狒狒尿酸氧化酶基因克隆及分子改造研究
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摘要
尿酸氧化酶是生物体内嘌呤代谢途径中最后一个关键酶，决定了嘌呤代谢的最终产物。由于尿酸氧化酶基因在进化过程中发生了无义突变，导致人类和大多数高等灵长目动物不能合成尿酸氧化酶，嘌呤代谢最终以尿酸形式排出体外,这造成了人体内的尿酸浓度明显高于其他物种。高浓度的尿酸是人类罹患高尿酸血症、痛风及其相关疾病的元凶。现今用于治疗痛风及相关疾病的药物主要是别嘌呤醇、秋水仙碱等化学药物，具有严重的副作用，这促使人们寻找更安全高效的降尿酸药物。尿酸氧化酶由于降解尿酸的高效率而受到人们的关注。现今利用DNA重组技术可以方便高效地获得各物种来源的尿酸氧化酶。但是作为一种异源蛋白，尿酸氧化酶在用于人体治疗时，其免疫原性成为人们日益关注的问题。单纯的尿酸酶重组产品其安全性受到人们的质疑。因此，如何消除或降低尿酸氧化酶的免疫原性成为制约其大规模应用于医学治疗的瓶颈。这也成为现今有关尿酸氧化酶研究的热点问题，即如何通过酶分子的修饰达到消除其免疫原性。
通过酶化学修饰将修饰分子与目的蛋白交联而达到消除尿酸氧化酶免疫原性的方法已获得成功。但是该法也有其缺点，如修饰后的酶分子稳定性下降等问题。利用基因工程技术，通过将修饰分子与目的蛋白融合表达以获得高纯度、高活力的尿酸氧化酶成为人们获得高效、安全的尿酸氧化酶的新途径。
本论文以狒狒尿酸氧化酶(rbUOX)作为蓝本，选取牛乳铁蛋白肽作为修饰分子，结合狒狒尿酸氧化酶的分子结构特点，从三个水平上设计了目的蛋白的修饰策略：N端修饰、N端C端同时修饰以及N端、C端和分子内综合修饰。以期达到消除或降低狒狒尿酸氧化酶的免疫原性目的。
在克隆策略上，通过密码子优化，设计了四个版本尿酸氧化酶基因的全序列，然后通过重叠延伸PCR克隆得到四个版本的狒狒尿酸氧化酶基因：rbUOX, rbUOX-LfcinB、rbUOX-LfcinB2、rbUOX-LfcinB3。接着将其导入到相应的大肠杆菌宿主菌中表达。结果证明，四个目的基因均成功得到表达。
其次，通过优化发酵条件，蛋白质表达定位，发现在25℃下目的蛋白为细胞内可溶性表达。目的蛋白经IPTG诱导下，于6h后产量达到最大。经过细胞破碎，Ni+亲和层析分离，BCA定量分析，目的蛋白的产量分别达到了136.0mg/L、127.5mg/L，116.6mg/L和111.7mg/L。回收率达70%以上。酶活检测的结果显示，由于修饰分子的存在，目的蛋白的酶活有所下降，其酶活力分别为：17.93、8.10、6.23、3.76U/mg。
通过MALDI-TOF-MS/MS对目的蛋白的一级结构进行分析。结果表明，四个版本的重组蛋白样品其蛋白质序列均与预期一致。
通过间接ELISA法对修饰分子在降低目的蛋白免疫原性的效果进行了定量分析。首先通过重组狒狒尿酸氧化酶作为抗原免疫正常人白细胞以获得人抗rbUOX抗体。然后用所制得的抗体对四个重组蛋白进行免疫吸附测定。结果表明，以重组狒狒尿酸氧化酶作为阳性对照，三个改造后的尿酸氧化酶其免疫反应发生率分别下降了12.09%、16.96%、29.06%。
本论文的研究结果表明通过牛乳铁蛋白肽的修饰，狒狒尿酸氧化酶的免疫原性得到降低，且其降低程度与修饰分子的数目成正相关。说明在目的蛋白分子中引入合适的活性小肽修饰分子并采取合适的修饰策略对于消除外源蛋白的免疫原性是可行的。
Uricase (UOX) is a key enzyme in the metabolic pathway of purine,which decides the final breakdown product of purine in living organism. Dueto the evolutionary fate that two non-sense mutations of uricase gene occurredin human genome, human can not produce uricase, which subsequently resultsin higher physiological concentration of uric acid in human body than in thosespecies who possess the active uricase gene. Excessive uric acid is consideredas the direct cause of diseases, such as hyperuricemia, gout, nephrolithiasis,and vascular diseases. Therefore, development of drugs for reducing uric acidhas keeping attracting interests from pharmaceutical industry and researchcycles. Despite the excellent performance on control of physiological uric acid,the drugs applied in clinic now, such as allopurinol, febuxostat andbenzbromarone have serious side effects, which actuates people to developmore effective and safer drugs. Uricase, as a living organism-derived enzyme,has a high efficiency on reduction of uric acid. However, as a heterogeneousprotein, it may trigger serious hypersensitive reactions when applied in humanbody. So, how to erase or decrease the immunogenicity of uricase becomes an issue that must be solved.
