CN109957003B - 一种稳定的saa突变体及其在疾病检测中的应用 - Google Patents
一种稳定的saa突变体及其在疾病检测中的应用 Download PDFInfo
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Abstract
本发明公开了一种稳定的SAA突变体及其在疾病检测中的应用，包括将SAA蛋白进行定向突变，使第10位的半胱氨酸突变为赖氨酸，第26位的甘氨酸突变为脯氨酸，第50位的丝氨酸突变为缬氨酸，第66位的甘氨酸突变为脯氨酸，得到突变型蛋白的氨基酸序列如序列1所示。该突变型SAA1具有很高的稳定性，可应用于体外诊断试剂，提高了在临床疾病检测过程中的准确性。
Description
技术领域
本发明属于生物技术领域，具体而言，涉及一种与血清淀粉样蛋白(SAA)的稳定性显著关联的突变位点，以及突变型SAA蛋白及其在疾病检测中的应用。
背景技术
血清淀粉样蛋白A(serum amyloidA,SAA)是由活化的单核/巨噬细胞释放的细胞因子合成的载脂蛋白家族，为组织淀粉样蛋白A的前体物质，属急性时相反应蛋白(陈长强，顾志冬，樊绮诗.血清淀粉样蛋白A在疾病应用中的研究进展[J].检验医学,2012,27(9):776-779.)。它与C反应蛋白(CRP)一样是急性期的敏感标志物，炎症、感染性和非感染性疾病期间，在血液中的浓度能在数小时内急剧升高。已有研究表明，在细菌、真菌及病毒感染、动脉粥样硬化、心血管疾病、急性移植排斥反应、肿瘤等疾病中均可检测到血清SAA升高。尤其是当病毒感染、心血管疾病、移植排斥反应时，SAA的敏感性可高于CRP，对临床诊断有更好的参考价值。
目前，SAA的检测方法主要有以下几种：(1)酶联免疫吸附法，采用抗原与抗体的特异反应将待测物与酶连接，然后通过酶与底物产生颜色反应，用于定量测定。(2)胶体金免疫层析法，该方法操作简单，通常用于定性或者半定量的测定。(3)免疫比浊法，利用抗原抗体反应形成一定结构的免疫复合物，根据复合物在溶液中具有的特殊光学性质进行检测。(4)荧光免疫层析法，利用荧光物质在特定的激发光照射下发射荧光的特点，针对急性炎症时SAA含量进行检测，目前已在医学上进行推广，并且发展迅猛。无论采用何种检测方法，SAA的稳定性在诊断过程中起着非常重要的作用，稳定的SAA蛋白可提高检测过程中的准确度。
然而，野生型SAA在体外的稳定性较差，导致疾病诊断技术应用中的SAA使用期限短、诊断效率低，因此如何提高SAA的稳定性显得尤为重要。
发明内容
鉴于现有技术的不足，本发明的目的在于提供一种与SAA稳定性显著关联的突变位点，以及一种具有更高稳定性的SAA突变体及其编码基因，更加适应在医疗诊断以及科研中的应用。
为了实现本发明的技术目的，发明人通过大量试验摸索和研究，最终以野生型SAA的基因为模板，采用定点突变技术获得编码突变体的基因，随后将编码突变体的基因位点进行重组表达，获得了一种稳定性显著提高的SAA突变体。具体地，本发明的技术方案概况如下：
一种与血清淀粉样蛋白A的稳定性显著关联的突变位点，该突变位点位于野生型血清淀粉样蛋白A的10、26、50和66氨基酸处，所述位点是将第10位的半胱氨酸突变为赖氨酸,第26位的甘氨酸突变为脯氨酸，第50位的丝氨酸突变为缬氨酸，以及第66位的甘氨酸突变为脯氨酸，突变后血清淀粉样蛋白A的稳定性显著提高。
由于按上述突变位点突变后得到的突变体SAA1的稳定性显著提高，因此本发明还保护上述的突变位点在提高野生型血清淀粉样蛋白A的稳定性中的应用。
一种稳定性提高的血清淀粉样蛋白A突变体，所述突变体具有：
(1)SEQ ID NO.1所示的氨基酸序列；或
(2)在SEQ ID NO.1所示的氨基酸序列基础上缺失、替换、插入或/和添加一个至几个氨基酸的保守性突变而获得的具有同样稳定性的氨基酸序列。
需要说明的是，本发明所提供的血清淀粉样蛋白A突变体属于载脂蛋白家族中的一员。SEQ ID NO.1所示的氨基酸序列由104个氨基酸残基组成，主要是将血清淀粉样蛋白A的第10位的半胱氨酸突变为赖氨酸，第26位的甘氨酸突变为脯氨酸，第50位的丝氨酸突变为缬氨酸，第66位的甘氨酸突变为脯氨酸。所得突变体命名为SAA1。
一种血清淀粉样蛋白A突变体的编码基因，该基因编码具有SEQ ID NO.1所示的氨基酸序列的蛋白质。
