CN114644714B - 一种非洲猪瘟病毒重组融合蛋白cpe、制备及其应用 - Google Patents
一种非洲猪瘟病毒重组融合蛋白cpe、制备及其应用 Download PDFInfo
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
本发明提供了一种非洲猪瘟病毒重组融合蛋白CPE、制备及其应用，属于生物技术制药领域，所述重组融合蛋白CPE由编码非洲猪瘟病毒CD2v的N段氨基酸序列的基因和pEP153R的C段氨基酸序列的基因融合并构建重组表达质粒，经大肠杆菌表达、纯化获得。本发明还提供了上述重组蛋白的制备方法和应用。实验结果证明，该重组蛋白表达量高，能被非洲猪瘟病毒阳性血清识别。利用本发明重组蛋白制备的疫苗，免疫动物可诱导产生显著抑制非洲猪瘟病毒感染的体液免疫和特异性细胞免疫，可用于非洲猪瘟亚单位疫苗的制备。
Description
技术领域
本发明属于生物技术制药领域，具体涉及一种非洲猪瘟病毒重组融合蛋白CPE、制备及其应用。
背景技术
非洲猪瘟（African swine fever, ASF）是由非洲猪瘟病毒（African swinefever virus,ASFV）引起猪的急性烈性传染病，死亡率可达100%，目前无商品化的疫苗。ASF疫苗研究进展表明：ASF灭活疫苗不能提供免疫保护；ASF减毒活疫苗接种后虽然能够提供针对同源或异源毒株的免疫保护，但接种的动物易出现不良反应，而且存在毒力返强的风险；一些ASFV抗原亚单诱导产生中和抗体和细胞免疫，保护部分动物抵抗病毒感染，但并不能满足防控需要。因此，通过不断发掘保护抗原、筛选适合的免疫增强剂及佐剂等多种手段可提升亚单位疫苗的免疫效果，有望设计出安全、有效的ASF亚单位疫苗。
CD2v是EP402R基因编码的ASFV囊膜蛋白，分子量约为45 kDa，与T淋巴细胞表面粘附受体CD2类似，是一种含有多个结构域的糖蛋白，从N端到C端依次为信号肽、免疫球蛋白样结构域、跨膜区和胞内区。由于猪红细胞表面具有CD2配体，可与CD2v发生特异性结合，是介导ASFV红细胞吸附的关键蛋白。研究表明：CD2v是诱导ASFV保护性免疫应答的重要抗原之一，其抗体能抑制ASFV感染猪肺泡巨噬细胞（PAM）细胞，也诱导保护性细胞免疫应答。此外，研究还发现pEP153R介导特异性血清学反应、并可能诱导针对同源毒株的免疫保护，这与pEP153R上富集T 淋巴细胞的抗原表位有关，提示pEP153R也是诱导保护性免疫应答的重要抗原。这两种蛋白的免疫学功能为设计安全、有效的ASF亚单位疫苗提供了重要理论依据。
发明内容
为了开发出安全、有效的ASF疫苗，本发明将编码非洲猪瘟病毒结构蛋白CD2v的N段氨基酸序列的基因和pEP153R的C段氨基酸序列的基因融合，并构建重组表达质粒，经大肠杆菌表达、纯化获得重组融合蛋白CPE，该重组蛋白表达量高，能被非洲猪瘟病毒阳性血清识别。利用本发明重组蛋白制备的疫苗，免疫动物可诱导产生中和抗体和特异性细胞免疫，是一种有应用前景的非洲猪瘟亚单位疫苗。
本发明采用的具体方案如下：
本发明的目的一是提供一种非洲猪瘟病毒重组融合蛋白CPE，所述重组融合蛋白CPE是由ASFV结构蛋白CD2v的N段氨基酸序列和pEP153R的C段氨基酸序列通过连接肽(Linker)连接形成，具有以下通式：CD2v (Asp17-Tyr206)-(Linker)n-pEP153R (Asn49-Lys158)；
其中，所述CD2v的N段氨基酸序列为SEQ ID NO：3；所述pEP153R的C段氨基酸序列为SEQ ID NO：4；所述连接肽序列为GGGGS，n为1、2、3或4。
作为对上述重组融合蛋白CPE的进一步优化，所述连接肽序列为GGGGS，n为2。
