CN115125215B - 分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用 - Google Patents
分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用 Download PDFInfo
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Abstract
本发明属于免疫学领域,涉及一株分泌猪IFN‑λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用。杂交瘤细胞株λ4‑1A2的保藏编号:CCTCC NO:C2022132。该单克隆抗体的轻链可变区包括SEQ ID NO:3所示的CDR1、氨基酸序列为LVS的CDR2和SEQ ID NO:4所示的CDR3;重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3。该单克隆抗体可以用在检测sIFN‑λ4中。本发明的杂交瘤细胞株λ4‑1A2所分泌的单克隆抗体具有良好特异性,可以特异性识别原核及真核表达的sIFN‑λ4重组蛋白。
Description
技术领域
本发明属于免疫学领域,具体涉及一株能分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用。
背景技术
干扰素(interferon,IFN)是一类具有抗病毒、抗肿瘤、免疫调节和维持细胞自身稳定等多种生物学活性的糖蛋白,根据其氨基酸序列与结构、识别受体的特异性及生物功能的不同,可将其分为Ⅰ型、Ⅱ型和Ⅲ型。I型IFN于1957年发现,由多个IFN-α亚型和单个的IFN-β、IFN-ε、IFN-κ、IFN-ω亚型组成,其中对IFN-α和IFN-β的研究最明确,IFN-α在机体抵抗病毒的感染中发挥重要作用,IFN-β的主要作用是免疫调节。Ⅱ型干扰素主要具有促炎和免疫调节的功能。III型干扰素也称为IFN-λ,是近几年才发现的一类干扰素,人的IFN-λ有IFN-λ1、IFN-λ2、IFN-λ3和IFN-λ4 4种亚型,猪只有IFN-λ1、IFN-λ3和IFN-λ4 3种亚型,没有IFN-λ2。IFN-λ在抗病毒方面的作用与I型干扰素相似。
干扰素不能直接发挥抗病毒作用,而是通过与细胞上的特异性受体结合后诱导一系列的信号传导从而发挥功能。在干扰素家族中,I型干扰素的受体IFNAR1和IFNAR2广泛分布于所有有核细胞(Zhou JH,Wang YN,Chang QY,Ma P,Hu Y,Cao X.Type IIIInterferons in Viral Infection and Antiviral Immunity.Cell PhysiolBiochem.2018;51(1):173-185.)。III型干扰素的受体由IFNLR1(也称为IL28Rα)和IL10Rβ组成,其中IL10Rβ和IFNAR一样,可在许多类型的细胞和组织中广泛表达(Lazear HM,NiceTJ,Diamond MS.Interferon-λ:Immune Functions at Barrier Surfaces andBeyond.Immunity.2015Jul 21;43(1):15-28.),但IFNLR1的表达有比较严格的组织特异性,主要表达于上皮细胞,虽然有研究表明某些免疫细胞(NK细胞、T细胞、B细胞和树突状细胞)也可表达IFNLR1,但目前尚未发现这些细胞在IFN-λ介导的抗病毒免疫中的作用(T,Hernandez P,Gronke K,Diefenbach A,Staeheli P.Leukocyte-derivedIFN-α/βand epithelial IFN-λconstitute a compartmentalized mucosal defensesystem that restricts enteric virus infections.PLoS Pathog.2015Apr7;11(4):e1004782.)。
由于IFNLR1主要表达于呼吸道、胃肠道和生殖道的黏膜上皮细胞,所以IFN-λ会在相关器官组织的黏膜屏障位点产生特异性抗病毒反应,但不会像I型干扰素那样引起有害的系统性炎症反应。III型干扰素中的IFN-λ4于2013年才发现,与其它几种IFN-λ的同源性较低。研究表明IFN-λ4与丙肝病毒的清除、慢性病毒感染、免疫抑制患者巨细胞病毒的激活有关,但其作用机制尚不清楚,特别是关于猪IFN-λ4国内外报道较少,市场上也未见有猪IFN-λ4定性定量检测的相关产品,这极大限制了其功能的研究和临床的应用,单克隆抗体的制备将为猪IFN-λ4的功能研究和检测方法的建立奠定基础。
发明内容