本发明还提供一种重组载体，该重组载体携带上述的突变体SAA1的编码基因。所述重组载体为将上述编码基因插入出发载体pET-30a的多克隆位点得到的重组表达载体。可用现有的表达载体构建含有所述基因的重组表达载体。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型启动子或组成型启动子，它们可单独使用或与其它的启动子结合使用。此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。
本发明还提供一种重组细胞或重组菌，该重组细胞或重组菌包含上述的重组载体。例如，将上述任一所述编码基因插入出发载体pET-30a的多克隆位点得到的重组表达载体转化至大肠杆菌BL21-801，得到重组细胞或重组菌。
本发明还提供一种制备突变体SAA1的方法，该方法包括：构建含编码突变型SAA1的核苷酸序列的载体，利用含有突变位点信息的引物，对含有SAA基因的质粒进行扩增得到目的条带后利用同源重组得到重组质粒或直接转化得到突变质粒，测序验证习各步骤得到的载体转化宿主细胞得到重组细胞将载体转化到宿主细胞中，利用抗生素筛选得到正确转化的重组细胞；培养上述的重组细胞并由培养产物中收集突变体SAA1。
本发明还提供一种用于上述血清淀粉样蛋白A突变体的保存液，该保存液的组成为：pH＝7.4-9.0硼酸-硼砂缓冲液+0.9％NaCl+0.8-1.2mM EDTA+0.08-0.12％ProClin300。
本发明还提供上述稳定性提高的血清淀粉样蛋白A突变体在制备体外诊断试剂中的应用。进一步优选地，所述的体外诊断试剂用于诊断细菌、真菌或病毒感染、动脉粥样硬化、心血管疾病、急性移植排斥反应、肿瘤。细菌或病毒感染包括甲型和乙型肝炎、巨细胞病毒感染、varicellae——带状疱疹、传染性单核细胞增多症，甲型流感，细菌性肺炎，streptococ-卡尔咽炎细菌败血症和严重的细菌性败血症。
本发明测试结果显示与未突变的SAA相比，使用本发明改造的SAA1稳定性明显比其它公司购买的SAA高。
本发明的优点和有益效果：本发明对野生型SAA进行定向突变优化，使之适应在临床检测技术中的应用要求，得到突变型SAA1。该突变型SAA1不易降解，增加了SAA1在炎症等疾病的体外检测中的稳定性，降低了试剂的批间差，可应用SAA Serum amyloid AReagentkits(Nephelometry)(此试剂盒由南京立顶生物科技有限公司提供)。
附图说明
图1为目的蛋白SAA的SDS-PAGE检测纯化效果图。
图2为DNA切割酶经过酶切的突变型SAA1与表达载体Pet-30a。
具体实施方式
下面结合实施例和附图来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的，不对本发明的保护范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神下可以对技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
实施例1
构建含编码突变型SAA1的核苷酸序列的载体，利用基因合成的方法，合成引物序列如下：
购买于山东山东维真生物科技有限公司的SAA序列为模板，利用对人SAA1基因进行扩增。扩增分为4个反应，分别以为引物(由擎科生物合成)，在50uL反应体系中，10umoL的引物各加入2uL，扩增试剂用Master Mix(擎科)。扩增条件94℃-4min，94℃-30s，60℃-30s，72℃-30s，72℃-10min为一共40个循环。
扩增结束后，以1.5％琼脂糖凝胶电泳，得到目的条带后切胶回收。将回收产物与Pet-30a先利用DAN切割酶进行剪切半小时，再以1.5％琼脂糖凝胶电泳，电泳结果为图2，然后进行胶回收，再利用DAN连接酶进行重组反应，37℃恒温孵育半小时。
取10uL冷却反应液，加入到时100uL DH5@感受态细胞中，轻弹管壁数下混匀，在冰上放置0.5h后42℃热激90秒，冰水浴孵育2min。加入500ul的LB培养基，37℃孵育10min充分复苏。37℃摇菌1h。取100ul菌液均匀涂布在含有氨苄青霉素的平板上。将平板倒置，于37℃过夜培养。第二天挑取单克隆测序。测序结果表明本发明合成的突变型酶的核苷酸序列如序列表中序列2所示。
取测序验证无误的载体以同样转化方法转化到BL21-801感受态细胞中，涂布在含卡那青霉素的平板上，37℃过夜培养。第二天挑取单克隆。
