CN106380516B - 一种特异性结合bvd病毒非结构蛋白ns5b的纳米抗体及其应用 - Google Patents
一种特异性结合bvd病毒非结构蛋白ns5b的纳米抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种特异性结合BVD病毒非结构蛋白NS5B的纳米抗体及其应用，属抗病毒研究技术领域。本发明通过构建NS5B特异的单域重链抗体文库，筛选抗NS5B特异性纳米抗体，构建稳定表达NS5B纳米抗体的MDBK细胞系并用BVDV感染，公开了特异性结合BVD病毒非结构蛋白NS5B纳米抗体和其氨基酸序列，以及编码该纳米抗体的基因序列，还公开了NS5B纳米抗体在制备抗BVD病毒药物中的应用。
Description
技术领域
本发明属于抗病毒研究技术领域，具体涉及一种特异性结合BVD病毒非结构蛋白NS5B的纳米抗体及其在抗BVD病毒中的应用。
背景技术
牛病毒性腹泻（Bovine viral diarrhea，BVD）是由牛病毒性腹泻病毒（BVDV）引起的一种病毒性传染病。BVDV是一种单股正链RNA病毒，全基因组由5’非翻译区、ORF和3’非编码区组成。其中，BVDV的ORF起始于5’端386位核苷酸，编码一个3988个氨基酸的前体多聚蛋白，该蛋白在宿主细胞和蛋白酶的共同作用下，经过翻译、加工后形成至少11个成熟的蛋白，包括4中结构蛋白（C、E0、E1、E2）和7种非结构蛋白（Npro、P7、NS2-3、NS4A、NS4B、NS5A、NS5B）。NS5B蛋白又称P75，是BVDV ORF编码的最后一个蛋白，由718个氨基酸残基组成，具有依赖RNA聚合酶活性，在病毒RNA的复制方面发挥着非常重要的作用。
牛病毒性腹泻病毒感染牛并引发以出现多症状、持续性感染和免疫抑制为主要特征的疾病，对养牛业造成重大损失和极大危害。对该疾病的防制主要采用疫苗和抗生素，在安全性、治疗效果等方面都不能对最终控制该疾病起到决定性的作用。随着分子生物学的发展和应用，科学家们致力于通过基因工程技术研究治疗牛病毒性腹泻的新途径。
1993 年，比利时科学家Hamers 等人在骆驼血液中抗体，偶然发现存在一种天然缺失轻链的抗体，将其称为重链抗体，更让人吃惊的是，这些天然缺失轻链的重链抗体像普通抗体一样能与抗原等靶位特异性结合。这种抗体有一个重链可变区和两个常规的CH2与CH3区，更重要是单独克隆并表达出来的VHH区具有很好的结构稳定性与抗原结合活性，VHH是目前己知的可特异性结合抗原的最小单位，所以VHH也称纳米抗体（Nanobody Nb）。纳米抗体有来源于成年骆驼体内重链抗体的最小的功能性抗原结合片段，具有高稳定性和与抗原结合的高亲和力，能与蛋白裂隙和酶活性位点的相互作用，使之作用类似于抑制剂。由于仅有重链，纳米抗体的制造也较为容易。纳米抗体结构简单，仅有重链的可变区，这决定其水溶性好、稳定性强、亲和力高等优点，纳米抗体的独特性质，如处于极端温度和pH环境中的稳定性，可以低成本制造大产量。因此，纳米抗体在疾病的预防、诊断和治疗等领域得到了广泛的应用。
发明内容
本发明所要解决的技术问题是提供一种针对BVDV非结构蛋白NS5B并可以抑制BVD病毒增殖的纳米抗体，同时提供该纳米抗体的编码序列。
本发明提供一种特异性结合BVD病毒非结构蛋白NS5B纳米抗体，所述纳米抗体的VHH链包括框架区FR和互补决定区CDR，其中，所述框架区FR选自下组的FR1~FR4的氨基酸序列：FR1如SEQ ID NO:1所示，FR2如SEQ ID NO:2所示，FR3如SEQ ID NO:3所示，FR4如SEQ IDNO:4所示；所述互补决定区CDR选自下组的CDR1~CDR3的氨基酸序列：CDR1如SEQ ID NO:5所示，CDR2如SEQ ID NO:6所示，CDR3如SEQ ID NO:7所示。
本发明提供一种DNA分子，所述DNA分子编码特异性结合BVD病毒非结构蛋白NS5B的纳米抗体，所述的DNA 分子具有SEQ ID NO:9所示的核苷酸序列。
本发明提供了所述纳米抗体在制备抗BVD病毒药物中的应用。
本发明提供了所述DNA分子在开发抗BVD转基因牛中的应用。
有益效果：本发明通过噬菌体展示技术筛选到了抗BVDV非结构蛋白NS5B的特异性纳米抗体，此纳米抗体可以特异性结合BVD病毒非结构蛋白NS5B，具有抑制BVD病毒增值的功能，该纳米抗体可以为开发治疗BVD病毒的药物提供依据，其基因序列可应用于检测牛病毒性腹泻疾病，为公共卫生安全提供保障。
