CN110585445A - 香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用 - Google Patents
香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，尤其涉及香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用。本发明中的Serpin蛋白序列见SEQ ID NO.2，为香菇来源的具有醒酒保肝作用蛋白，是首次报道具有醒酒保肝功能的香菇蛋白类物质。本发明进行生物信息学分析，从基因水平上确定了蛋白的组成，以及进行了体外重组表达和纯化，确定了醒酒活性物质，研究了它的体内醒酒活性以及保肝作用，该研究成果将为改善饮酒引起的醉酒及酒精性疾病的预防提供新的研究思路和方法，为研发醒酒类的功能性产品提供一定的理论基础。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用。
背景技术
近年来由于人们的生活水平的不断提高，我国的饮酒者基数亦在逐年递增。少量的饮酒或是饮用乙醇含量较低的酒，可增加唾液和胃液的分泌，促进胃肠道的消化和吸收。而过量饮酒则会引起人的中枢神经系统由兴奋转变为抑制的状态。这种急性的酒精中毒，不仅会严重影响人的身体健康，而且极易引发出一系列的社会问题。根据世界卫生组织全球疾病负担项目，酒精因素约占全世界每年所有死亡人数的3.2％(相当于180万人)，并与60多种不同的疾病有因果关系(Varela-Rey,2013)。因此，对急性酒精中毒的预防和治疗需要引起人们的高度重视。
目前的醒酒产品原料多以中草药和植物蛋白为主。其中前者多以葛根、葛花和茶叶为主料，另外添加甘草、生姜、党参以及糖、维生素、琥珀酸和延胡索酸等，其活性成分多为葛根素、茶多酚，维生素、糖琥珀酸和延胡索酸等(张自然，2009)，天然牛磺酸也具有很好的醒酒效果(高加龙，2007)；蛋白则主要以玉米蛋白为原料酶解制备醒酒肽为多。Yamaguchi等率先报道了玉米肽具有促进乙醇代谢的醒酒作用，可明显降低原发性高血压大鼠以及健康人血液中乙醇及其氧化产物乙醛的浓度。究其原因可能是玉米蛋白中丙氨酸和亮氨酸含量较高达32.16％左右(王淑嫒，2007)，它们有助于产生稳定的辅酶(NAD+)，维持正常的三羧酸循环活动，抑制血液中乙醇浓度的升高(Yamaguchi，1997)。隋玉杰等在研究玉米肽醒酒机理时发现玉米肽可激活ADH，具有良好的抑制·OH的能力(隋玉杰，2008)。郭辉等利用NAD法测定肝脏中ADH的活力，进一步证明玉米肽能够激活ADH，从而起到醒酒作用(郭辉，2011)。周凤娟等(2007)通过测定小鼠血清中乙醇含量的降低程度得出丝素肽具有明显的解酒和防醉作用。杨波等(2008)通过枯草杆菌蛋白酶对花生蛋白进行水解制成多肽，最后调配成具有醒酒活性的花生蛋白醒酒肽饮料，其中分子质量在6 000u左右的肽中含有丙氨酸，对于减轻麻痹，防止酒醉有良好效果。葛如振等根据瓦勒一霍赫法对黑豆醒酒饮料进行体外试验，得出酶解后的黑豆蛋白肽能够激活ADH，且激活率达到31.98％(葛如振，2013)。
香菇(Lentinula edodes)，属于真菌门，担子菌纲，伞菌目，口蘑科。香菇含有丰富的营养，是一种食药同源的药用真菌，研究香菇的有效成分，表明它们可以在免疫调节，抗肿瘤，抗衰老，保护肝脏和抗呼吸道感染等具有良好作用。目前国内外对药用真菌蛋白的临床试验以及实际应用的报道还非常少，但实验表明药用真菌蛋白同药用真菌多糖一样具有很大的开发空间和挖掘潜力。香菇C91-3菌株的发酵液蛋白对诱导肿瘤细胞凋亡具有良好的作用(董晓宇，2006)。
现有技术的缺点：目前的醒酒产品原料多以中草药和植物蛋白多肽为主。其中前者多以葛根、葛花和茶叶为主料，另外添加甘草、生姜、党参以及糖、维生素、琥珀酸和延胡索酸等，其活性成分多为葛根素、茶多酚，维生素、糖琥珀酸和延胡索酸等；蛋白多肽则主要是蛋白酶解多肽为主，如玉米肽、丝素肽、花生蛋白水解多肽、黑豆蛋白肽等。这些产品的核心活性成分多不是单一物质，而且在原料中活性成分含量非常低微，提取需要耗费大量原材料。
发明内容