This thesis aims at erasing or decreasing the immunogenicity of baboonuricase (bUOX) by way of modification of uricase with a proper, selectedmodifier. We took bovine lactoferricin (LfcinB) as a modifier and designedthree modified uricase fusions with LfcinB: rbUOX-LfcinB, rbUOX-LfcinB2,rbUOX-LfcinB3, in which rbUOX were connected by LfcinB at N-terminal,N-and C-terminal, and in molecule, respectively.
Four genes encoding target proteins (rbUOX and three modified uricases)were first amplified by splicing by overlap extension PCR (SOE), andtransformed into proper E. coli host cell. After induction by IPTG, four geneshad expressed successfully the target proteins.
By way of lowering induction temperature to25℃, the target proteinswere found in cytoplasm in a soluble form. After cell lysis, the target proteinswere separated effectively by Ni2+-His Bind Resin. The quantities of theexpressed proteins were detected by BCA method and results indicated thefields of the four proteins: rbUOX, rbUOX-LfcinB, rbUOX-LfcinB2, andrbUOX-LfcinB3were136.0mg/,127.5mg/L,116.6mg/L, and111.7mg/L,respectively. The recoveries of the proteins were over70%. Enzyme activitydetection on the acquired proteins revealed that the four proteins possessedactivities of17.93,8.10,6.23, and3.76U/mg, respectively. This result showedthat the activity of baboon uricase decreased after modified by LfcinB.
For the characterization of the expressed proteins, MALDI-TOF-MS/MS was performed, and the results confirmed the amino acid sequences of the fourseparated proteins agreed very well with the theoretical proteins.
As to the determination of immunogenicity of the modified rbUOX,rbUOX was first utilized to immune human leukocyte for7d to preparehuman anti-rbUOX antibody. And then a modified indirect ELISA wasoperated to detect the immunogenicity of modified uricases using rbUOX aspositive control and human serum albumin as negative control. Resultsrevealed that the immunogenicity of modified uricases declinedcorrespondingly with the amount of modifiers.
Results presented here reveal it is helpful to decrease the immunogenicityof uricase by way of introduction of LfcinB and subsequently provide a novelstrategy on protein modification in enzyme engineering cycle.
通过酶化学修饰将修饰分子与目的蛋白交联而达到消除尿酸氧化酶免疫原性的方法已获得成功。但是该法也有其缺点，如修饰后的酶分子稳定性下降等问题。利用基因工程技术，通过将修饰分子与目的蛋白融合表达以获得高纯度、高活力的尿酸氧化酶成为人们获得高效、安全的尿酸氧化酶的新途径。
本论文以狒狒尿酸氧化酶(rbUOX)作为蓝本，选取牛乳铁蛋白肽作为修饰分子，结合狒狒尿酸氧化酶的分子结构特点，从三个水平上设计了目的蛋白的修饰策略：N端修饰、N端C端同时修饰以及N端、C端和分子内综合修饰。以期达到消除或降低狒狒尿酸氧化酶的免疫原性目的。
在克隆策略上，通过密码子优化，设计了四个版本尿酸氧化酶基因的全序列，然后通过重叠延伸PCR克隆得到四个版本的狒狒尿酸氧化酶基因：rbUOX, rbUOX-LfcinB、rbUOX-LfcinB2、rbUOX-LfcinB3。接着将其导入到相应的大肠杆菌宿主菌中表达。结果证明，四个目的基因均成功得到表达。
其次，通过优化发酵条件，蛋白质表达定位，发现在25℃下目的蛋白为细胞内可溶性表达。目的蛋白经IPTG诱导下，于6h后产量达到最大。经过细胞破碎，Ni+亲和层析分离，BCA定量分析，目的蛋白的产量分别达到了136.0mg/L、127.5mg/L，116.6mg/L和111.7mg/L。回收率达70%以上。酶活检测的结果显示，由于修饰分子的存在，目的蛋白的酶活有所下降，其酶活力分别为：17.93、8.10、6.23、3.76U/mg。
通过MALDI-TOF-MS/MS对目的蛋白的一级结构进行分析。结果表明，四个版本的重组蛋白样品其蛋白质序列均与预期一致。
通过间接ELISA法对修饰分子在降低目的蛋白免疫原性的效果进行了定量分析。首先通过重组狒狒尿酸氧化酶作为抗原免疫正常人白细胞以获得人抗rbUOX抗体。然后用所制得的抗体对四个重组蛋白进行免疫吸附测定。结果表明，以重组狒狒尿酸氧化酶作为阳性对照，三个改造后的尿酸氧化酶其免疫反应发生率分别下降了12.09%、16.96%、29.06%。
本论文的研究结果表明通过牛乳铁蛋白肽的修饰，狒狒尿酸氧化酶的免疫原性得到降低，且其降低程度与修饰分子的数目成正相关。说明在目的蛋白分子中引入合适的活性小肽修饰分子并采取合适的修饰策略对于消除外源蛋白的免疫原性是可行的。