作为对上述重组融合蛋白CPE的进一步优化，所述重组融合蛋白CPE的氨基酸序列为SEQ ID NO：2。优选地，编码所述重组融合蛋白CPE的核苷酸序列为SEQ ID NO：1。
本发明的目的二是提供一种表达载体，所述表达载体由骨架质粒可修饰地连接编码上述重组蛋白的核苷酸序列形成；优选地，所述骨架质粒为pET-32a(+)。
本发明的目的三是提供上述重组蛋白的制备方法，所述方法包括：
(1) 重组表达质粒构建：构建如上述的重组表达质粒；
(2) 转化及阳性克隆的筛选：将步骤(1)中的表达载体转化至宿主菌，经诱导表达、SDS-PAGE鉴定得到能够表达目的蛋白的重组表达菌；
(3) 诱导表达：将步骤(2)中的阳性重组表达菌接种新鲜培养基生长至一定浓度，添加IPTG进行诱导表达；
(4) 蛋白纯化：收集步骤(3)中的菌体，经超声破碎、包涵体洗涤及溶解、亲和层析纯化和透析复性得到目的蛋白；
(5) 重组蛋白的鉴定：采用SDS-PAGE和Western blotting 对步骤(4)中获得的重组蛋白进行鉴定。
本发明的目的四是提供上述重组蛋白在ASF疫苗制备中的应用。
本发明的目的五是提供一种ASF亚单位疫苗，包含上述的重组融合蛋白CPE。优选地，将所述重组融合蛋白CPE与ISA 206佐剂配伍后所得的ASF亚单位疫苗的效果更佳。
有益效果：
1、重组蛋白CPE可在原核表达系统（大肠杆菌）中诱导表达，而且具有表达量高、易于纯化的优点；
2、选择pET-32a(+)载体自身携带硫氧还蛋白标签，具有促进重组蛋白CPE可溶性表达并提高表达量的作用；此外，重组蛋白的N端还携带一个6×His标签，简化了纯化过程，经一步纯化得到的重组蛋白CPE的纯度不低于90％；
3、重组蛋白CPE制备的疫苗免疫动物后，既能诱发高水平的特异性抗体，又能激发良好的特异性细胞免疫。
4、利用本发明制备的重组蛋白CPE可通过皮下途径免疫，诱导机体产生高滴度的特异性IgG抗体。并经中和试验证实，所述重组蛋白疫苗诱导产生的抗体在体外显著抑制ASFV感染猪肺泡巨噬细胞，为制备ASF亚单位疫苗打下基础。
附图说明
图1为本发明实施列中重组表达载体pET-32a(+)-CPE双酶切鉴定结果；其中，1：pET-32a(+)-CPEBamHI-XhoI双酶切，2：pET-32a(+)-CPE；M：DNA分子量标准(Marker)；
图2为本发明实施列中重组蛋白CPE诱导表达及纯化的SDS-PAGE检测；其中，泳道1：阳性菌诱导前；泳道2：蛋白分子量标准（Marker）；泳道3：阳性菌诱导后；泳道4：菌体裂解沉淀；泳道5：包涵体溶解；泳道6：纯化的重组蛋白CPE；
图3为本发明实施列中利用6×His单克隆抗体(A)和ASFV阳性血清(B)对重组蛋白CPE进行鉴定的Western blotting结果；
图4本发明实施列中首次免疫小鼠后不同时间点血清中抗CD2v特异性IgG的消长趋势；
图5本发明实施列中首次免疫小鼠后不同时间点血清中抗pEP153R特异性IgG的消长趋势；
图6为本发明实施列中首次免疫小鼠后28天，脾淋巴细胞经抗原体外刺激后表达细胞因子类型及水平；
图7为本发明实施列中首次免疫小鼠后28天，脾淋巴细胞经抗原体外刺激后增殖水平检测结果；
图8为本发明实施列中首次免疫小鼠28天的血清与ASFV孵育后培养的病毒基因组拷贝数。
具体实施方式
本发明的发明人在研究中发现，将编码ASFV结构蛋白CD2v的N段氨基酸序列的基因与pEP153R的C段氨基酸序列的基因串联，构建重组表达质粒，转化大肠杆菌感受态细胞，构建重组表达菌、表达纯化重组ASFV抗原融合蛋白，用试制的疫苗免疫动物后，产生高水平的抗CD2v和抗pEP153R IgG抗体和特异性细胞免疫，特异性抗体能阻断ASFV感染PAM细胞，提示该重组ASFV抗原融合蛋白可用于ASF亚单位疫苗的制备。
