CN112553233A - 一种Tulp2多克隆抗体及其制备方法和应用 - Google Patents
一种Tulp2多克隆抗体及其制备方法和应用 Download PDFInfo
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- CN112553233A CN112553233A CN202011279339.1A CN202011279339A CN112553233A CN 112553233 A CN112553233 A CN 112553233A CN 202011279339 A CN202011279339 A CN 202011279339A CN 112553233 A CN112553233 A CN 112553233A
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- tulp2
- protein
- prokaryotic
- polyclonal antibody
- recombinant
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Abstract
本发明公开了一种重组表达载体、重组菌、Tulp2原核蛋白及其制备方法，本发明还公开了一种Tulp2多克隆抗体及其制备方法和应用。本发明还公开了Tulp2原核蛋白、Tulp2蛋白多克隆抗体在制备诊断或治疗男性不育症试剂或药物中的应用。该Tulp2多克隆抗体能特异性识别小鼠及人组织中的Tulp2蛋白，可应用于小鼠及人多种组织ELISA、Western Blot、组织免疫荧光等技术分析，具有特异性高，适用广泛等特点。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种Tulp2多克隆抗体及其制备方法和应用。
背景技术
Tulp（tubby-like protein，Tulp）家族是在渐进性肥胖Tubby突变小鼠中首先发现的蛋白，Tubby常染色体隐形遗传突变小鼠患有肥胖、听力缺失、视网膜变性、雄性不育、胰岛素抵抗等多种疾病，提示Tulp 家族成员具有多种功能。Tulp2是Tulp家族成员之一，Tulp2羧基端含有与其他成员相似的tubby结构域，氨基端具有IFT-A结合结构域，其基因敲除雄性小鼠不育，提示其在小鼠睾丸组织中发挥重要的作用。
为探索Tulp2的功能，需对其进行功能分析及作用机制研究。经检索，目前未发现有商品化的抗小鼠Tulp2抗体出售，同时，经文献检索，也未发现抗小鼠Tulp2的多克隆及单克隆抗体的任何文献报道。
发明内容
发明目的：本发明应用大肠杆菌原核蛋白表达系统进行Tulp2原核蛋白表达时发现，所获得的原核蛋白比预计蛋白分子量大，同时，在真核蛋白表达系统中获得的Tulp2真核蛋白也有类似的结果。因此，应用Tulp2的哪一种抗原进行动物免疫，进而获得特异性识别小鼠内源性Tulp2蛋白是本发明的决定性因素。为研究Tulp2的功能，阐明其在正常生理情况下的作用机制，研究其在精子发生过程中的具体作用，本发明首先应用大肠杆菌蛋白表达系统进行了小鼠Tulp2全长原核蛋白的表达和纯化，然后应用纯化的原核蛋白免疫新西兰大白兔，从而获得抗小鼠Tulp2多克隆抗体。在此基础上，应用制备的Tulp2多克隆抗体，对小鼠血液进行ELISA分析，对小鼠睾丸组织进行免疫荧光分析及Western Blot分析，结果发现，该抗体能够特异性识别小鼠睾丸组织中的Tulp2蛋白。本发明为研究Tulp2的功能提供了物质基础，同时也有可能为临床男性不育症的诊断和治疗提供新的靶标。
