CN103901209B - 一种重组蛋白ie1包被酶标反应板的制备方法及定量检测人血浆hcmv中和抗体试剂盒 - Google Patents
一种重组蛋白ie1包被酶标反应板的制备方法及定量检测人血浆hcmv中和抗体试剂盒 Download PDFInfo
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Abstract
本发明公开了一种重组蛋白IE1包被酶标反应板的制备方法及定量检测人血浆HCMV中和抗体试剂盒，涉及一种新的重组抗原IE1包被酶标反应板的制法，以及在其基础上制备ELISA定量检测人血浆HCMV中和抗体试剂盒。所述方法包括用基因工程方法制备特异性重组HCMV？IE1抗原、蛋白纯化、酶标反应板包被以及定量检测人血浆HCMV中和抗体ELISA试剂盒的组装。与其它市售ELISA试剂盒相比，本发明能够高度特异、敏感、快速地检测血浆中具有中和作用的HCMV特异性IgG抗体滴度，检测的结果与微量中和试验的结果高度一致。本发明主要供实验室研究、临床筛查和血制品企业筛选高滴度HCMV静脉注射丙种球蛋白生产原料使用。
Description
技术领域
本发明涉及基因工程技术、诊断试剂和血液制品研发领域，特别是涉及一种重组蛋白IE1包被酶标反应板的制备方法及定量检测人血浆HCMV中和抗体试剂盒。
背景技术
人巨细胞病毒(humancytomegalovirus，HCMV)属于人类疱疹病毒β亚科病毒，在人群中的感染极为普遍，呈世界性分布，血清流行病学调查表明，40～100%成人有HCMV抗体。目前的研究表明HCMV是人类先天性感染最常见的病原体之一，对孕妇及胎儿影响巨大，常造成流产、死胎、胎儿畸形、发育迟缓及迟发性中枢神经系统缺陷等疾病。
孕妇在孕期（尤其是妊娠早期）因激素水平的急剧变化、心理和免疫水平等改变，使原发性感染机会增加，激活潜伏的HCMV、或感染新的HCMV毒株产生再发感染，病毒经胎盘传给胎儿，引起胎儿先天性感染。先天性HCMV感染是引起出生缺陷最常见和最重要的病毒病因，主要表现为神经损伤如引起小脑畸形、室周钙化、脑水肿、小眼畸形等。非遗传性感音神经听觉丧失（sensorineuralhearingloss,SNHL）是先天性HCMV感染最常见的后遗症表现。研究发现，发达国家先天性HCMV感染率比发展中国家低，如意大利为0.47%(CI:0.22–1.0%)；瑞典为0.46%(CI:0.37–0.58%)；而冈比亚则高达13.6%。在美国，每年约有40000新生儿发生先天性HCMV感染，其中10%～15%发生迟发性后遗症，主要为SNHL、智力障碍、学习能力低下等神经系统损伤性疾病，其中因SNHL每年付出高达10亿美元的医疗帐单。2011年，我国出生人口约1615万，按照发达国家先天性HCMV感染率来计算，我国每年约有16.2万例先天性HCMV感染，家庭和国家都将为此支出高昂的成本。根据目前的临床资料显示，实际上先天性HCMV感染的人数可能是16.2万人/年的2～3倍。此外，HCMV感染也是器官移植失败和艾滋病病人并发感染死亡的主要原因之一。
鉴于HCMV对人类的巨大危害，人们一直在寻找更好的治疗方法。最常用的抗病毒药物为更昔洛韦，但是由于长期使用目前已经出现了很多耐药毒株，尤其是在移植患者和艾滋病患者中更为常见。具有高中和滴度的特异性HCMVIgG已被证明具有良好的治疗作用，而且不产生耐药性。因此，筛选含高HCMV中和滴度的血浆，制备静脉丙种球蛋白制剂，将有助于对HCMV感染的治疗。HCMV虽然在人群中的感染率很高，HCMV特异性IgG的含量也不低，但是体外病毒中和实验证明只有约5%的高滴度血浆才具中和病毒的能力。目前虽有部分用于检测人血浆中HCMVIgG滴度的ELISA试剂盒，但其结果与病毒中和实验的结果不一致。因此，用这些试剂盒筛选的高滴度血浆并不能用于特异性静脉丙种球蛋白制剂生产。这与ELISA试剂盒选用的包被抗原有关，HCMV感染机体主要由pp150蛋白诱导机体产生体液免疫，因此市售的ELISA试剂盒多采用pp150蛋白作为包被抗原，但针对pp150蛋白的抗原并不具有中和病毒的能力。IE1基因是HCMV的立即早期基因，控制病毒DNA的复制，它在病毒感染2h即开始表达，体外实验显示IE1的单克隆抗体可以阻断HCMV感染易感细胞。鉴于此，我们选取了编码IE1基因的部分序列基因(1006～1521nt)，采用基因重组技术，原核表达，亲和层析后获得一种融合IE1重组蛋白。经过改造过的IE1重组蛋白既保留了其抗原性，又提高原核表达量。采用该重组蛋白包被ELISA板，我们成功地用间接ELISA法，快速筛查出中和抗体含量更高的人血浆，其结果与中和实验结果一致率大于96.0％