针对现有技术存在的问题，本发明提供了香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用。
本发明是这样实现的，香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物中的应用。
进一步，所述香菇丝氨酸蛋白酶抑制剂的核苷酸序列见SEQ ID NO.1。
进一步，所述SEQ ID NO.1的核苷酸序列编码的蛋白序列见SEQ ID NO.2。
香菇丝氨酸蛋白酶抑制剂在制备防治酒精性肝损伤药物中的应用，所述香菇丝氨酸蛋白酶抑制剂的核苷酸序列见SEQ ID NO.1，蛋白序列见SEQ ID NO.2。
综上所述，本发明的优点及积极效果为：
1、本发明中的Serpin蛋白，为香菇来源的具有醒酒保肝作用蛋白，是首次报道具有醒酒保肝功能的香菇蛋白类物质，具有首创性。2、本技术获得的具有醒酒保肝活性作用蛋白为利用基因工程技术，采用大肠杆菌表达系统对目的蛋白进行体外重组表达，表达产物特异以及适用于大规模生产和发酵，能产生重要的经济价值和重大的社会意义。
本发明进行生物信息学分析，从基因水平上确定了蛋白的组成，以及进行了体外重组表达和纯化，确定了醒酒活性物质，研究了它的体内醒酒活性以及保肝作用。与现有技术中已知的一类丝氨酸蛋白酶抑制剂苯甲基磺酰氟用于防治药物性肝损伤，或者市面现有的合成类醒酒保肝药物相比，本发明主要目的是研究一种对酒精性肝损伤具有保护作用的丝氨酸蛋白酶抑制剂，由于生物合成具有安全，环保等特点，而且蛋白类物质更加能转变为多肽和氨基酸等营养物质，所以有着较广泛的应用价值和前景。本发明的研究成果将为改善饮酒引起的醉酒及酒精性疾病的预防提供新的研究思路和方法，为研发醒酒类的功能性产品提供一定的理论基础。
附图说明
图1是PCR基因鉴定和重组LeSPI的表达纯化；
图2是rLeSPI对(A)AST和(B)ALT活性的影响，将样品分为空白对照组、模型组(乙醇处理组)、HWJZ组、rLeSPI组。统计分析采用graphpad prism软件进行数据处理和分析，*p<0.05代表具有显著性差异。图中**代表p<0.01，***代表p<0.001，****代表p<0.0001，ns代表没有显著性差异。
图3是rLeSPI对GSH(A)、ALDH(B)、MDA(C)的影响，将样品分为空白对照组、模型组(乙醇处理组)、HWJZ组、rLeSPI组。统计分析采用graphpad prism软件进行数据处理和分析，*p<0.05代表具有显著性差异。图中**代表p<0.01，***代表p<0.001，****代表p<0.0001，ns代表没有显著性差异。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例对本发明进行进一步详细说明，各实施例及试验例中所用的设备和试剂如无特殊说明，均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
本发明披露了香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用，具体如下各实施例所示。
实施例1目标基因的合成和表达及目的蛋白的纯化
Lentinus edodes的丝氨酸蛋白酶抑制剂(LeSPI)基因序列进行了密码子优化后序列如下：AGCCTGGAAACCGGTCGTTATCTGATTCATAATGGCAATAACATTGTGAGCCGTAATCTGGCAGAAGATCGTAGCCTGAATCCGAAACGTATTGTTCTGCTGGAACCGACCGATAAAATTCAGCTGACCTGGATTATTGAGAAAAGCGGTGATGAATACATCCTGAATAATCGTGGTGCACCGACCGCACATATTGAAGATCATGTTTTTGCACTGCTGATCCATCAAGAGGGTGCAACCAAATGGTCAATTGAAGCAGTTCCGCGTCATGGTCGTAATGCCTATATCATTAAAGGTAGTGATGGTAAAGGTTGGGTTGCACCGGATAAAGCCGGTGAGCAGATTATCTATCGTACCCTGATTGTTGGTCCGAGCGAACCGCCTACCTTTCCGCTGAATCAGGTTTTTCAGATTATCAAACTGGAA(426bp)，见SEQ ID NO.1。