技术方案：为了解决上述问题，本发明的技术方案是提供了一种重组表达载体，所述重组表达质粒是将Tulp2基因插入到原核表达载体中得到重组表达载体。
其中，所述原核表达载体为大肠杆菌表达载体，具体的，所述大肠杆菌表达载体为pET-30a（+）。
本发明内容还包括一种重组菌，所述重组菌是将所述的重组表达载体导入宿主菌中，筛选得到重组菌。
其中，所述的宿主菌为大肠杆菌JM109或E.coli BL21。
本发明内容还包括一种Tulp2原核蛋白，所述Tulp2原核蛋白是将所述的重组菌进行诱导表达后获得。
其中，本发明所述的Tulp2原核蛋白分子量约90KD。
本发明内容还包括一种Tulp2原核蛋白的制备方法，包括以下步骤：
1）、Tulp2基因的获得；
2）、Tulp2原核表达载体构建：将Tulp2基因插入到原核表达载体中获得；
3）、重组菌的获得：将步骤2）得到的原核表达载体导入宿主菌中即得重组菌；
4）、Tulp2原核蛋白的诱导表达：将重组菌进行诱导即可得到Tulp2原核蛋白；
5）、Tulp2原核蛋白的纯化。
本发明内容还包括一种Tulp2蛋白多克隆抗体，将所述的Tulp2原核蛋白免疫动物即得，所述动物包括但不仅限于新西兰大白兔。
本发明内容还包括一种Tulp2蛋白多克隆抗体的制备方法，包括以下步骤：将所述的Tulp2原核蛋白多次免疫新西兰大白兔即得。
Tulp2蛋白多克隆抗体的制备方法具体包括以下步骤：首先，构建原核蛋白表达重组质粒pET-30a-Tulp2；然后在大肠杆菌原核表达系统中表达具有His标签的小鼠Tulp2原核蛋白；对大肠杆菌中的Tulp2原核蛋白进行提取及纯化；以纯化的全长小鼠Tulp2原核蛋白作为免疫原免疫新西兰大白兔；最后对制备的兔抗小鼠Tulp2多克隆抗体进行应用分析。
其中，所述免疫频率为每隔2周免疫1次，次数为5次，免疫剂量为200μg/只。
本发明的兔抗小鼠Tulp2多克隆抗体，其可应用于人及小鼠睾丸组织ELISA、Western Blot及免疫组织荧光分析。
本发明内容还包括所述的Tulp2原核蛋白、所述的Tulp2蛋白多克隆抗体在制备诊断或治疗男性不育症试剂或药物中的应用。
有益效果：本发明相对于现有技术，具有以下优点：
1、本发明提供的制备抗小鼠Tulp2多克隆抗体的制备方法。应用Tulp2全长编码阅读框蛋白进行原核蛋白的表达，该蛋白在大肠杆菌表达过程中可能进行了蛋白质翻译后修饰，获得的原核蛋白较预计蛋白（约62KD）大，约90KD，其纯化后纯度高。应用其作为免疫原进行新西兰大白兔免疫后，经过5次免疫过程，获得的抗小鼠Tulp2多克隆抗体效价达到1:105。这种抗体制备方法优于Tulp2短片段多肽作为免疫原后制备的多克隆抗体。
2、本发明提供的抗小鼠Tulp2多克隆抗体的应用范围较广，能特异性识别小鼠和人睾丸组织中的内源性Tulp2蛋白，且识别蛋白的大小与预计大小一致，为62KD左右，可用于Tulp2蛋白的ELISA、Western Blot及免疫组织荧光化学分析，更多的应用领域还有待验证。
3、本发明提供的这种多克隆抗体将为研究小鼠Tulp2的功能及作用机制提供有力的技术支持，为临床男性不育症的诊断和治疗提供新的靶标。
附图说明
图1、pET-30a-Tulp2重组质粒双酶切琼脂糖凝胶电泳结果；M：DNA marker；1~2：重组质粒双酶切电泳结果；
图2、SDS-PAGE分析Tulp2重组蛋白的诱导表达；M：蛋白质Mr标准；1：未诱导大肠杆菌；2~4：IPTG诱导后的大肠杆菌；