本发明提供了一株能稳定分泌猪IFN-λ4(sIFN-λ4)单克隆抗体的杂交瘤细胞株,并提供了sIFN-λ4单克隆抗体及其在IFA、Western-Blot及间接ELISA方法检测sIFN-λ4中的应用。
本发明具体采用以下技术方案:
1.分泌sIFN-λ4单克隆抗体的杂交瘤细胞株的建立
将原核表达并纯化的sIFN-λ4重组蛋白与快速免疫佐剂等比混合后免疫BALB/c小鼠,利用间接ELISA检测小鼠血清中的sIFN-λ4抗体,对ELISA抗体效价达到1:12800的小鼠加强免疫后,取小鼠脾细胞与骨髓瘤细胞融合,经三轮克隆与筛选,最终获得1株可稳定分泌sIFN-λ4单克隆抗体的杂交瘤细胞,将其命名为杂交瘤细胞株λ4-1A2,将杂交瘤细胞株λ4-1A2保藏于中国典型培养物保藏中心,保藏时间:2022年6月7日,保藏编号:CCTCC NO:C2022132。
2.sIFN-λ4单克隆抗体λ4-1A2的特异性鉴定
以原核表达并纯化的sIFN-λ1重组蛋白、sIFN-λ3重组蛋白和sIFN-λ4重组蛋白分别包被ELISA板,以杂交瘤细胞λ4-1A2株的培养上清为一抗,羊抗鼠IgG-HRP为二抗进行间接ELISA检测。结果显示杂交瘤细胞λ4-1A2株分泌的单克隆抗体λ4-1A2只与sIFN-λ4重组蛋白发生特异性反应,与sIFN-λ1重组蛋白和sIFN-λ3重组蛋白无特异性反应,说明单克隆抗体λ4-1A2具有良好的特异性。
3.sIFN-λ4单克隆抗体λ4-1A2的大量制备与纯化
将弗氏不完全佐剂经腹腔注射BALB/c小鼠,7d后经腹腔注射λ4-1A2株杂交瘤细胞悬液,10~14d后视小鼠腹部膨胀情况及时采集腹水。将收集的腹水离心后,取中间层淡黄色清亮液体,用Protein G柱进行纯化,经SDS-PAGE检测显示获得了纯度较高的单克隆抗体,ELISA检测显示抗体效价达100×211。
4.单克隆抗体λ4-1A2可变区的扩增与分析
提取杂交瘤细胞株λ4-1A2的总RNA并反转为cDNA,设计简并引物扩增单克隆抗体λ4-1A2轻链可变区和重链可变区。
得到的单克隆抗体λ4-1A2轻链可变区大小为339bp,核苷酸序列如SEQ ID NO:1所示,该轻链可变区的氨基酸序列如SEQ ID NO:2所示,包括SEQ ID NO:3所示的CDR1、氨基酸序列为LVS的CDR2和SEQ ID NO:4所示的CDR3。单克隆抗体λ4-1A2重链可变区大小为330bp,核苷酸序列如SEQ ID NO:5所示,该重链可变区的氨基酸序列如SEQ ID NO:6所示,包括SEQID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3。
5.sIFN-λ4单克隆抗体在检测sIFN-λ4中的应用
将表达sIFN-λ4重组蛋白的真核质粒pCAGGS-Flag-sIFN-λ4转染HEK293T细胞,24h后收集样品,用单克隆抗体λ4-1A2为一抗进行IFA或Western-Blot检测,结果显示单克隆抗体λ4-1A2与转染pCAGGS-Flag-sIFN-λ4的细胞有特异性荧光反应或特异性反应条带出现,与转染载体质粒pCAGGS-Flag的细胞无特异性反应。
将纯化的sIFN-λ4原核表达蛋白梯度稀释后作为抗原包被酶标板,同时包被等比稀释的空载体转化菌表达蛋白作为对照。以单克隆抗体λ4-1A2为一抗,羊抗鼠IgG-HRP为二抗进行间接ELISA分析,结果显示单克隆抗体λ4-1A2可应用于ELISA检测原核表达的sIFN-λ4重组蛋白,并且该ELISA方法对原核表达的sIFN-λ4重组蛋白的最低检测限为6.25ng。
本发明的有益效果在于:
(1)本发明制备的杂交瘤细胞株λ4-1A2可以分泌特异性针对sIFN-λ4的单克隆抗体,填补了市场上无sIFN-λ4单克隆抗体的空白。
(2)本发明制备的单克隆抗体λ4-1A2可应用于间接ELISA、Western-Blot和IFA方法检测sIFN-λ4中,具有良好特异性,可以特异性识别原核及真核表达的sIFN-λ4重组蛋白,为sIFN-λ4检测方法的建立奠定了基础。
(3)本发明制备的腹水型sIFN-λ4单克隆抗体的效价可达100×211,其应用于间接ELISA方法中对原核表达的sIFN-λ4重组蛋白的最低检测限为6.25ng。
(4)本发明制备的单克隆抗体可为研发sIFN-λ4其他检测方法如直接ELISA、夹心ELISA和胶体金诊断试剂等提供材料。
附图说明
图1:为猪IFN-λ1和IFN-λ3基因的PCR扩增结果。图A:M1:DL 2000DNA Marker;1、2:猪IFN-λ1的PCR产物;图B:M2:DL 2000DNA Marker;1、2:猪IFN-λ3的PCR产物。