一种重组ASFV抗原融合蛋白，所述重组ASFV抗原融合蛋白由CD2v的N段氨基酸序列和pEP153R的C段氨基酸序列通过连接肽(Linker)连接成的ASFV抗原融合蛋白的编码基因构建重组表达载体，转化大肠杆菌构建重组表达菌，表达纯化获得，以下简称重组蛋白CPE，所述重组蛋白具有以下通式：CD2v (Asp17-Tyr206)-(Linker)n-pEP153R (Asn49-Lys158)；其中所述连接肽序列为GGGGS，；n为1、2、3或4，优选为2。
在本发明的实施方案中，所述ASFV结构蛋白CD2v的N段氨基酸序列为SEQ ID NO：3。所述ASFV非结构蛋白pEP153R的C段氨基酸序列为SEQID NO：4。
在本发明的实施方案中，所述重组蛋白的氨基酸序列为SEQ ID NO：2；优选地，编码所述重组蛋白的核苷酸序列为SEQ ID NO：1。
上述重组蛋白可用于制备ASF亚单位疫苗。
为了更好的理解本发明的内容，下面结合具体实施方法对本发明内容作进一步说明，但发明的保护内容不局限于以下实施例。
1、重组表达质粒的构建与鉴定
将ASFV CD2v (蛋白ID：AYW34030.1) 的N段氨基酸序列(Asp17-Tyr206)与pEP153R (蛋白ID：AYW34029.1)的C段氨基酸序列(Asn49-Lys158)通过(GGGGS)2柔性Linker串联，将串联后的氨基酸序列按照大肠杆菌的密码子偏好转换为基因序列，并在其5＇端和3＇端分别引入特异性酶切位点BamHI和XhoI，委托南京金斯瑞生物科技有限公司合成，并将其克隆到pET-32a(+)表达载体上，命名为pET-32a(+)-CPE。将该重组质粒转化DH5α，挑取阳性克隆接种于LB（Kan+），37 ℃ 220 rpm过夜培养。
重组表达质粒的酶切鉴定：采用快速质粒小提试剂盒(Omega Bio-Tek)提取阳性克隆的质粒。通过BamHI和XhoI进行酶切，37 ℃水浴1.5 h。体系如下：重组质粒7 µL、BamHI酶 1 µL、XhoI酶 1µL、10×Cutsmart buffer 1 µL。将上述酶切反应体系中各加入2.5 µL5×Loading buffer，经1.0％琼脂糖凝胶电泳，UV扫描仪观察酶切的结果。
如图1所示：重组质粒pET-32a(+)-CPE经BamHI和XhoI双酶切后，在琼脂糖凝胶电泳图显示两条清晰的条带，其中一条950 bp的DNA片段与目的基因大小完全相符，提示pET-32a(+)-CPE重组质粒构建成功，测序结果显示，重组基因与设计目的基因的同源性为100%，读码框完全正确。
2、重组蛋白CPE的诱导表达及纯化
将鉴定阳性的重组质粒pET-32a(+)-CPE转化大肠杆菌BL21(DE3) 感受态细胞，挑取单菌落接种于6 mL卡那抗性LB培养基中，37 ℃ 220 rpm培养。待菌液的OD600达到0.4-0.6时，留取2 mL菌液转至灭菌EP管中作为扩大培养的菌种，将剩余4 mL菌液平均分成2份，一份加入终浓度为0.5 mM IPTG进行诱导，另一份作为对照，16 ℃ 200 rpm培养20 h，收集菌体进行SDS-PAGE检测。结果显示（如图2），阳性菌经IPTG诱导后在47 kDa处有明显的条带，与重组蛋白CPE预期大小相符。