图3、SDS-PAGE分析不同浓度咪唑溶液洗脱Tulp2原核蛋白及纯化后蛋白；M：蛋白质Mr标准；1-2：纯化后蛋白；3：25 mmol/L咪唑洗脱液；4：50 mmol/L咪唑洗脱液；5：100mmol/L咪唑洗脱液；6：200 mmol/L咪唑洗脱液；
图4、ELISA检测Tulp2多克隆抗体的效价，1-4代表新西兰大白兔的编号；
图5、应用Tulp2多克隆抗体Western Blot分析对野生型（Tulp2+/+）和Tulp2基因敲除纯合子（Tulp2-/-）小鼠睾丸组织中内源性Tulp2蛋白表达情况；
图6、应用Tulp2多克隆抗体对野生型及Tulp2基因敲除纯合子小鼠睾丸组织进行免疫荧光组织化学染色法分析（Bar=100um）；
图7、pET-30a-Tulp2-C 重组质粒双酶切琼脂糖凝胶电泳结果；M：DNA marker；1：重组质粒双酶切鉴定结果；
图8、SDS-PAGE分析Tulp2-C重组蛋白诱导表达情况；M：蛋白质Mr标准；1，3：未诱导大肠杆菌；2，4：IPTG诱导后的大肠杆菌；
图9、SDS-PAGE分析不同浓度咪唑溶液洗脱Tulp2-C原核蛋白情况；M：蛋白质Mr标准；1-2：0 mmol/L咪唑洗脱液；3：25 mmol/L咪唑洗脱液；4：50 mmol/L咪唑洗脱液；5：100mmol/L咪唑洗脱液；6：200 mmol/L咪唑洗脱液；
图10、ELISA检测Tulp2-C多克隆抗体的效价，1-3代表新西兰大白兔的编号；
图11、应用Tulp2-C多克隆抗体Western Blot分析对野生型（Tulp2+/+）和Tulp2基因敲除纯合子（Tulp2-/-）小鼠睾丸组织中内源性Tulp2蛋白表达情况；
图12、应用Tulp2-C多克隆抗体对野生型及Tulp2基因敲除纯合子小鼠睾丸组织进行免疫荧光组织化学染色法分析（Bar=100um）。
具体实施方式
下面结合附图对本发明作更进一步的说明。
实施例1 原核蛋白表达重组质粒pET-30a-Tulp2的构建
应用Gene Runner软件设计Tulp2全长开放阅读框（NM_001045555）序列特异引物，序列为：前向引物：5’-GGTACCATGGACCGTGAGGGGCCAAGAG-3’，反向引物：5’-GAGCTCAAAAAACGCCAGTTTCCCATCG-3’。以Tulp2真核表达质粒pCDNA3.0-Tulp2（pCDNA3.0-Tulp2该质粒的制备方法：以小鼠睾丸组织cDNA为模板，逆转录PCR扩增获得Tulp2全长开放阅读框序列，然后将PCR产物插入真核表达质粒pCDNA3.0中，获得的重组质粒经测序证实插入片段无误）为模板，利用Tulp2特异性引物，进行PCR扩增。PCR反应条件为：95°C，5min；95°C，30s、58°C 45s、72°C 110s，共35个循环；72°C 10min，4°C保存PCR扩增产物。应用12g/L琼脂糖凝胶电泳分离PCR扩增产物并切胶纯化，利用T4-DNA连接酶将纯化的PCR产物与pGEM-T-Easy载体相连接，16°C过夜。将连接产物转化入大肠杆菌JM109感受态细胞中，与IPTG、X-gal一并涂布于氨苄青霉素抗性的LB固体培养板上。利用蓝白斑筛选原理，挑选白色单克隆菌落，摇菌过夜，提取质粒后，使用限制性内切酶EcoRI 37°C水浴过夜，酶切鉴定。用12g/L琼脂糖凝胶电泳检测酶切产物。将连接成功的pGEM-T-Easy-Tulp2重组质粒进行测序鉴定。