蛋白序列为：
SLETGRYLIHNGNNIVSRNLAEDRSLNPKRIVLLEPTDKIQLTWIIEKSGDEYILNNRGAPTAHIEDHVFALLIHQEGATKWSIEAVPRHGRNAYIIKGSDGKGWVAPDKAGEQIIYRTLIVGPS EPPTFPLNQVFQIIKLE(142aa)，见SEQ ID NO.2。
使用核苷酸合成仪(Biolytic Lab Performance，Inc)合成DNA序列，并插入到载体pET28a-JT(武汉华美生物工程有限公司)，转化到大肠杆菌Rosetta。通过PCR(引物为通用引物T7:5'-TAATACGACTCACTATAGGG-3'，见SEQ ID NO.3；T7t:5'-GCTAGTTATTGCTCAGCGG-3'，见SEQ ID NO.4)鉴定并筛选了阳性克隆。PCR体系为20μL体系，如下：
PCR程序为：
结果显示PCR产物的长度约为500bp，含有靶基因和部分载体序列。阳性克隆经过DNA测序证实了目的基因正确插入到载体pET28a-JT上，与设计的一致，见图1A，泳道1，PCR产物；泳道2，DNA分子量标记。基因翻译的蛋白质序列与Lentinula edodes中首次报道的丝氨酸蛋白酶抑制剂LeSPI(UniProtKB/Swiss-Prot：P81639.1)具有100％的一致性。
然后将该基因在大肠杆菌中进行诱导表达。本技术使用大肠杆菌Rosetta作为宿主菌株，在Luria-Bertani(LB)培养基中培养，添加卡那霉素(25μg/mL)。培养重组大肠杆菌菌株直至培养物达到OD600为0.6，并使用0.5mM IPTG诱导培养物3小时，然后通过离心收获细胞，在4℃下保持15分钟。将沉淀物重新悬浮于含有500mM NaCl的20mM Tris-HCl缓冲液(pH8.0)中。收集样品通过SDS-PAGE检测目的蛋白表达。结果表明，目标蛋白表达明显，计算分子量为20kDa，见图1，B1，泳道1和2分别为大肠杆菌的粗细胞提取物，IPTG诱导之前(泳道1)和诱导之后(泳道2)。
收集细菌液体的过量表达用于离心，倒入500mL离心管中，并以10000g离心3分钟。保留沉淀物，加入STET缓冲液并用DTT溶解至终浓度为5mM。将溶菌酶(1：200)加入上述样品中，混合并在冰中反应30分钟。然后使用Scientz JY92-IIN超声波仪在4℃(工作时间：3秒，间隙时间：4秒，操作99次，功率不超过200w)超声处理细胞破碎并离心(16,000g，4℃，30分钟)，再次重复16000g，在4℃下保持20分钟。收集上清液和沉淀物并制备样品用于SDS-PAGE以检查蛋白质表达。结果表明，rLeSPI蛋白在细胞内大量表达形成包涵体沉淀，见图1，B2，泳道1，细胞裂解物的不溶性部分，泳道2，细胞裂解物的可溶性部分。
由于靶蛋白表达形成不可溶的包涵体，因此需要进行复性。复性方法如下：1)将洗涤的包涵体用20mM NaHCO3，50mM NaCl，0.5mm EDTA，0.5％SKL(十二烷基肌氨酸钠)和5mMDTT溶解，然后在37℃振荡器中摇动约1小时。2)离心并保持上清液中加入0.2％PEG 4000,1mM氧化型谷胱甘肽和2mM还原型谷胱甘肽；3)将其放入透析袋中，在4℃下用10mM Tris-HCl，1mM EDTA处理72h。在此过程中更换透析液一次；4)再次离心16000g，20min，取上清液，用0.22微米过滤器过滤；5)复性后获得蛋白质并通过SDS-PAGE检测。结果表明纯化后能获得高纯度的目的蛋白，见图1，B3，泳道1，纯化的重组LeSPI，M，蛋白质分子量标记物。
实施例2小鼠体内醒酒活性测定
将雄性Wistar小鼠随机分成5组(每组10只大鼠)：1)对照组，用蒸馏水灌胃小鼠；2)阳性组，剂量依据产品说明书和动物药物标准用海王金尊(50×9.1mg/Kg)灌胃；3)rLeSPI-H组，用rLeSPI蛋白(5mg/kg)对小鼠进行灌胃；4)rLeSPI-M组，用rLeSPI蛋白(2mg/kg)对小鼠进行灌胃；5)rLeSPI-L组，用rLeSPI蛋白(0.5mg/kg)对小鼠进行灌胃。半小时后对每个试验剂量组灌胃饮用酒，然后将小鼠翻转放置在平板上，观察记录其翻正反射消失时间(即将其翻转放置，若背向下的姿势保持30S以上，则判为翻正反射消失，小鼠进入醉酒状态)和醉酒持续时间(即小鼠翻正反射消失到翻正反射恢复的时间)。