将留取的2 mL菌液扩繁后，按1:80接种到1.5 L卡那抗性的LB培养基中，37 ℃220 rpm培养。待菌液的OD600达到0.4-0.6时，加入终浓度为0.5 mM IPTG进行诱导，16 ℃200 rpm培养20 h。7000 rpm离心6 min收集菌体，加入45 mL缓冲液1（300 mM NaCl, 20 mMNaH2PO4, 5 mM咪唑; pH 8.3）重悬菌体，将菌体超声裂解35 min，经10000rpm离心25min分别收集沉淀和上清，并进行SDS-PAGE检测。结果显示，重组蛋白CPE为包涵体形式表达。用含8M尿素的缓冲液1溶解包涵体，10000 rpm离心25 min，收集的上清与Ni螯合亲和介质在4℃条件下结合1.5 h，然后用30 mL（10倍柱体积）缓冲液2（8 M尿素，300 mM NaCl,20 mMNaH2PO4, 20 mM咪唑; pH 8.3）洗涤杂蛋白，最后用15 mL缓冲液3（8 M尿素，300 mM NaCl,20 mM NaH2PO4, 500 mM咪唑; pH 7.5）洗脱目的蛋白。洗脱的重组蛋白依次通过8 M，6 M，4M，2 M，0 M的缓冲液4 (20 mM NaH2PO4, 300 mM NaCl, 2 mM β-mercaptoethanol, 0.4%arginine, 10%glycerol, pH 7.5)透析复性。透析液每隔8 h换一次液，直到透析至尿素浓度降为0 M，取少量复性重组蛋白进行SDS-PAGE检测，结果显示，复性蛋白约47 kDa，与包涵体目的条带大小一致（图2）。
3、重组蛋白CPE的Western blotting鉴定
采用Bradford法对重组CPE进行浓度测定，取300ng重组蛋白CPE与5×上样缓冲液混合后煮沸10分钟，进行SDS-PAGE（80 V 0.5 h，120V 1.25 h），，然后电转印于硝酸纤维素膜（PVDF）上，加入适量含5%脱脂奶粉的PBST封闭液，室温封闭2 h；分别加入适量鼠抗6×His单克隆抗体（1:5000）或ASFV阳性猪血清（1:300），4 ℃过夜，然后用PBST洗涤6次，每次5min；分别加入过氧化物酶标记的羊抗鼠IgG（1:5000）和过氧化物酶标记羊抗猪IgG（1:5000），室温孵育1 h，并用PBST洗涤6次，每次5min；配制酶标底物显色液（ECL）并均匀铺至PVDF膜上，采用多功能成像仪进行图像采集。
Western blotting鉴定重组蛋白结果如图3所示，重组蛋白CPE被His单克隆抗体识别，且与ASFV阳性血清反应性良好，在ASF诊断试剂或疫苗开发方面具有潜在的应用价值。
4、疫苗制备及小鼠免疫方案
将重组蛋白CPE的浓度稀释成300 µg/mL。取2份等体积稀释好的重组蛋白，其中1份与ISA 206按照50g：50g混合，乳化成疫苗（水/油/水），另一份与等体积PBS混匀制成无佐剂疫苗。设置ISA 206佐剂与等体积PBS混合并乳化作为阴性对照，PBS作为空白对照。将20只6-8周龄SPF级雌性BALB/c小鼠随机分为4组（5只/每组），将上述制备好的疫苗经皮下多点免疫，按照表1中的免疫分组以及免疫剂量，初次免疫1次，14天后加强免疫1次。
表1 疫苗免疫方案
| 组别 | 免疫组分/只 | 注射体积uL/只 |
| 实验组1 | 30 µg CPE | 200 |
| 实验组2 | 30 µg CPE+ISA206 | 200 |
| 阴性对照 | ISA 206 | 200 |
| 空白对照 | PBS | 200 |
5、免疫效果评价
5.1特异性抗体检测
首次免疫前及免疫后7、14、21和28天，对所有实验小鼠尾静脉采血，分离血清，以本实验室建立的间接ELISA检测免疫后不同时间点血清中特异性抗体水平。即分别以纯化的CD2v（2 µg/mL）和pEP153R（2 µg/mL）包被96孔酶标板，100μL/孔，4 ℃过夜；弃掉包被液，加入含5%脱脂奶粉的PBST，200μL/孔，37 ℃封闭2 h；PBST洗涤6次并拍干；加入免疫小鼠血清（1∶100），100 µL/孔，每个样品3孔重复，37 ℃孵育1 h；弃掉酶标板中的液体，用PBST洗板6次后拍干。加入1:10000 稀释的HRP标记的羊抗小鼠IgG，100 µL/孔，37 ℃孵育1 h；PBST洗板6次并拍干，加入显色液，100μL/孔，室温避光15 min；加入2 M H2SO4终止反应，每孔100 μL，测450 nm处的OD值，绘制抗体消长曲线图。