将pET-30a（+）质粒与pGEM-T-Easy-Tulp2质粒用限制性内切酶KpnI和SacI进行酶切，应用12g/L琼脂糖凝胶电泳分离并回收酶切产物，将回收的Tulp2目的片段、pET-30a（+）表达载体相连接，转化到大肠杆菌JM109感受态细胞中，涂布于卡那霉素抗性的LB固体平板上培养。挑选单克隆菌落，应用限制性内切酶KpnI和SacI双酶切进行鉴定。重组质粒双酶切鉴定结果如图1所示，泳道1-2酶切后可见两个片段，其中5422bp为线性载体片段，小片段为插入片段，与预计大小1686bp相一致，结果提示pET-30a-Tulp2重组质粒构建成功。
实施例2 Tulp2原核蛋白的表达与纯化
将pET-30a-Tulp2重组质粒转化入E.coli BL21感受态细胞中，在卡那霉素抗性的LB固体培养基上培养。挑选阳性单克隆菌落于5mL卡那霉素抗性的LB液中，37℃，225r/min，培养过夜。次日，将过夜菌液加入500mL卡那霉素抗性的LB液中，37℃，225r/min，培养3h。取1mL菌液于试管中，作为阴性对照，在其余菌液中加入诱导剂IPTG至终浓度为1mmol/L，37℃，225r/min，诱导4h。SDS-PAGE检测阴性对照、诱导后菌液，判断原核蛋白是否表达。
将诱导后表达蛋白的大肠杆菌离心收集菌体，加入60mL重悬缓冲液(50mmol/LTris-HCl、1mmol/L EDTA、250mL/L蔗糖、10mmol/L DTT，pH=8.0)充分重悬。重悬后加入4.2mL细菌裂解液(1mg/mL溶菌酶、5mmol/L MgCl2、0.05mg/mL DNaseI、10mL/L TritonX-100、10mmol/mL DTT)，4℃，磁力搅拌仪半速旋转裂解30min。将裂解后菌液置冰上间断超声3次，收集上清与沉淀，进行SDS-PAGE检测。将沉淀用15mL洗涤液(50mmol/L Tris-HCl、100mmol/L NaCl、1mmol/L EDTA、10mmol/mL DTT，pH=8.0)洗涤，最终溶于10mL 8mol/L尿素，4°C过夜。次日离心，收集上清。原核蛋白诱导及裂解结果如图2所示，图中可见，泳道1为未诱导大肠杆菌，泳道2为诱导后大肠杆菌，可见在约90KD处看到有Tulp2原核蛋白的表达，比预计蛋白（62KD）大。泳道3为含原核蛋白的大肠杆菌经超声后的上清，其中未见到Tulp2原核蛋白，泳道4为超声后沉淀，可见大量Tulp2原核蛋白。这些结果提示Tulp2原核蛋白以包涵体的形式存在于大肠杆菌沉淀中。诱导后表达的Tulp2原核蛋白比预计蛋白分子量大，可能是蛋白表达后进行了翻译后修饰或蛋白质折叠。
由于重组蛋白带有His标签，因此，使用能够吸附His标签的亲和层析柱纯化含有Tulp2重组蛋白的上清液。再利用咪唑与His标签相似的性质，从而可以竞争性洗脱目的蛋白的原理，分别用不同浓度的咪唑溶液进行洗柱。将上清液过Ni-NTA亲和层析柱，然后用25、50、100及200mmol/L咪唑分别洗脱层析柱，收集过柱洗脱液，SDS-PAGE检测纯化及洗脱效率。将纯化效率最高洗脱液装入处理好的透析袋中，置入7M、6M、5M、4M、3M及2M尿素溶液中进行梯度复性，收集复性后的蛋白溶液。Tulp2原核蛋白纯化结果如图3所示，泳道1-2为纯化后蛋白，3-6为25、50、100及200mmol/L咪唑洗脱过柱液，结果可见200mmol/L咪唑洗脱能够获得最大且纯度高（>90%）的原核蛋白。
实施例3 Tulp2多克隆抗体的制备
以复性后的Tulp2重组蛋白作为免疫原，免疫成年雄性新西兰大白兔。初次免疫时将复性后Tulp2原核与等体积福氏完全佐剂混合，冰上乳化，背部皮下多点注射免疫成年新西兰大白兔（~200μg/只）。此后，将福氏完全佐剂替换为不完全佐剂，每隔2周免疫1次，共5次。末次免疫后，取兔耳缘静脉血血清检测抗体效价。
实施例4 ELISA检测 Tulp2多克隆抗体效价