结果见表1，实验结果表明小鼠喝醉后会进入睡眠状态，有效的抗醉物质可以大大缩短睡眠时间。与生理盐水组相比，海王金樽组和rLeSPI-H组均能显著减少醉酒小鼠的数量(分别为4只和3只)，延长小鼠的醉酒时间，缩短清醒时间。两者的醉酒时间分别延长了58.18％和37.66％，两者的清醒时间分别缩短了37.41％和19.23％。结果表明，海王金尊组和rLeSPI-H组均具有显著的抗醉酒作用。因此，rLeSPI-H组在结论中具有最有效的抗醉酒效果。rLeSPI-L组也可以延长醉酒时间并缩短清醒时间，但它们并不比其他三组有效，这也表明抗酗酒效果与蛋白质剂量有正相关。
表1小鼠预防醉酒效果试验结果
实施例3香菇Serpin的肝脏保护作用研究
将小鼠随机分为4组(空白对照组，模型对照组，HWJZ组和rLeSPI组)，每组6只小鼠，按照实施例3的剂量灌胃相应的活性蛋白及阳性对照药物海王金樽，模型对照组灌胃蒸馏水，空白对照组未做处理。半小时后，按照小鼠醉酒剂量(56°二锅头，0.15ml/10g)灌胃饮用酒，1h后测定小鼠的AST/ALT、GSH、ALDH、MAD等肝损伤和保护作用的相关生化指标。各组分别取6只小鼠眼球取血处死，无菌取肝。将肝组织置于生理盐水中机械匀浆制成肝匀浆，按照试剂盒说明书分别测定肝组织中的肝损伤生物指标。采用试剂盒测定天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)在血清中的活性，以及肝脏中谷胱甘肽过氧化物酶(GSH)，乙醛脱氢酶、丙二醛(MDA)的含量。
结果见图2，显示模型组ALT、AST酶活性明显高于空白对照组(p<0.0001)，而rLeSPI组和HWJZ预处理组较模型组ALT、AST活性明显降低(p<0.01)。表明rLeSPI能有效抑制肝细胞损伤引起的ALT和AST活性升高，对乙醇诱导的肝损伤具有明显的保护作用。经酒精预处理的模型组GSH含量较空白对照组显著降低(p<0.0001)，rLeSPI组和HWJZ组较模型组GSH含量显著升高(p<0.01)，见图3A。经酒精预处理的模型组ALDH活性明显高于空白对照组(p<0.0001)，说明酒精可以激活ALDH，但rLeSPI和HWJZ组活性更强，较模型组显著提高了酒精代谢(p<0.01)，图3B。经酒精预处理后模型组MDA含量明显高于空白对照组(p<0.0001)，而rLeSPI组和HWJZ组较模型组MDA含量明显下降(p<0.01)，表明rLeSPI能够有效抑制肝细胞损伤引起的MDA含量升高，见图3。以上结果均表明rLeSPI对乙醇诱导的肝损伤具有明显的保护作用，见图3C。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 武汉工程大学
<120> 香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物及防治酒精性肝损伤药物中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 426
<212> DNA
<213> 丝氨酸蛋白酶抑制剂(serpin protease inhibitor)
<400> 1
agcctggaaa ccggtcgtta tctgattcat aatggcaata acattgtgag ccgtaatctg 60
gcagaagatc gtagcctgaa tccgaaacgt attgttctgc tggaaccgac cgataaaatt 120
cagctgacct ggattattga gaaaagcggt gatgaataca tcctgaataa tcgtggtgca 180
ccgaccgcac atattgaaga tcatgttttt gcactgctga tccatcaaga gggtgcaacc 240