结果及分析：如图4和图5所示，佐剂免疫组和PBS组在整个实验期间，均未检测到抗CD2v和pEP153R特异性IgG抗体。重组蛋白CPE单独免疫组和重组蛋白CPE+ISA 206组，在首次免疫后7天，均检测到抗CD2v和抗pEP153R的特异性IgG抗体，且这两种IgG抗体水平在加强免疫后均明显升高，但重组蛋白CPE+ISA 206组所诱导的抗CD2v和抗pEP153R特异性IgG抗体水平显著高于重组蛋白CPE单独免疫组（p<0.001）。结果说明，重组蛋白CPE具有良好的免疫原性，ISA 206佐剂明显提高抗原特异性IgG水平。
5.2 细胞因子水平测定
首次免疫后28天，从每组随机选取3只小鼠脱臼处死，在75%酒精中浸泡约 3 min；无菌条件下摘取脾脏并浸润于RPMI-1640培养基中，用5 mL注射器活塞轻轻研磨成脾细胞悬液；500×g离心5 min弃上清，向细胞沉淀中加入2 mL红细胞裂解液重悬，室温裂解3min；随后加入15 mL PBS终止反应，500×g离心5 min，收集细胞并用PBS洗涤2次；将收集的脾细胞用含10%胎牛血清的RPMI-1640培养基重悬，调整细胞密度至1×106/mL，接种12孔板，每孔1 mL。然后加入终浓度为5 μg/mL的重组蛋白CD2v和pEP153R，37 ℃ 5% CO2培养72h，收集上清，检测培养细胞上清中IL-2，IFN-γ和TNF-α的含量。
结果及分析：如图6所示，重组蛋白CPE单独免疫组和重组蛋白CPE+ISA 206免疫组的脾淋巴细胞经抗原体外刺激后都能分泌IL-2，IFN-γ和TNF-α，但重组蛋白CPE+ISA 206免疫组显著高于重组蛋白CPE免疫组（p<0.001），且这两组的3种细胞因子水平均显著高于佐剂免疫组和PBS组（p<0.001）。结果说明，重组蛋白CPE与ISA 206佐剂配伍免疫后能够显著活化淋巴细胞，提高细胞因子的分泌水平。
5.3 淋巴细胞增殖实验
脾细胞分离如5.2所述，将脾细胞浓度调整为5×105/mL，接种96孔细胞培养板（100 µL/孔），加入终浓度为2.5 μg/mL的纯化的重组蛋白CD2v和pEP153R。同时，设培养基对照、正常细胞对照和ConA刺激对照（5 μg/mL），37 ℃ 5% CO2培养72 h后，每孔加入10 µL的CCK8，37 ℃ 5% CO2孵育3.5 h后，测定OD450nm的吸光度值，计算刺激指数。计算公式为：
SI=(实验组OD450-空白对照OD450)/(阴性对照OD450-空白对照OD450)。
结果及分析：如图7所示，重组蛋白CPE+ISA 206免疫组，体外刺激后淋巴细胞的增殖水平最高，刺激指数平均为3.46，明显高于其他三组（p<0.001）。重组蛋白CPE免疫组的淋巴细胞刺激指数平均值为2.53，增殖水平显著高于佐剂免疫组和PBS组（p<0.001）。佐剂免疫组和PBS对照组的淋巴细胞刺激指数均<2，无明显增殖。结果说明，重组蛋白CPE与ISA206佐剂配伍免疫后能够显著活化细胞免疫，激发良好的免疫记忆，提高淋巴细胞的增殖水平。
5.4 病毒中和实验