将纯化后的Tulp2原核蛋白稀释至8μg/mL，96孔酶标板每孔加入100μL进行包被，放入湿盒中，4°C过夜。次日，去除板内液体，用PBS-T溶液洗涤5min×3次。然后加入含1g/L脱脂奶粉的PBS-T溶液，37°C封闭1h。去除封闭液，扣干，用PBS-T溶液洗涤5min×3次。加入稀释比例为1：10~1：1000000的抗Tulp2血清和正常兔血清，37°C孵育90min。用PBS-T溶液洗涤5min×3次。每孔加入100μL HRP标记的山羊抗兔IgG（1：2000稀释），37°C孵育40min。去除二抗，用PBS-T溶液洗涤5min×3次。每孔加入100μL TMB显色液，室温避光反应10min，每孔加入100μL终止液终止反应。酶标仪测定波长490nm处各孔吸光度值。抗体效价检测结果如图4所示，1-4为新西兰大白兔的编号，由图可见，多克隆抗体的效价达1:106。
实施例5 应用Tulp2多克隆抗体进行睾丸组织Western Blot分析
取成年Tulp2基因敲除纯合子小鼠（南京医科大学祝辉课题组惠赠）及野生型小鼠（购自扬州大学比较医学中心）睾丸总蛋白与上样蛋白缓冲液混匀后，沸水浴10min，冰浴5min后，进行SDS-PAGE。电泳后，将蛋白电转至PVDF膜上，用含50 g/L脱脂奶粉TBS室温封闭2h。加Tulp2抗血清（1∶2000稀释），4℃孵育过夜。次日，TBST洗膜10 min×3次，加入HRP标记的山羊抗兔IgG（1:2000稀释），室温孵育1 h；TBST洗膜10 min×3次。用ECL显影试剂盒在Tanon-2500化学发光成像系统中显影。Western Blot实验结果如图5所示，结果可见在野生型小鼠睾丸组织中可见预计大小的内源性Tulp2蛋白条带，而在Tulp2基因敲除纯合子小鼠睾丸组织中未见Tulp2蛋白，而内参GAPDH在所有小鼠中均有相应的条带。这些实验结果表明，本发明获得的Tulp2多克隆抗体能够有效识别小鼠睾丸组织中的Tulp2蛋白，其蛋白大小与内源性蛋白大小相一致。
实施例6 应用Tulp2多克隆抗体进行睾丸组织免疫荧光分析
取成年野生型小鼠睾丸组织石蜡切片，常规脱蜡并水化。置于封闭液中(63mL甲醇、7mL双氧水)，37°C水浴10min，去除内源性过氧化物酶。PBST洗涤5min×3次，置于枸橼酸盐缓冲液(pH=6.0)中，微波炉高火2.5min，低火7.5min进行修复，室温自然冷却。PBST洗涤5min×3次，10g/L BSA室温封闭1h。加兔抗Tulp2多克隆抗体(1:250)，4°C孵育过夜。次日，切片置于37°C温箱复性30min。PBST洗涤5min×3次。加入AlexaFluor555标记的驴抗兔IgG(1∶1000稀释，赛默飞世尔科技(中国)有限公司)，室温避光孵育1h。PBST洗涤5min×3次。DAPI室温避光染核10min，PBST洗涤5min×3次。甘油封片，正置荧光显微镜下观察。睾丸组织免疫荧光染色实验结果如图6所示，图中可见，在野生型小鼠（Tulp2+/+）中Tulp2蛋白主要定位于生精小管圆形精子及其后的变形中精子细胞胞质中（图A-C)，在Tulp2基因敲除纯合子小鼠（Tulp2-/-）中未见Tulp2蛋白信号（图D-F），阴性对照组也未见非特异性信号（图G-I）。这些实验结果表明，本发明中的Tulp2多克隆抗体能有效识别小鼠内源性Tulp2蛋白，可用于睾丸组织免疫荧光分析。
对比例1 原核蛋白表达重组质粒pET-30a-Tulp2-C（Tulp2羧基端蛋白序列，以下简称Tulp2-C）的构建、蛋白的表达与纯化、多克隆抗体的指标以及应用
1、原核蛋白表达重组质粒pET-30a-Tulp2-C的构建
