activity of the truncated body is as follows: MCT-hPDE4B2 (1.25 U mg-1). The yield of egg white in the whole length and the truncated body weight group is significantly higher than the first two schemes. The requirements for the activity of target proteins by selective inhibitors, MCF-h PDE4B2 and MCT-hPDE4B2 as target proteins are more advantageous in cost. To sum up, this study successfully obtained the fusion expression of hPDE4B2 full length and truncated mutants and three different labels: the activity yield of hPDE4B2 truncated mutant with 6His-SUMO tag is more effective than hPD The full length of E4B2 is good; the effect of the fusion GST tag active yield is not ideal; the activity yield of the hPDE4B2 full length and the truncated mutant of the N terminal fusion MBP tag C terminal fusion 6His tag is the best. Therefore, the PDE4 inhibitor which recommends the potential anti-inflammatory activity of the high throughput screening is used to confirm the PDE4 inhibitor for the potential anti-inflammatory activity. Using MCF-hPDE4B2.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R3411

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