培养并收集重组细胞，提取SAA1：挑取BL21-801-Pet30a-SAA1单菌落各一个，接种到5mL LB液体培养基中，37℃，200rpm震荡培养5h。将5mL LB培养的菌液接种到330mL LB培养基，共接2L，37℃，200rpm震荡培养至OD600至0.6-0.8时，加入50mg/ml的IPTG 79.2μL，28℃，100rpm摇床继续培养18h，。将菌体离心收集，10000rpm离心5min，去掉上清，加48mL非变性裂解液，加PSMF(1:50)960μL，加溶菌酶(1:100)480μL至1mg/ml，涡旋混匀后，4度摇床上放置30min，随后超声20min，4度10500rpm离心40min，取上清进行蛋白分离纯化。
树脂纯化SAA1蛋白：将Ni IDABeads装入合适的层析柱，用5倍体积的Lysisbuffer平衡树脂。将上述上清蛋白重悬树脂，放置摇床孵育1h，使得树脂充分与目的蛋白SAA1结合，再装入合适的层析柱过柱。用10倍体积的Wash buffer对树脂清洗，将非特异性杂蛋白清洗干净。用5倍体积的Elution buffer，收集洗脱液，既SAA1蛋白。用5倍体积的Elutionbuffer对树脂清洗，保证树脂的重复利用率。使用3倍体积Lysis buffer与5倍体积的ddH2O平衡填料，最后用5倍体积的20％乙醇平衡，然后等体积20％乙醇中4℃保存。将目的蛋白SAA1使用SDS-PAGE检测纯化效果如图1所示。对纯化蛋白进行浓度测定(BCA法测定)，SAA1浓度大于500mg/L。
SAA1保存液：pH＝9(硼酸-硼砂缓冲液)+0.9％NaCl+1mM EDTA+0.1％Pc300；
对纯化蛋白进行透析处理：透析袋(透过分子量3.5kD)，透析液为200mM的硼酸-硼砂缓冲液，透析液与洗脱的SAA1蛋白液比例为1：10，4℃透析6h换一次透析液，共透析12h。
SAA1稳定性测试(胶乳增强免疫比浊)：
用免疫比浊的方法测定对比SAA的稳定性，测得SAA1的稳定性明显比其它公司的稳定性要高。
测试方法：增强免疫比浊法。
测试仪器：半自动特定蛋白分析仪
测定原理：抗原与抗体特异性结合
测定步骤：
1.测试前试剂盒中的R1(缓冲稀释液)与R2(胶乳抗血清)平衡至室温后再进行测试。
2.将突变SAA1和未突变SAA以及购买的申基生物公司的SAA和北京春雷杰创SAA按适当比例(40倍，80倍稀释)。
3.取出测量杯，先加入缓冲液R1400ul，然后将2u1抗原稀释液加入测量杯中，放入仪器上的测量通道后10s加入抗血清R2，同时按指示键。
4.仪器将自动开始检测，测试完毕后，自动显示吸光度结果。
5.对每个的第一次测试的吸光度记为0d的试验结果，之后放值37℃培养箱进行加速，之后按操作依次测试第1d，3d，5d，7d，11d，15d的吸光度进行分析。
数据分析：由表1的测试结果可以看出，突变SAA1的吸光度从0-15d是几乎不变，40倍稀释与80倍稀释的CV≤1％，未突变SAA的CV≤15％，而购买的SAA在加速条件下极度不稳定，CV≥20％，并且稀释倍数越高越不稳定。
表1：SAA稳定性测试结果
序列表
<110> 南京立顶生物科技有限公司
<120> 一种稳定的SAA突变体及其在疾病检测中的应用
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Claims (8)
1.一种提高野生型血清淀粉样蛋白A的稳定性的方法，其特征在于，该方法包括将野生型血清淀粉样蛋白A第10位的半胱氨酸突变为赖氨酸,第26位的甘氨酸突变为脯氨酸，第50位的丝氨酸突变为缬氨酸，以及第66位的甘氨酸突变为脯氨酸，得到氨基酸序列如SEQ IDNO.1所示的血清淀粉样蛋白A突变体。
2.一种稳定性提高的血清淀粉样蛋白A突变体，其特征在于，所述突变体的氨基酸序列如SEQ ID NO.1所示。
3.一种血清淀粉样蛋白A突变体的编码基因，其特征在于，该基因编码SEQ ID NO.1所示的氨基酸序列的蛋白质。
4.一种携带权利要求3所述编码基因的载体或重组细胞。
5.根据权利要求4所述的载体或重组细胞，其特征在于，所述重组细胞所使用的宿主细胞为BL21-801。
6.权利要求2所述稳定性提高的血清淀粉样蛋白A突变体在制备体外诊断试剂中的应用。
7.根据权利要求6所述的应用，其特征在于，所述的体外诊断试剂用于诊断：（1）细菌、真菌或病毒感染；或（2）心血管疾病；或（3）急性移植排斥反应；或（4）肿瘤。
8.根据权利要求7所述的应用，其特征在于，所述的心血管疾病为动脉粥样硬化。
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