应用Gene Runner软件设计Tulp2-C特异引物，序列为：前向引物：5′-GGTACCATGACCTGCCCCCAGCCTC-3′，反向引物：5’-GAGCTCAAAAAACGCCAGTTTCCCATCG-3’。同样以pCDNA3.0-Tulp2为模板，利用上述特异性引物，进行PCR扩增。PCR反应条件为：95°C，5min；95°C，30s、58°C 45s、72°C 110s，共35个循环；72°C 10min，4°C保存PCR扩增产物。应用12g/L琼脂糖凝胶电泳分离PCR扩增产物并切胶纯化，利用T4-DNA连接酶将纯化的PCR产物与pGEM-T-Easy载体相连接，16°C过夜。将连接产物转化入大肠杆菌JM109感受态细胞中，与IPTG、X-gal一并涂布于氨苄青霉素抗性的LB固体培养板上。利用蓝白斑筛选原理，挑选白色单克隆菌落，摇菌过夜，提取质粒后，使用限制性内切酶EcoRI 37°C水浴过夜，酶切鉴定。用12g/L琼脂糖凝胶电泳检测酶切产物。将连接成功的pGEM-T-Easy-Tulp2-C重组质粒进行测序鉴定。
将pET-30a（+）质粒与pGEM-T-Easy-Tulp2-C质粒用限制性内切酶KpnI和SacI进行酶切，应用12g/L琼脂糖凝胶电泳分离并回收酶切产物，将回收的Tulp2-C目的片段、pET-30a（+）表达载体相连接，转化到大肠杆菌JM109感受态细胞中，涂布于卡那霉素抗性的LB固体平板上培养。挑选单克隆菌落，经限制性内切酶KpnI和SacI双酶切鉴定，鉴定结果参见图7，图中泳道1可见，重组质粒经双酶切后有两个片段，其中小片段为插入序列，与预计大小813bp相一致，提示pET-30a-Tulp2-C重组质粒构建成功。
2、Tulp2-C原核蛋白的表达与纯化
将pET-30a-Tulp2-C重组质粒转化入E.coli BL21感受态细胞中，在卡那霉素抗性的LB固体培养基上培养。挑选阳性单克隆菌落于5mL卡那霉素抗性的LB液中，37℃，225r/min，培养过夜。次日，将过夜菌液加入500mL卡那霉素抗性的LB液中，37℃，225r/min，培养3h。取1mL菌液于试管中，作为阴性对照，在其余菌液中加入诱导剂IPTG至终浓度为1mmol/L，37℃，225r/min，诱导4h。将诱导后表达蛋白的大肠杆菌离心收集菌体，加入60mL重悬缓冲液(50mmol/L Tris-HCl、1mmol/L EDTA、250mL/L蔗糖、10mmol/L DTT，pH=8.0)充分重悬。重悬后加入4.2mL细菌裂解液(1mg/mL溶菌酶、5mmol/L MgCl2、0.05mg/mL DNaseI、10mL/LTritonX-100、10mmol/mL DTT)，4℃，磁力搅拌仪半速旋转裂解30min。将裂解后菌液置冰上间断超声3次，收集上清与沉淀，进行SDS-PAGE检测。原核蛋白诱导及裂解结果如图8所示，图8中可见，泳道1为未诱导大肠杆菌，泳道2为诱导后大肠杆菌，可见在约37KD处看到有Tulp2-C原核蛋白的表达，与预计蛋白大小相一致。泳道3为含原核蛋白的大肠杆菌经超声后的上清，其中未见Tulp2-C原核蛋白，泳道4为超声后沉淀，可见大量主要Tulp2-C原核蛋白。这些结果提示Tulp2-C原核蛋白以包涵体的形式存在于大肠杆菌沉淀中。