发明内容
本发明目的就是为了弥补已有技术的缺陷，提供一种重组蛋白IE1包被酶标反应板的制备方法及定量检测人血浆HCMV中和抗体试剂盒。
本发明是通过以下技术方案实现的：
一种重组蛋白IE1包被酶标反应板的制备方法，包括以下步骤：
（1）将IE1蛋白抗原用pH为9-10的碳酸盐缓冲液稀释后包被96孔板，100ng/孔，置湿盒中4℃，22-24小时，PBST洗板3次，每次2-4min，然后拍干；
（2）每孔加入100μl含1%BSA和10%小牛血清的PBS，37℃孵育1h，洗板3次后密封，即得所述的重组蛋白IE1包被酶标反应板。
一种重组蛋白IE1包被酶标反应板的制备方法，所述的重组蛋白IE1的氨基酸残基序列如SEQNo.1所示。
一种重组蛋白IE1包被酶标反应板的制备方法，所述的重组蛋白IE1的DNA核苷酸序列如SEQNo.2所示。
一种ELISA定量检测人血浆HCMV中和抗体试剂盒，所述的试剂盒使用的酶标反应板为权利要求1所述的重组蛋白IE1包被酶标反应板。
本发明中所述的IE1重组蛋白的氨基酸序列（SEQNo.1）：
MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQLSLGDPLFPELAEESLKTFEQVTEDCNENPEKDVLAELVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMGGCLQNALDILDKVHEPFEEMKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDELRRKMMYMCYRNIEFFTKNSAFPKTTNGCSQAMAALQNLPQCSPDEIMAYAQKIFKILDEERDKVLTHIDHIFMDILTTCVETMCNEYKVTSDACMMTMYGGISLLSEFCRVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSVDDLRAIAEESDEEEAIVAYTLATAGVSSSDSLVSPPESPVPATIPLSSVIVAENSDQEESEQSDEEEEEGAQEEREDTVSVKSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ
本发明中所述的IE1重组蛋白的DNA核苷酸序列（SEQNo.2）：
TTACTGGTCAGCCTTGCTTCTAGTCACCATAGGGTGGGTGCTCTTGCCTCCAGAGGCGGTGGGTTCCTCAGCACCATCCTCCTCTTCCTCTGGGGCAACTTCCTCTATCTCAGACACTGGCTCAGACTTGACAGACACAGTGTCCTCCCGCTCCTCCTGAGCACCCTCCTCCTCTTCCTCATCACTCTGCTCACTTTCTTCCTGATCACTGTTCTCAGCCACAATTACTGAGGACAGAGGGATAGTCGCGGGTACAGGGGACTCTGGGGGTGACACCAGAGAATCAGAGGAGCTGACACCAGCGGTGGCCAAAGTGTAGGCTACAATAGCCTCTTCCTCATCTGACTCCTCGGCGATGGCCCGTAGGTCATCCACACTAGGAGAGCAGACTCTCAGAGGATCGGCCCCCAGAATGTACTGGGCAAAGACCTTCATGCAGATCTCCTCAATGCGGCGCTTCATTACACTGATAACCTCAGGCTTGGTTATCAGAGGCCGCTTGGCCAGCATCACACTAGTCTCCTCTAAGACATAGCAGCACAGCACCCGACAGAACTCACTTAAGAGAGAGATGCCCCCGTACATGGTCATCATACAAGCGTCACTAGTGACCTTGTACTCATTACACATTGTTTCCACACATGTAGTGAGGATATCCATAAATATGTGATCAATGTGCGTGAGCACCTTGTCTCTCTCCTCATCCAAAATCTTAAATATTTTCTGGGCATAAGCCATAATCTCATCAGGGGAGCACTGAGGCAAGTTCTGCAGTGCCGCCATGGCCTGACTGCAGCCATTGGTGGTCTTAGGGAAGGCTGAGTTCTTGGTAAAGAACTCTATATTCCTGTAGCACATATACATCATCTTTCTCCTAAGTTCATCCTTTTTAGCACGGGCCTTAGCCTGCAGTGCACCCCCCAACTTGTTAGCGGCGCCCTTGCTCACATCATGCAGCTCCTTAATACAAGCCATCCACATCTCCCGCTTATCCTCAGGTACAATGTAGTTCTCATACATGCTCTGCATAGTTAGCCCAATACACTTCATCTCCTCGAAAGGCTCATGAACCTTATCTAAGATATCTAAGGCATTCTGCAAACATCCTCCCATCATATTAAAGGCGCCAGTGAATTTCTCTTCCGTCTGGGTATATTTTTTCAGCATGTGCTCCTTGATTCTATGCCGCACCATGTCCACTCGAACCTTAATCTGTTTGACTGTAGAGGAGGATAACAACACATATAAGTATCCGTCCTCCTGACTCATTTATCGCTATCTCGATGCCCCGCTCACATGCAAGAGTTAATCTTTACTCTATCTGACATACACAAGTAAATCCACGTCCCATGCAGGTTAGTATACATCACATACATGTCAACAGACTTACCGAGTTCTGCCAGGACATCTTTCTCGGGGTTCTCGTTGCAATCCTCGGTCACTTGTTCAAAAGTTTTGAGGGATTCTTCGGCCAACTCTGGAAACAGCGGGTCTCCCAGACTCAGCTGACTGTTAACCTCCTTCCTCAACATAGTCTGCAGGAACGTCGTGGCCTTGGTCACGGGTGTCTCGGGCCTAAACACATGAGAAATAGAGTCATAAGCACATGGGTCACATACAGGAGATATGTATATAACATTAATACAATTTTATTAAAAAAAAAGGGGGGGCACAAACCCCGACACGTACCGTGGCACCTTGGAGGAAGGGCCCTCGTCAGGATTATCAGGGTCCATCTTTCTCTTGGCAGAGGACTCCATCGTGTCAAGGACGGTGACTGCAGAAAAGACCCATGGAAAG
本发明的优点是：
本发明可以准确、快速筛查出中和抗体含量更高的人血浆，便于血液制品企业原料筛选。
本法筛选得到的高滴度血浆与微量中和实验得到的结果一致性达到96%，可进行规模化、标准化操作，更便利、经济。
附图说明
图1表达IE1重组蛋白的大肠杆菌经IPTG诱导后菌体蛋白经SDS-PAGE蛋白电泳鉴定；泳道1为不含表达载体的BL21(DE3)空菌对照；泳道2为含表达载体的BL21(DE3)；泳道3为纯化后的IE1重组蛋白；
图2为IE1重组蛋白的westernblot鉴定；泳道1为含表达载体的BL21(DE3)；泳道2为不含表达载体的BL21(DE3)空菌对照；
图3为市售某品牌ELISA试剂盒和微量中和试验检测相同标本结果的一致性较低（r2=0.7171）；
图4为本发明ELISA试剂盒和微量中和试验检测相同标本结果的一致性较高（r2=0.9670）。
具体实施方式
实施例1
一、IE1重组蛋白制备
1.重组前准备
根据Genebank中已公布的HCMV基因序列，确定IE1基因编码序列，设计用于扩增IE1基因1、2外显子的PCR引物。
2.目的DNA片段的RT-PCR获取
从HCMVAD169株病毒培养物中提取RNA作为模板，通过逆转录形成cDNA，再通过PCR扩增出不含内含子的IE1基因。
3.重组DNA片段插入表达载体
将pET32a载体和目的DNA片段经过双酶切，酶切产物纯化后经T4DNA连接酶连接，连接产物转化转化大肠杆菌BL21(DE3)感受态，涂布与含Amp的LB平板上37℃导致培养过夜，次日挑取平板上的单菌落进行菌落PCR和测序鉴定。
4.重组IE1蛋白的诱导表达及纯化
将筛选出的菌株，接种到含Amp的液体LB培养基中，培养至OD值为0.5左右，加入IPTG诱导表达5h，收集菌体经超声破碎、镍柱和离子交换柱层析得到纯化的重组IE1蛋白。