用免疫前和免疫后28天的血清进行中和试验，具体操作如下：将血清分别用灭菌的PBS进行1：5稀释，并用0.22 m的针头滤器过滤除菌，56℃水浴灭能30分钟，然后与ASFV-CN/SC/2019（MOI=0.01，中国农业科学院兰州兽医研究所 BSL-3实验室保存）等体积混合，37℃过夜，取200 μL血清/病毒混合物接种已长成单层的猪肺泡巨噬细胞（24孔板），37 ℃孵育1 h，吸弃血清/病毒混合物，用无菌PBS洗2次，加入500 μL含5% FBS的RPMI-1640继续培养48 h，收集培养物并提取ASFV DNA，qPCR试剂盒检测ASFV拷贝数，评价免疫血清对病毒增殖的抑制水平。
如图8所示，与免疫前血清相比，重组蛋白CPE免疫组和重组蛋白CPE+ISA 206组免疫血清均显著降低了PAM细胞内ASFV的拷贝数（p<0.001），说明重组蛋白CPE疫苗诱导产生的抗体具有阻断病毒进入PAM、或/和抑制ASFV在猪肺泡巨噬细胞内的复制。重组蛋白CPE+ISA 206组ASFV基因拷贝数显著低于重组蛋白CPE免疫组（p<0.001），提示ISA 206明显增强了重组蛋白CPE诱导中和抗体水平。而佐剂免疫组和PBS对照组的ASFV基因拷贝数与免疫前血清无显著性差异（p>0.05）。结果说明，重组蛋白CPE能诱导产生的中和抗体，ISA206佐剂显著提高中和抗体滴度，该重组蛋白及其疫苗是一种具有应用前景的ASF亚单位疫苗。
需要说明的是，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> 一种非洲猪瘟病毒重组融合蛋白CPE、制备及其应用
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Claims (8)
1.一种非洲猪瘟病毒重组融合蛋白CPE，其特征在于：所述重组融合蛋白CPE是由ASFV结构蛋白CD2v的N段氨基酸序列和pEP153R的C段氨基酸序列通过连接肽(Linker)连接形成，具有以下通式：CD2v (Asp17-Tyr206)-(Linker)n-pEP153R (Asn49-Lys158)；
其中，所述CD2v的N段氨基酸序列为SEQ ID NO：3；所述pEP153R C段氨基酸序列为SEQID NO：4；所述连接肽序列为GGGGS，n为2；
所述重组融合蛋白CPE的氨基酸序列如SEQ ID NO：2所示。
2.根据权利要求1所述的一种非洲猪瘟病毒重组融合蛋白CPE，其特征在于：编码所述重组融合蛋白CPE的核苷酸序列如SEQ ID NO：1所示。
3.一种表达载体，所述表达载体由骨架质粒可修饰地连接编码权利要求1所述的重组融合蛋白CPE的核苷酸序列形成。
4.一种转化体，所述转化体携带编码权利要求1所述重组融合蛋白CPE的核苷酸序列并表达所述重组融合蛋白CPE。
5.一种重组融合蛋白CPE的制备方法，其特征在于：所述制备方法包括以下步骤：
(1) 重组表达质粒构建：构建如权利要求3所述的表达载体；
(2) 转化及阳性克隆的筛选：将步骤(1)中的表达载体转化至宿主菌，经诱导表达、SDS-PAGE鉴定得到能够表达目的蛋白的重组表达菌；
(3) 诱导表达：将步骤(2)中的阳性重组表达菌接种新鲜培养基生长至一定浓度，添加IPTG进行诱导表达；
(4) 蛋白纯化：收集步骤(3)中的菌体，经超声破碎、包涵体洗涤及溶解、亲和层析纯化和透析复性得到目的蛋白；
(5) 重组蛋白的鉴定：采用SDS-PAGE和Western blotting 对步骤(4)中获得的重组蛋白进行鉴定。
6.根据权利要求1或2所述的重组融合蛋白CPE在ASF疫苗制备中的应用。
7.一种ASF亚单位疫苗，其特征在于：包含如权利要求1或2所述的重组融合蛋白CPE。
8.根据权利要求7所述的ASF亚单位疫苗，其特征在于：是将所述重组融合蛋白CPE与ISA 206佐剂配伍后所得。
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