将沉淀用15mL洗涤液(50mmol/L Tris-HCl、100mmol/L NaCl、1mmol/L EDTA、10mmol/mL DTT，pH=8.0)洗涤，最终溶于10mL 8mol/L尿素，4°C过夜。次日离心，收集上清；将含Tulp2-C原核蛋白的上清液过Ni-NTA亲和层析柱，然后分别用25、50、100及200mmol/L咪唑分别洗脱层析柱，收集过柱洗脱液，SDS-PAGE检测纯化及洗脱效率。将纯化效率最高洗脱液装入处理好的透析袋中，置入7M、6M、5M、4M、3M及2M尿素溶液中进行梯度复性，收集复性后的蛋白溶液。Tulp2-C原核蛋白纯化结果如图9所示，泳道1-2为0mmol/L咪唑洗脱过柱液，3-6为25、50、100及200mmol/L咪唑洗脱过柱液，结果可见200mmol/L咪唑洗脱能够获得最大且纯度高（>90%）的原核蛋白。
3、Tulp2-C多克隆抗体的制备
以复性后的Tulp2-C重组蛋白作为免疫原，免疫成年雄性新西兰大白兔。初次免疫时将复性后Tulp2-C原核与等体积福氏完全佐剂混合，冰上乳化，背部皮下多点注射免疫成年新西兰大白兔（~200μg/只）。此后，将福氏完全佐剂替换为不完全佐剂，每隔2周免疫1次，共5次。末次免疫后，取兔耳缘静脉血血清检测抗体效价。
4、ELISA检测 Tulp2-C多克隆抗体效价
将纯化后的Tulp2-C原核蛋白稀释至8μg/mL，96孔酶标板每孔加入100μL进行包被，放入湿盒中，4°C过夜。次日，去除板内液体，用PBS-T溶液洗涤5min×3次。然后加入含1g/L脱脂奶粉的PBS-T溶液，37°C封闭1h。去除封闭液，扣干，用PBS-T溶液洗涤5min×3次。加入稀释比例为1：10~1：1000000的抗Tulp2-C血清和正常兔血清，37°C孵育90min。用PBS-T溶液洗涤5min×3次。每孔加入100μL HRP标记的山羊抗兔IgG（1：2000稀释），37°C孵育40min。去除二抗，用PBS-T溶液洗涤5min×3次。每孔加入100μL TMB显色液，室温避光反应10min，每孔加入100μL终止液终止反应。酶标仪测定波长490nm处各孔吸光度值。抗体效价检测结果如图10所示，1-3为新西兰大白兔的编号，由图可见，多克隆抗体的效价达1:106。
5、应用Tulp2-C多克隆抗体进行睾丸组织Western Blot分析
取成年Tulp2基因敲除纯合子小鼠及野生型小鼠睾丸总蛋白与上样蛋白缓冲液混匀后，沸水浴10min，冰浴5 min后，进行SDS-PAGE。电泳后，将蛋白电转至PVDF膜上，用含50g/L脱脂奶粉TBS室温封闭2 h。加Tulp2-C抗血清（1∶2000稀释），4℃孵育过夜。次日，TBST洗膜10 min×3次，加入HRP标记的山羊抗兔IgG（1:2000稀释），室温孵育1 h；TBST洗膜10 min×3次。用ECL显影试剂盒在Tanon-2500化学发光成像系统中显影。Western Blot实验结果如图11所示，结果可见在野生型小鼠睾丸组织中可见预计大小的内源性Tulp2蛋白条带，但Tulp2基因敲除纯合子小鼠睾丸组织中也可以识别到相应大小的条带，提示，以Tulp2-C原核蛋白作为免疫原制备的多克隆抗体缺乏特异性。
6、应用Tulp2-C多克隆抗体进行睾丸组织免疫荧光分析
取成年野生型小鼠睾丸组织石蜡切片，常规脱蜡并水化。置于封闭液中(63mL甲醇、7mL双氧水)，37°C水浴10min，去除内源性过氧化物酶。PBST洗涤5min×3次，置于枸橼酸盐缓冲液(pH=6.0)中，微波炉高火2.5min，低火7.5min进行修复，室温自然冷却。PBST洗涤5min×3次，10g/L BSA室温封闭1h。加兔抗Tulp2-C多克隆抗体(1:250)，4°C孵育过夜。次日，切片置于37°C温箱复性30min。PBST洗涤5min×3次。加入AlexaFluor555标记的驴抗兔IgG(1∶1000稀释)，室温避光孵育1h。