5.重组IE1蛋白的Westernblot验证
为验证重组IE1蛋白的抗原性，我们用商品化的HCMVIE1单抗，对重组IE1蛋白进行Westernblot检测。以感染HCMV的人胚肺成纤维细胞作为阳性对照，检测结果见图1。
二、IE1重组蛋白酶标反应板制备
1.棋盘滴定法选择包被抗原最适含量
（1）用包被液将抗原作一系列稀释后进行包被，洗涤。
（2）将强阳性参考血清、弱阳性参考血清和阴性参考血清用稀释液按10倍递增系列稀释，加样，保温，洗涤。
（3）加按最适滴度稀释的酶标抗人IgG单抗，保温，洗涤。
（4）加底物显色，加酸终止反应后读取OD450值。
（5）选择强阳性参考血清的OD450值为0.8～1.0间、阴性参考血清的A值小于0.1的包被抗原的稀释度作为最适滴度为100g/孔。
2.IE1重组蛋白酶标反应板包被
（1）将IE1重组蛋白作为抗原，用pH9.6的碳酸盐缓冲液稀释成适宜浓度后按100ng/孔包被96孔酶标板，置湿盒中4℃24小时，PBST洗板3次，每次3min，然后拍干。（2）用含1%BSA和10%小牛血清的PBS100μl/孔，37℃孵育，封闭1h，洗板3次后密封；该板即为IE1包被酶标反应板。
三、ELISA试剂盒的装配及应用
1.酶标二抗最适滴度的选择
（1）用100ng/ml人IgG进行包被，洗涤。
（2）将山羊抗人IgG-HRP用稀释液作一系列稀释后分别加入已包被的孔中，保温、洗涤。
（3）加底物显色。加酸终止反应后，读取吸光度OD450值，取OD450值在1.0时的酶标抗体稀释度，得到作为酶标二抗的最适滴度为1：1000。
2.试剂盒的组装
所述试剂盒由IE1包被酶标反应板、阳性对照血清、标本稀释液、洗涤液、显色液、终止液构成，具体组分为：
（1）用购自德国实验的标准HCMVIgG阳性血清通过微量中和实验标定滴度为50AU的人血浆
（2）标本稀释液：含10％小牛血清和血浆显色剂的0.01MpH7.4的磷酸盐缓冲液（PBS）；
（3）洗涤液：含有0.05％Tween20的磷酸盐缓冲液（PBST）；
（4）酶标二抗：辣根过氧化物酶标记的山羊抗人IgG单抗；
（5）显色液：TMB-H2O2尿素溶液；
（6）终止液：2mol/LH2SO4溶液。
3.利用上述试剂盒未知血浆样品中HCMV中和抗体滴度：
（1）待检血清，加入1：100稀释的待检血清到包被好的酶标反应板，100μl/
孔，将阳性对照做1:10、1:20、1:40和1:80稀释，每孔加入100μl，37℃孵
育1h，洗板3次；
（2）加酶标二抗，加入工作浓度的山羊抗人IgG-HRP，100μl/孔，37℃孵育30min，（3）显色，洗板后每孔加入TMB-H2O2系统底物100μl，37℃或室温避光显色
15分钟；
（4）每孔加入2mol/LH2SO450μl，终止反应；
（5）结果判断，空白孔调零，读取450nm波长处吸光度，即OD450值。
（6）计算结果，用阳性标本的稀释倍数和OD450值制作标准曲线，将不同血浆标本的测值代入标准曲线，计算中和抗体滴度，如果测得OD450值在不同稀释度阳性对照OD450值范围之外，则调整标本稀释比例后重新测定。
Claims (3)
1.一种ELISA定量检测人血浆HCMV中和抗体试剂盒，其特征在于，试剂盒使用的酶标反应板为重组蛋白IE1包被酶标反应板；
所述的重组蛋白IE1包被酶标反应板的制备方法，包括以下步骤：
（1）将IE1蛋白抗原用pH为9-10的碳酸盐缓冲液稀释后包被96孔板，100ng/孔，置湿盒中4℃，22-24小时，PBST洗板3次，每次2-4min，然后拍干；
（2）每孔加入100μl含1%BSA和10%小牛血清的PBS，37℃孵育1h，洗板3次后密封，即得；
所述试剂盒由IE1包被酶标反应板、阳性对照血清、标本稀释液、洗涤液、酶标二抗、显色液、终止液构成。
2.根据权利要求1所述的ELISA定量检测人血浆HCMV中和抗体试剂盒，其特征在于，所述的重组蛋白IE1的氨基酸残基序列如SEQNo.1所示。
3.根据权利要求1所述的ELISA定量检测人血浆HCMV中和抗体试剂盒，其特征在于，所述的重组蛋白IE1的DNA核苷酸序列如SEQNo.2所示。
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