【摘要】：精氨酸脫亞胺酶(Arginine deiminase，ADI，EC3.5.3.6)能將L-精氨酸不可逆地水解為L(zhǎng)-瓜氨酸和氨，可用于治療精氨酸營(yíng)養(yǎng)缺陷型癌癥。PEG修飾蛋白質(zhì)藥物可以提高蛋白藥物的半衰期和藥理學(xué)特性，并可獲得更加良好的療效和較低的副作用。為解決ADI在體內(nèi)生理?xiàng)l件下的半衰期短和免疫原性強(qiáng)等問題，本研究采用PEG對(duì)ADI進(jìn)行修飾并研究其藥理學(xué)特性。 在前期研究中，通過蛋白質(zhì)工程改造獲得了一株來源于變形假單胞菌(Pseudomonasplecoglossicida)，在生理?xiàng)l件下具有高比酶活和改良的酶學(xué)性質(zhì)的ADI優(yōu)良突變株M13-3。首先，采用單因素優(yōu)化與響應(yīng)面法優(yōu)化M13-3菌株發(fā)酵培養(yǎng)基，提高產(chǎn)酶效率。優(yōu)化后培養(yǎng)基經(jīng)搖瓶發(fā)酵，ADI酶活達(dá)到5.7U mL-1發(fā)酵液，比LB培養(yǎng)基提高了2.01倍。在3L發(fā)酵罐中發(fā)酵8h后，ADI酶活最大值為17.38U mL-1發(fā)酵液，較LB培養(yǎng)基提高了3.51倍。 對(duì)M13-3菌株發(fā)酵后的粗酶液進(jìn)行陰離子交換層析和凝膠過濾層析純化獲得ADI純酶，選用七種不同PEG修飾劑對(duì)ADI進(jìn)行修飾，獲得了三種產(chǎn)率較高的修飾產(chǎn)物：ADI-SS-PEG20kDa，ADI-SC-PEG20kDa，ADI-SPA-PEG20kDa。經(jīng)過體外穩(wěn)定性分析，三種PEG修飾酶較好地保留了ADI在生理?xiàng)l件下的酶活，且pH穩(wěn)定性和熱穩(wěn)定性都略有提高。 選擇ADI-SPA-PEG20kDa(ADI-SPA)進(jìn)行藥理學(xué)實(shí)驗(yàn)，，由藥效/藥代動(dòng)力學(xué)結(jié)果分析可得，ADI-SPA修飾酶在小鼠體內(nèi)表現(xiàn)出增強(qiáng)的藥物效應(yīng)與延長(zhǎng)的藥效時(shí)間，5U注射劑量均能有效降解血液中精氨酸并維持5天以上，大大高于未修飾的ADI。藥代動(dòng)力學(xué)參數(shù)整體上也優(yōu)于未修飾酶，其中經(jīng)由靜脈注射比肌肉注射和皮下注射具有更好的藥代動(dòng)力學(xué)表現(xiàn)：靜脈注射5UADI-SPA，半衰期為53.2h，較ADI提高了11倍；而通過肌肉注射則為34.5h，提高了6倍多。 在H22腫瘤模型小鼠體內(nèi)對(duì)ADI-SPA進(jìn)行藥物評(píng)估，結(jié)果顯示通過肌肉注射高劑量組ADI-SPA，即每5天注射5U ADI-SPA，15天內(nèi)共注射3次共計(jì)15U，對(duì)H22肝癌的抑制率可以達(dá)到95.02%，高于ADI-SPA中、低劑量組和ADI組，說明總劑量相同的ADI-SPA比未修飾ADI具有更強(qiáng)更持久的療效。同時(shí)高劑量ADI-SPA組抑制率接近5-FU化療藥物治療的陽性組(98.34%)。本研究結(jié)果表明，PEG修飾的ADI酶(如：ADI-SPA)可以作為有效的抗肝癌藥物進(jìn)行進(jìn)一步研究。
[Abstract]:Arginine deiminase (Arginine deiminase,ADI,EC3.5.3.6) can hydrolyze L-arginine irreversibly into L-citrulline and ammonia. PEG modified protein drugs can improve the half-life and pharmacological properties of protein drugs and obtain better curative effect and lower side effects. In order to solve the problems of short half-life and strong immunogenicity of ADI under physiological conditions in vivo, PEG was used to modify ADI and its pharmacological properties were studied. In the previous study, we obtained an excellent mutant M13-3 of ADI derived from Pseudomonas mutans (Pseudomonasplecoglossicida), with high specific enzyme activity and improved enzymatic properties under physiological conditions. First, single factor optimization and response surface method were used to optimize the fermentation medium of strain M13-3 to improve the efficiency of enzyme production. After fermentation in shaking flask, the enzyme activity of ADI reached 5.7 U mL-1 fermentation broth, which was 2.01 times higher than that of LB medium. After 8 h fermentation in a 3L fermenter, the maximum activity of ADI was 17.38 U mL-1 fermentation broth, which was 3.51 times higher than that of LB medium. ADI pure enzyme was purified by anion exchange chromatography and gel filtration chromatography from the fermented crude enzyme solution of strain M13-3. Seven different PEG modifiers were used to modify ADI. Three modification products with higher yield were obtained: ADI-SS-PEG20kDa, ADI-SC-PEG20kDa,ADI-SPA-PEG20kDa. After in vitro stability analysis, the three PEG modifiers retained the enzyme activity of ADI under physiological conditions, and the stability and thermal stability of pH were improved slightly. ADI-SPA-PEG20kDa (ADI-SPA) was selected for pharmacological experiment. The results of pharmacodynamics / pharmacokinetics showed that ADI-SPA modified enzyme showed enhanced drug effect and prolonged pharmacodynamic time in mice. The injection dose of 5U could effectively degrade arginine in blood for more than 5 days, which was much higher than that of unmodified ADI.. The pharmacokinetic parameters were also better than that of unmodified enzyme on the whole. The pharmacokinetics of 5UADI-SPA by intravenous injection was better than that by intramuscular injection and subcutaneous injection. The half-life of 5UADI-SPA was 53.2 h, which was 11 times higher than that of ADI. But the intramuscular injection was 34.5 h, which increased by more than 6 times. ADI-SPA was evaluated in H22 tumor model mice. The results showed that ADI-SPA, was injected three times every 5 days with 5 U ADI-SPA,15 in high dose group. The inhibition rate of H22 liver cancer was 95.02, which was higher than that of ADI-SPA, low dose group and ADI group, indicating that ADI-SPA with the same total dose had stronger and more lasting curative effect than unmodified ADI. At the same time, the inhibitory rate of high dose ADI-SPA group was similar to that of 5-FU positive group (98.34%). The results showed that PEG modified ADI enzyme (such as ADI-SPA) could be used as an effective antitumor drug for further study.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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