CN114675040B - 一种检测胰岛素受体胞内段抗体的试剂盒 - Google Patents
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Abstract
本发明公开了一种检测胰岛素受体胞内段抗体的试剂盒，属于生物医药技术领域。所述试剂盒包括胰岛素受体蛋白胞内段表达质粒、TNT SP6快速转录翻译试剂、S35‑蛋氨酸、TBST缓冲液、蛋白A琼脂糖和蛋白G琼脂糖。本发明基于对胰岛素受体胞内段质粒的设计，在现有的放射配体检测法基础上，首次建立了胰岛素受体胞内段抗体的检测方法，实现了对胰岛素受体蛋白胞内段抗体的检测。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种检测胰岛素受体胞内段抗体的试剂盒。
背景技术
B型胰岛素抵抗综合征(Type B insulin resistance syndrome，TBIRS)是由于体内产生针对胰岛素受体的自身抗体所致的一种临床综合征，严重的高血糖和胰岛素抵抗是该病的主要临床特征。B型胰岛素抵抗综合征的诊断主要依靠临床和免疫两个方面，免疫诊断即检测胰岛素受体抗体。胰岛素受体抗体阳性是确诊的标准。由于实验条件限制，胰岛素受体抗体的检测尚未在临床广泛开展，尚无成熟的试剂盒，所以目前多数病例研究仍依靠临床诊断。
胰岛素受体抗体的检测在过去缺少一个高效、准确的方法。较早出现的一种检测方法是通过采用胰岛素的竞争性结合实验来间接的推测胰岛素受体抗体的存在，此方法较为耗时，且只适用于少量样品的分析，目前只能在世界范围内的少数实验室中进行。2008年出现了使用S35标记的胰岛素受体与待测样本结合(Ping Zhou et al.2008)，通过免疫沉淀法进行胰岛素受体抗体的检测，但该方法使用了胰岛素受体的全长蛋白进行标记，而胰岛素受体蛋白作为一个跨膜蛋白，具有疏水性，在翻译过程中会不可避免的出现聚集，影响翻译的效率与实验的稳定，此后未见进一步的相关报道。
发明内容
本发明的目的是提供一种检测胰岛素受体胞内段抗体的试剂盒，通过构建胰岛素受体蛋白胞内段表达质粒，该质粒不仅可以稳定转录翻译出胰岛素受体蛋白胞内段，还可携带足够的放射信号，实现对胰岛素受体蛋白胞内段抗体的检测。
为了实现上述目的，本发明采用以下技术方案：
一种检测胰岛素受体胞内段抗体的试剂盒，包括胰岛素受体蛋白胞内段表达质粒、TNT SP6快速转录翻译试剂、S35-蛋氨酸、TBST缓冲液、蛋白A琼脂糖和蛋白G琼脂糖；
所述胰岛素受体蛋白胞内段表达质粒是将序列XhoI+kozak+ATG+目的序列+终止密码子+XbaI导入载体pTnTTMVector 84-107位点得到；
所述目的序列的核苷酸序列如SEQ ID NO.1所示。
本发明综合考虑胰岛素受体蛋白跨膜的结构特点，首次将胰岛素受体拆解为两部分，一部分为胞外段，一部分为胞内段，该拆解有效的解决了过往检测中因跨膜区域不溶于水带来的蛋白质聚集等不良影响，也对针对不同位点的抗体进行了有效区分。
本发明构建的质粒不仅可以稳定转录翻译出胰岛素受体蛋白胞内段，亦可以携带足够的放射信号，从而用于制备针对胰岛素受体蛋白胞内段抗体INRbA的检测试剂盒。
附图说明
图1为载体pTnTTMVector的模式图。
图2为不同模拟样本添加量下INRbA检测的放射读数。
图3为健康人群RBA INRbA检测的结果。
图4为INRbA分别在非T1DM、T1DM及健康人群中的分布结果。
具体实施方式
胰岛素受体抗体的检测对临床诊断B型胰岛素抵抗综合征具有极高的参考意义，鉴于过去的检测方法的低效以及不稳定，本发明在申请人自有的S35放射配体法检测平台上，评估了胰岛素受体蛋白的整体结构，单独拆分出胰岛素受体蛋白胞内段结构域，避开跨膜的疏水区域后，其具有完全的亲水性，优化密码子后插入载体，构建出全新的质粒。构建出的全新质粒不仅可以稳定转录翻译出胰岛素受体蛋白胞内段，亦可以携带足够的放射信号，从而设计得到首个针对胰岛素受体蛋白胞内段抗体(INRbA)的检测试剂盒。
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
一、实验材料
1.样本来源：胰岛素受体胞内段抗体(INRbA)检测阳性质控样本来自于购买的针对胰岛素受体胞内段蛋白(INRb)的商品化抗体。阴性质控血清标本取自于无糖尿病家族史的健康志愿者。糖尿病(DM)血清504例，其中1型糖尿病(T1DM 195例)、非1型糖尿病(非T1DM309例)。健康人319例来自招募人群[年龄(29.7±5.9)岁；男150例，女169例]；糖耐量检测(OGTT)空腹及2h血糖正常，排除心、脑、肝、肾等慢性及内分泌疾病，无糖尿病家族病史及自身免疫疾病史。所有研究对象均签署知情同意。