PBST洗涤5min×3次。DAPI室温避光染细胞核10min，PBST洗涤5min×3次。甘油封片，正置荧光显微镜下观察。睾丸组织免疫荧光染色实验结果如图12所示，图中可见，在野生型小鼠（Tulp2+/+）中Tulp2-C抗体未能看到明显的阳性信号，不能有效识别内源性Tulp2蛋白（图A-C)。在Tulp2基因敲除纯合子小鼠（Tulp2-/-）中有少量的非特异性信号（图D-F），而阴性对照组未见非特异性信号（图G-I）。这些结果表明，以Tulp2-C原核蛋白为免疫原制备的多克隆抗体不能用于小鼠睾丸组织免疫荧光分析。
因此，从以上实施例和对比例可以看出，同等条件下，本发明的多克隆抗体Tulp2优于应用小鼠Tulp2-C作为免疫原产生的多克隆抗体。对比例同样构建了Tulp2羧基端（Tulp2-C）原核蛋白表达重组质粒，并进行了原核蛋白的表达与纯化，得到了预计大小的Tulp2-C原核蛋白(约37KD)，并应用纯化后复性的Tulp2-C原核表达蛋白进行新西兰大白兔5次加强免疫，同样获得高滴度的抗血清，但是该种抗血清并不能识别小鼠睾丸组织中的内源性Tulp2蛋白，Western Blot 和免疫组织荧光技术分析都不能特异性地识别内源性Tulp2蛋白。从以上实施例和对比例可以看出，本发明在进行克隆表达时蛋白质可能发生了特殊的折叠或修饰使得其蛋白大小达到90kD，远远超过了理论值62KD，相比对比例中的同样条件制备的原核蛋白(约37KD)却并不能特异性识别内源性蛋白，本发明的该分子量更大的蛋白质（90KD）却能特异性识别内源性真核蛋白（62KD），这也是值得我们更加深入的研究和思考的。
序列表
<110> 扬州大学
<120> 一种Tulp2多克隆抗体及其制备方法和应用
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Claims (10)
1.一种重组表达载体，其特征在于，所述重组表达质粒是将Tulp2基因插入到原核表达载体中得到重组表达载体。
2.根据权利要求1所述的重组表达载体，其特征在于，所述原核表达载体为大肠杆菌表达载体。
3.一种重组菌，其特征在于，所述重组菌是将权利要求1或2所述的重组表达载体导入宿主菌中，筛选得到重组菌。
4.根据权利要求3所述的重组菌，其特征在于，所述的宿主菌为大肠杆菌JM109或E.coli BL21。
5.一种Tulp2原核蛋白，其特征在于，所述Tulp2原核蛋白是将权利要求3或4所述的重组菌进行诱导表达后获得。
6.一种Tulp2原核蛋白的制备方法，其特征在于，包括以下步骤：
1）、Tulp2基因的获得；
2）、Tulp2原核表达载体构建：将Tulp2基因插入到原核表达载体中获得；
3）、重组菌的获得：将步骤2）得到的原核表达载体导入宿主菌中即得重组菌；
4）、Tulp2原核蛋白的诱导表达：将重组菌进行诱导即可得到Tulp2原核蛋白；
5）、Tulp2原核蛋白的纯化。
7.一种Tulp2蛋白多克隆抗体，其特征在于，将权利要求5所述的Tulp2原核蛋白免疫动物即得。
8.一种Tulp2蛋白多克隆抗体的制备方法，其特征在于，包括以下步骤：将权利要求5所述的Tulp2原核蛋白多次免疫新西兰大白兔即得。
9.根据权利要求8所述的Tulp2蛋白多克隆抗体的制备方法，其特征在于，所述免疫频率为每隔2周免疫1次，次数为5次，免疫剂量为200μg/只。
10.权利要求5所述的Tulp2原核蛋白、权利要求7所述的Tulp2蛋白多克隆抗体在制备诊断或治疗男性不育症试剂或药物中的应用。
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