aaatggtcaa ttgaagcagt tccgcgtcat ggtcgtaatg cctatatcat taaaggtagt 300
gatggtaaag gttgggttgc accggataaa gccggtgagc agattatcta tcgtaccctg 360
attgttggtc cgagcgaacc gcctaccttt ccgctgaatc aggtttttca gattatcaaa 420
ctggaa 426
<210> 2
<211> 142
<212> PRT
<213> 丝氨酸蛋白酶抑制剂(serpin protease inhibitor)
<400> 2
Ser Leu Glu Thr Gly Arg Tyr Leu Ile His Asn Gly Asn Asn Ile Val
1 5 10 15
Ser Arg Asn Leu Ala Glu Asp Arg Ser Leu Asn Pro Lys Arg Ile Val
20 25 30
Leu Leu Glu Pro Thr Asp Lys Ile Gln Leu Thr Trp Ile Ile Glu Lys
35 40 45
Ser Gly Asp Glu Tyr Ile Leu Asn Asn Arg Gly Ala Pro Thr Ala His
50 55 60
Ile Glu Asp His Val Phe Ala Leu Leu Ile His Gln Glu Gly Ala Thr
65 70 75 80
Lys Trp Ser Ile Glu Ala Val Pro Arg His Gly Arg Asn Ala Tyr Ile
85 90 95
Ile Lys Gly Ser Asp Gly Lys Gly Trp Val Ala Pro Asp Lys Ala Gly
100 105 110
Glu Gln Ile Ile Tyr Arg Thr Leu Ile Val Gly Pro Ser Glu Pro Pro
115 120 125
Thr Phe Pro Leu Asn Gln Val Phe Gln Ile Ile Lys Leu Glu
130 135 140
<210> 3
<211> 20
<212> DNA
<213> 人工序列(T7)
<400> 3
taatacgact cactataggg 20
<210> 4
<211> 19
<212> DNA
<213> 人工序列(T7t)
<400> 4
gctagttatt gctcagcgg 19
Claims (4)
1.香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物中的应用。
2.根据权利要求1所述的香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物中的应用，其特征在于：所述香菇丝氨酸蛋白酶抑制剂的核苷酸序列见SEQ ID NO.1。
3.根据权利要求2所述的香菇丝氨酸蛋白酶抑制剂在制备醒酒保肝药物中的应用，其特征在于：所述SEQ ID NO.1的核苷酸序列编码的蛋白序列见SEQ ID NO.2。
4.香菇丝氨酸蛋白酶抑制剂在制备防治酒精性肝损伤药物中的应用，所述香菇丝氨酸蛋白酶抑制剂的核苷酸序列见SEQ ID NO.1，蛋白序列见SEQ ID NO.2。
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| CN116474070A (zh) * | 2023-04-23 | 2023-07-25 | 江苏恰瑞生物科技有限公司 | 一种治疗急性酒精性肝损伤的血液灌流填料的制备方法 |
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| CN116474070A (zh) * | 2023-04-23 | 2023-07-25 | 江苏恰瑞生物科技有限公司 | 一种治疗急性酒精性肝损伤的血液灌流填料的制备方法 |
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