2.主要试剂及仪器：TNT SP6快速转录翻译试剂盒(L2080，Promega)；S35-蛋氨酸(NEG709A 5mCi，PerkinElmer)；蛋白A琼脂糖PA(17-5280-02，GE)；蛋白G琼脂糖PG(17061805，GE)；96孔PVDF平板(3504，Corning)；Microscint-20闪烁液(6013621，PerkinElmer)；TBST缓冲液(Tris-Base 2.424g，NaCl 8.70g，Tween-20 1.5mL，BSA 1.0g加蒸馏水定容至1000mL，pH7.4)；βCounter液体闪烁计数仪(2450Microplate Counter，Perkin-Elmer)；蛋白纯化柱NAP Column(17-0853-02，GE)；INRbA(ab227831，abcam)；载体pTnTTMVector(L5610，Promega)。
二、实验方法
1.构建INRb质粒
a.按照哺乳蛋白表达体系进行目的序列密码子优化(避开两个酶切位点)。
b.将载体pTnTTMVector(L5610，Promega)84-107位点替换为设计好的序列：XhoI(ctcgag)+kozak序列(gccacc)+ATG+目的序列+终止密码子+XbaI。
c.载体模式图如图1所示。
目的序列(SEQ ID NO.1)如下：
agaaagaggcagccagatgggccgctgggaccgctttacgcttcttcaaaccctgagtatctcagtgccagtgatgtgtttccatgctctgtgtacgtgccggacgagtgggaggtgtctcgagagaagatcaccctccttcgagagctggggcagggctccttcggcatggtgtatgagggcaatgccagggacatcatcaagggtgaggcagagacccgcgtggcggtgaagacggtcaacgagtcagccagtctccgagagcggattgagttcctcaatgaggcctcggtcatgaagggcttcacctgccatcacgtggtgcgcctcctgggagtggtgtccaagggccagcccacgctggtggtgatggagctgatggctcacggagacctgaagagctacctccgttctctgcggccagaggctgagaataatcctggccgccctccccctacccttcaagagatgattcagatggcggcagagattgctgacgggatggcctacctgaacgccaagaagtttgtgcatcgggacctggcagcgagaaactgcatggtcgcccatgattttactgtcaaaattggagactttggaatgaccagagacatctatgaaacggattactaccggaaagggggcaagggtctgctccctgtacggtggatggcaccggagtccctgaaggatggggtcttcaccacttcttctgacatgtggtcctttggcgtggtcctttgggaaatcaccagcttggcagaacagccttaccaaggcctgtctaatgaacaggtgttgaaatttgtcatggatggagggtatctggatcaacccgacaactgtccagagagagtcactgacctcatgcgcatgtgctggcaattcaaccccaagatgaggccaaccttcctggagattgtcaacctgctcaaggacgacctgcaccccagctttccagaggtgtcgttcttccacagcgaggagaacaaggctcccgagagtgaggagctggagatggagtttgaggacatggagaatgtgcccctggaccgttcctcgcactgtcagagggaggaggcggggggccgggatggagggtcctcgctgggtttcaagcggagctacgaggaacacatcccttacacacacatgaacggaggcaagaaaaacgggcggattctgaccttgcctcggtccaatccttcc。
2.快速转录翻译质粒，获得放射性标记的抗原