Uricase (UOX) is a key enzyme in the metabolic pathway of purine,which decides the final breakdown product of purine in living organism. Dueto the evolutionary fate that two non-sense mutations of uricase gene occurredin human genome, human can not produce uricase, which subsequently resultsin higher physiological concentration of uric acid in human body than in thosespecies who possess the active uricase gene. Excessive uric acid is consideredas the direct cause of diseases, such as hyperuricemia, gout, nephrolithiasis,and vascular diseases. Therefore, development of drugs for reducing uric acidhas keeping attracting interests from pharmaceutical industry and researchcycles. Despite the excellent performance on control of physiological uric acid,the drugs applied in clinic now, such as allopurinol, febuxostat andbenzbromarone have serious side effects, which actuates people to developmore effective and safer drugs. Uricase, as a living organism-derived enzyme,has a high efficiency on reduction of uric acid. However, as a heterogeneousprotein, it may trigger serious hypersensitive reactions when applied in humanbody. So, how to erase or decrease the immunogenicity of uricase becomes an issue that must be solved.
This thesis aims at erasing or decreasing the immunogenicity of baboonuricase (bUOX) by way of modification of uricase with a proper, selectedmodifier. We took bovine lactoferricin (LfcinB) as a modifier and designedthree modified uricase fusions with LfcinB: rbUOX-LfcinB, rbUOX-LfcinB2,rbUOX-LfcinB3, in which rbUOX were connected by LfcinB at N-terminal,N-and C-terminal, and in molecule, respectively.
Four genes encoding target proteins (rbUOX and three modified uricases)were first amplified by splicing by overlap extension PCR (SOE), andtransformed into proper E. coli host cell. After induction by IPTG, four geneshad expressed successfully the target proteins.
By way of lowering induction temperature to25℃, the target proteinswere found in cytoplasm in a soluble form. After cell lysis, the target proteinswere separated effectively by Ni2+-His Bind Resin. The quantities of theexpressed proteins were detected by BCA method and results indicated thefields of the four proteins: rbUOX, rbUOX-LfcinB, rbUOX-LfcinB2, andrbUOX-LfcinB3were136.0mg/,127.5mg/L,116.6mg/L, and111.7mg/L,respectively. The recoveries of the proteins were over70%. Enzyme activitydetection on the acquired proteins revealed that the four proteins possessedactivities of17.93,8.10,6.23, and3.76U/mg, respectively. This result showedthat the activity of baboon uricase decreased after modified by LfcinB.
For the characterization of the expressed proteins, MALDI-TOF-MS/MS was performed, and the results confirmed the amino acid sequences of the fourseparated proteins agreed very well with the theoretical proteins.
As to the determination of immunogenicity of the modified rbUOX,rbUOX was first utilized to immune human leukocyte for7d to preparehuman anti-rbUOX antibody. And then a modified indirect ELISA wasoperated to detect the immunogenicity of modified uricases using rbUOX aspositive control and human serum albumin as negative control. Resultsrevealed that the immunogenicity of modified uricases declinedcorrespondingly with the amount of modifiers.
Results presented here reveal it is helpful to decrease the immunogenicityof uricase by way of introduction of LfcinB and subsequently provide a novelstrategy on protein modification in enzyme engineering cycle.
引文
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[2] Nobutoshi K, Keisuke S, Takao M, et al. Determination of Uric Acid in Plasma byClosed-loop FIA with a Coimmo bilized Enzyme Flow Cell[J]. Anal. Sci.,2000,16:1203-1205
[3] Yamamoto K, Kojima Y, Kikuchi T, et al. Nucleotide sequence of the uricase genefrom Bacillus sp. TB-90[J]. J. Biochem.1996,119:80-84
[4] Liu JG, Li GX, Liu H et al. Purification and properties of uricase from Candida sp. andits application in uric acid analysis in serum[J]. Appl. Biochem. Biotech.,1994,47(1):57-63
[5] Richette P, Bardin T. Gout[J]. Lancet,2010,375:318-328
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