附图说明
图1是VHH基因电泳图；其中泳道1是PCR扩增重链抗体可变区基因片段，泳道M是DNA分子量标准。
图2是ELISA鉴定NS5B纳米抗体结果图。
图3是Western Blot检测MDBK细胞中表达的纳米抗体-eGFP融合蛋白的结果图。
图4是细胞生长曲线图。
图5是BVDV接种MDBK细胞经不同时间培养后上清中子代病毒滴度结果图。
具体实施方式
下面通过具体的实施例对本发明所述一种特异性结合BVD病毒非结构蛋白NS5B的纳米抗体做进一步的解释说明。
实施例1 BVD病毒非结构蛋白 NS5B 纳米抗体文库的构建
取5mL浓度为1mg/mL的NS5B重组蛋白，与弗氏佐剂等体积混合并乳化均匀，免疫一只阿拉善双峰驼，每两周1次，共免疫6次，其中，第一次免疫使用弗氏完全佐剂，其余5次免疫全部使用弗式不完全佐剂。当第 6 次免疫结束后4天，分离骆驼外周血淋巴细胞并提取总RNA，提取步骤参照RNA 提取试剂盒说明书。按照Invitrogen SuperScript® III 第一链合成系统试剂盒说明书，将提取的RNA 反转录成cDNA 并利用巢式PCR 扩增VHH 链，第一轮PCR ：
上游引物：GTCCTGGCTGCTCTTCTACAAGG
下游引物：GGTACGTGCTGTTGAACTGTTCC
扩增重链抗体信号肽和抗体CH2之间的片段，55℃退火，28个循环；回收700bp 附近目的条带。
第二轮PCR：
以第一轮 PCR 回收产物为模板，
上游引物：CAGGTGCAGCTGCAGGAGTCTGGGGGAGR
下游引物：CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
扩增重链抗体可变区（VHH）基因，55℃ 退火，18个循环，回收目的片段，结果如图1显示，目的条带约为500bp。
使用pCANTAB 5E 噬菌体展示载体20μg、PCR回收产物8μg分别使用限制性内切酶PstI 、NotI 双酶切，并用T4 DNA 连接酶连接两个片段。将连接产物电转化至电转感受态细胞TG1中，活化后涂布于LB-AMP琼脂平板，37℃过夜培养，收集菌苔-80℃保存。与此同时，通过菌落PCR 检测所建文库的阳性率，并测定库容大小及多样性，检测结果阳性率为98%，库容大小为4.2×108，具有较好的多样性。
取50μL -80℃保存的甘油菌，接种于100mL LB-AMP培养基，待细菌生长至对数期，用20 MOI（感染复数）的 M13KO7辅助噬菌体感染TG1细胞，过夜培养后纯化噬菌体，得到骆驼纳米抗体噬菌体展示基因库。
实施例2 NS5B 纳米抗体的筛选
将纯化的 NS5B 重组蛋白用 PBS 缓冲液稀释至浓度为4μg/mL，包被96 孔酶标板，每孔100μL，同时选取一个孔直接加入 PBS 缓冲液作为无抗原对照孔，4℃包被过夜。用2%脱脂奶粉封闭 ，每孔200 µL，25℃孵育 2 h。用2%的脱脂奶粉稀释0代浓缩产物至5×1010pfu/mL，以每孔100 μL将其加入96孔酶标版中，25℃孵育 2 h。用PBST溶液洗涤8次，洗去不结合的噬菌体，再加入新鲜配制的 0.1 mol/L的三乙胺，每孔100 µL，洗脱与NS5B特异性结合的噬菌体。将噬菌体感染处于对数期生长的大肠杆菌TG1，生产并纯化噬菌体用于下一轮的筛选。经过3轮筛选，富集阳性克隆。
实施例3 噬菌体ELISA鉴定单个阳性克隆
经过3轮筛选后，将感染噬菌体的TG1细胞按一定稀释比例涂布于LB-AMP琼脂平板，随机挑取121个单克隆进行测序分析共筛选出8株不同的纳米抗体并进行亲和力鉴定。接种于LB-AMP培养基中生长至对数期后，加入终浓度1 mmol/L的异丙基-β-D-硫代半乳糖苷（IPTG），37℃培养过夜；收集菌体，-20℃冻融一次，上清中含有纳米抗体片段；取100μL上清加入包被NS5B的ELISA板中，同时分别取100μL上清加入对照蛋白Nsp4包被的和未包被的ELISA板孔中，室温放置1小时；用PBST溶液洗3次，加入兔抗 E-tag 多克隆抗体室温放置1小时；用PBST溶液洗涤3次，加入HRP 标记的鼠抗 M13 噬菌体单克隆抗体，在室温下放置1小时；用PBST溶液 洗涤3次，加入TMB显色底物，读取OD值。最终得到抗NS5B特异性纳米抗体，ELISA结果如图2所示，氨基酸序列为SEQ ID NO:8 所示。
实施例4 稳定表达NS5B纳米抗体的MDBK细胞系的建立