S35-蛋氨酸和TNT SP6混合液解冻后置于冰上，依次加入40μL TNT混合液、1μL(1μg/μL)构建的质粒和5μL S35-蛋氨酸，用4μL无核酸酶水补充至总反应体系为50μL，充分混匀后置于30℃水浴箱孵育90min，随后取出置于冰上准备过NAP-5柱。
取出1只NAP-5柱置于试管架上，将其上下盖子均打开，弃去平衡液后加入1mL的TBST缓冲液平衡NAP-5柱，洗脱3次。将反应混合物小心加在NAP-5柱填充物表面，再用100μLTBST缓冲液清洗反应管后仍将其加入NAP-5柱中，待红色液体缓慢下移至柱2/3后，加入500μL TBST缓冲液，仔细观察柱下液滴的颜色变化，收集红色过柱液约500μL，从中取出2μL过柱抗原于闪烁管中与1mL闪烁液混匀，置96孔βCounter闪烁计数仪上计数其每分钟脉冲数(countsperminute，CPM)值。
3.待测样本与抗原的结合与检测
每孔各加8μL样本血清或模拟样本，每个标本和质控血清均为双复孔，取适量标记抗原，用6mL TBST缓冲液稀释标记抗原至20000CPM/60μL，向其中每孔加60μL稀释后的标记抗原，每孔CPM值要求≥20000，标记抗原与血清混匀振荡1小时，4℃冰箱中过夜。孵育PVDF板，150μL TBST/孔，4℃冰箱中过夜。次日，倒去PVDF板中的液体，每孔中加25μL的蛋白A/G混合琼脂糖(62.5％的PA与20％的PG按体积比4:1配制)，依次从96孔平板各孔中取出50μL混合液转移至96孔PVDF过滤板上，4℃冰箱中混匀1h沉淀抗原-抗体复合物后取出，真空泵抽吸去液体，先向PVDF过滤板各孔中加200μL TBST缓冲液洗涤沉淀物，真空泵抽吸去液体留取沉淀物，再加150μL缓冲液重复洗涤7次，置于烘箱烘干后每孔加入60μL闪烁液，置96孔βCounter计数仪上计数，每孔计数1min。
按下式计算结果：
放射指数(Index)＝(标本血清CPM－阴性质控CPM)/(阳性质控CPM－阴性质控CPM)。
所有数据均采用SPASS26软件进行统计，对所有计量资料符合正态分布的以用均数±标准差
表示，组间比较采用t检验、单因素方差分析、方差分析趋势性检验。P<0.05为差异显著，具有统计学意义。
三、实验结果
1.INRb抗原蛋白携带放射性信号捕获INRbA的有效性判断
转录翻译INRb质粒，获得带有放射性信号的胰岛素受体胞内段蛋白INRb作为抗原，将购买的INRbA作为模拟样本，使用INRb抗原进行捕获，根据上述实验方法，检测不同模拟样本量下的放射读数。
如图2所示，放射性读数随着模拟样本加样量的递减而递减，经过方差分析趋势性检验P<0.0001，趋势性极显著。因此该INRb抗原可有效捕获INRbA，其携带的放射信号随着抗体含量的变化而变化。
后续实验中使用购买的抗体作为阳参，根据信噪比(S/N)>10(S/N：不同INRbA添加量的CPM值/阴参的CPM值)以及成本综合考虑，选用0.5μg抗体，CPM约1900作为阳参的加样量，如表1。
表1不同INRbA添加量下信噪比(S/N)
2.RBA检测INRbA的正常人界值判定
取160例健康人血清进行RBA INRbA的测定，计算放射指数，取99％百分位点为界值，经计算阳性界值为0.036，阳性判断标准为≥0.036，如图3所示。
3.RBA检测INRbA的批内批间差异
根据INRbA指数低、中、高在正常人和患者中选择3份血清在批内和批间各重复检测5次(n＝5)，批内和批间变异系数(coefficient of variance，CV)见表2。结果显示RBAINRbA检测指数的批内CV为1.88％～9.51％，批间CV为5.56％～13.15％，阴阳性结果判断重复性100％。
表2 RBA INRbA检测的批内批间差异
4.INRbA在不同人群中的分布
检测INRbA分别在T1DM、非T1DM及健康人群中的百分比，分别为3.08％(6/195)、6.80％(21/309)及0.63％(1/159)，经单因素方差分析P<0.01，差异极显著。如图4所示。
根据以上结果可知，本发明构建的质粒可有效携带放射性信号，该质粒转录得到的抗原经标记后可有效识别INRbA，批内批间CV均在有效范围内，有效填补了INRbA检测的空白。
序列表
<110> 江苏省人民医院（南京医科大学第一附属医院）
<120> 一种检测胰岛素受体胞内段抗体的试剂盒
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1209
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agaaagaggc agccagatgg gccgctggga ccgctttacg cttcttcaaa ccctgagtat 60
ctcagtgcca gtgatgtgtt tccatgctct gtgtacgtgc cggacgagtg ggaggtgtct 120
cgagagaaga tcaccctcct tcgagagctg gggcagggct ccttcggcat ggtgtatgag 180
ggcaatgcca gggacatcat caagggtgag gcagagaccc gcgtggcggt gaagacggtc 240
aacgagtcag ccagtctccg agagcggatt gagttcctca atgaggcctc ggtcatgaag 300
ggcttcacct gccatcacgt ggtgcgcctc ctgggagtgg tgtccaaggg ccagcccacg 360
ctggtggtga tggagctgat ggctcacgga gacctgaaga gctacctccg ttctctgcgg 420
ccagaggctg agaataatcc tggccgccct ccccctaccc ttcaagagat gattcagatg 480
gcggcagaga ttgctgacgg gatggcctac ctgaacgcca agaagtttgt gcatcgggac 540
ctggcagcga gaaactgcat ggtcgcccat gattttactg tcaaaattgg agactttgga 600
atgaccagag acatctatga aacggattac taccggaaag ggggcaaggg tctgctccct 660
gtacggtgga tggcaccgga gtccctgaag gatggggtct tcaccacttc ttctgacatg 720
tggtcctttg gcgtggtcct ttgggaaatc accagcttgg cagaacagcc ttaccaaggc 780
ctgtctaatg aacaggtgtt gaaatttgtc atggatggag ggtatctgga tcaacccgac 840
aactgtccag agagagtcac tgacctcatg cgcatgtgct ggcaattcaa ccccaagatg 900
aggccaacct tcctggagat tgtcaacctg ctcaaggacg acctgcaccc cagctttcca 960
gaggtgtcgt tcttccacag cgaggagaac aaggctcccg agagtgagga gctggagatg 1020
gagtttgagg acatggagaa tgtgcccctg gaccgttcct cgcactgtca gagggaggag 1080
gcggggggcc gggatggagg gtcctcgctg ggtttcaagc ggagctacga ggaacacatc 1140
ccttacacac acatgaacgg aggcaagaaa aacgggcgga ttctgacctt gcctcggtcc 1200
aatccttcc 1209
Claims (4)
1.一种检测胰岛素受体胞内段抗体的试剂盒，其特征在于：包括胰岛素受体蛋白胞内段表达质粒、TNT SP6快速转录翻译试剂、S35-蛋氨酸、TBST缓冲液、蛋白A琼脂糖和蛋白G琼脂糖；
所述胰岛素受体蛋白胞内段表达质粒是将目的序列导入载体pTnT™ Vector得到，具体是将载体pTnTTMVector的84-107位点替换为设计好的序列：XhoI ctcgag+kozak序列gccacc+ATG+目的序列+终止密码子+XbaI，所述目的序列的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的试剂盒，其特征在于：还包括阳性对照和阴性对照。
3.根据权利要求2所述的试剂盒，其特征在于：所述阴性对照为健康人血清。
4.权利要求1所述的试剂盒在制备B型胰岛素抵抗综合征诊断试剂中的应用。
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| CN104126122A (zh) * | 2012-02-24 | 2014-10-29 | 鲁兹·舒姆伯格 | 与胰岛素受体族反应抗体结合性能调节剂之识别 |
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