（1）以pCANTAB 5E—VHH 质粒为模板扩增 VHH 基因，将回收的 PCR 产物和pTrip-CMV-Nb41EGFP-Puro 载体分别用Xba I和EcoR I进行双酶切，并用T4DNA 连接酶连接，得到pTrip-CMV-Nb1EGFP-Puro慢病毒真核表达载体。
（2）用0.9μg的psPAX2、0.5μg 的pMD2.G和0.6μg 的pTrip-CMV-NbxEGFP-Puro三质粒共转染293T细胞，包装慢病。
（3）将包装好的慢病毒转导MDBK细胞，利用嘌呤霉素筛选和有限稀释法筛选稳定表达纳米抗体的MDBK细胞系。
（4）Western-Blot检测纳米抗体-eGFP融合蛋白的表达，结果见图3显示，在40kDa附近出现特异性的目的条带，说明融合蛋白表达。
（5）为了检测纳米抗体在细胞内表达是否具有细胞毒性，将正常 MDBK 细胞和MDBK-NbsEGFP 细胞系分别接种于 96 孔细胞培养板，每孔约1 × 103 个细胞，每天用胰酶消化收集细胞并计数，绘制细胞增殖曲线，结果显示这株纳米抗体在胞内的表达并不影响MDBK 细胞的增殖，如图4所示，表明胞内表达的纳米抗体没有细胞毒性，将这株纳米抗体命名为MDBK-Nb1eGFP。
实施例5 病毒感染实验
将MDBK细胞和MDBK-Nb1eEGFP细胞铺在24孔细胞培养板，培养至细胞汇合度达到60-80%，接种0.1MOI的BVDV。分别感染24hpi、36hpi、48hpi后收集细胞上清，检测子代病毒滴度（TCID50），结果如图5所示，MDBK-Nb1eEGFP这株纳米抗体对子代病毒显著抑制，表明纳米抗体Nb1具有抑制BVD病毒增值的功能，为抗BVD病毒药物的研发制备提供了新的思路。
实施例中所用材料来源：
pCANTAB 5E噬菌粒载体购自GE公司；辅助噬菌体 M13KO7 购自 NEB 公司；E.coliTG1购自碧云天公司；HRP 标记的鼠抗 M13 噬菌体单克隆抗体购自 Sino Biological 公司；兔抗 E-tag 多克隆抗体购自南京金斯瑞公司；限制性内切酶PstI 和NotI、 T4 DNA 连接酶购自NEB公司；嘌呤霉素购自晨诺生物科技有限公司；RNA 提取试剂 RNAiso Plus、反转录试剂盒购自 Takara公司；
BVDV VEDEVAC 毒株由中国兽药监察所提供；MDBK细胞由西北农林科技大学兽医免疫生物学实验室保存；成年雄性阿拉善双峰驼购自甘肃民勤。
以上所述的实施方案应理解为说明性的，而非限制本发明的保护范围，本发明的保护范围以权利要求书为准。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对本发明作出的一些非本质的改进和调整仍属于本发明的保护范围。
SEQUENCE LISTING
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<120> 一种特异性结合BVD病毒非结构蛋白NS5B的纳米抗体及其应用
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Claims (5)
1.一种特异性结合BVD病毒非结构蛋白NS5B的纳米抗体，所述纳米抗体的VHH链包括框架区FR和互补决定区CDR，其特征在于：所述框架区FR1~FR4的氨基酸序列分别为：FR1如SEQID NO:1所示，FR2如SEQ ID NO:2所示，FR3如SEQ ID NO:3所示，FR4如SEQ ID NO:4所示；所述互补决定区CDR1~CDR3的氨基酸序列分别为：CDR1如SEQ ID NO:5所示，CDR2如SEQ IDNO:6所示，CDR3如SEQ ID NO:7所示。
2.一种DNA分子，其特征在于，所述DNA分子编码权利要求1所述的纳米抗体。
3.如权利要求2所述的一种DNA分子，其特征在于，具有SEQ ID NO:9所示的核苷酸序列。
4.根据权利要求1所述的纳米抗体在制备用于治疗和预防BVD药物中的应用。
5.根据权利要求2或3所述的DNA分子在开发抗BVD转基因牛中的应用。
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| BVDV纳米抗体的筛选、表达及抗病毒效果的研究;张国奇;《中国优秀硕士学位论文全文数据库》;20160415;全文 * |
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