图2:为重组质粒pET-15b-sIFN-λ1和pET-15b-sIFN-λ3的酶切鉴定结果。图A:M1:250bp DNA Marker;1:pET-15b/NcoI+Xho I;2、3:pET-15b-sIFN-λ1/NcoI+Xho I;图B:M2:250bp DNA Marker;1:pET-15b/NdeI+Xho I;2、3:pET-15b-sIFN-λ3/NdeI+Xho I。
图3:为sIFN-λ4重组蛋白免疫小鼠血清效价检测结果。1号、2号、3号、4号、5号表示小鼠编号。
图4:为制备的单克隆抗体λ4-1A2的特异性检测结果。
图5:为纯化的腹水型单克隆抗体λ4-1A2的SDS-PAGE电泳检测图;附图标记说明:M:Protein Marker;1:纯化的腹水型单克隆抗体λ4-1A2。
图6:为猪IFN-λ4的PCR扩增结果。M:DL 2000DNA Marker;1:猪IFN-λ4的PCR产物。
图7:为重组质粒pCAGGS-Flag-sIFN-λ4的酶切鉴定结果。M:250bp DNA Marker;1:pCAGGS-Flag/EcoRI+XhoI;2,3:pCAGGS-Flag-sIFN-λ4/EcoRI+XhoI。
图8:为真核表达的sIFN-λ4重组蛋白的Western-Blot检测结果。M:ProteinMarker;1:pCAGGS-Flag-sIFN-λ4转染细胞的裂解上清;2:pCAGGS-Flag转染细胞的裂解上清。
图9:为单克隆抗体λ4-1A2用于IFA检测真核表达的sIFN-λ4的结果。
图10:为单克隆抗体λ4-1A2用于Western-Blot检测真核表达的sIFN-λ4重组蛋白的结果。M:Protein Marker;1:pCAGGS-Flag-sIFN-λ4转染细胞的裂解上清;2:pCAGGS-Flag转染细胞的裂解上清。
保藏信息
分泌sIFN-λ4单克隆抗体的杂交瘤细胞株:
保藏时间:2022年6月7日;
保藏单位名称:中国典型培养物保藏中心;
保藏编号:CCTCC NO:C2022132;
保藏单位地址:中国,武汉,武汉大学;
分类命名:杂交瘤细胞株λ4-1A2。
具体实施方式
下面通过具体实施方式对本发明进行更加详细的说明,以便于对本发明技术方案的理解,但并不用于对本发明保护范围的限制。
实施例1:sIFN-λ1、sIFN-λ3、sIFN-λ4重组蛋白的原核表达与纯化
sIFN-λ1、sIFN-λ3、sIFN-λ4重组蛋白的原核表达与纯化参考申请号为202210515788.4的发明专利申请“一种猪干扰素λ4重组蛋白的制备方法及其应用”中记载的猪干扰素λ4重组蛋白的制备方法。具体如下:
1.sIFN-λ4重组蛋白的原核表达
首先,根据NCBI上公布的猪IFN-λ4(sIFN-λ4)的基因序列(GeneID:102161030),对sIFN-λ4的氨基酸序列进行优化,具体是:去掉sIFN-λ4蛋白的信号肽(MGPRGTAAVAMGLWVFVTAVFA),且在sIFN-λ4的C端引入6个组氨酸。培养LLC-PK1细胞,待培养的LLC-PK1细胞长至单层时,接种仙台病毒,于接毒12h后收集细胞。提取细胞总RNA作为模板,设计特异性引物λ4-P1(如SEQ ID NO:13所示)和λ4-P2(如SEQ ID NO:14所示)扩增优化后的sIFN-λ4基因,对获得的扩增产物和原核表达载体pET-15b(+)分别用NcoⅠ和XhoⅠ进行双酶切后,用T4 DNA连接酶进行连接,获得重组质粒pET-15b-sIFN-λ4。将pET-15b-sIFN-λ4转化大肠杆菌BL21(DE3),经IPTG诱导和SDS-PAGE检测证实sIFN-λ4在大肠杆菌中获得高效表达,并主要以包涵体形式存在。
2.sIFN-λ4重组蛋白的大量表达与纯化
大量诱导pET-15b-sIFN-λ4表达菌,离心收集菌体,经高压破碎仪破碎后,离心收集沉淀。用洗涤液(1%v/v Triton X-100,5mmol/L EDTA,20mmol/L Tris-HCl,0.1mol/LNaCl,pH 8.5)洗涤2次,离心后收集沉淀获得包涵体。
用溶解液(8mol/L脲素,50mmol/L Tris-HCl,300mmol/L NaCl,pH 8.5)溶解包涵体,再经离心收集上清,用0.45μm滤膜过滤。对滤液用Ni Focurose 6FF(IMAC)重力柱纯化。再用稀释液(6mol/L脲素,100mmol/L NaCl,50mmol/L Tris-HCl,pH 8.5)稀释后,依次用1号复性液(4mol/L脲素,0.5mol/L L型精氨酸,2mmol/L还原型谷胱苷肽,0.5mmol/L氧化型谷胱苷肽,20mmol/L Tris-HCl,pH 8.5)、2号复性液(3mol/L脲素,0.5mol/L L型精氨酸,2mmol/L还原型谷胱苷肽,0.5mmol/L氧化型谷胱苷肽,20mmol/L Tris-HCl,pH 8.5)、3号复性液(2mol/L脲素,0.5mol/L L型精氨酸,2mmol/L还原型谷胱苷肽,0.5mmol/L氧化型谷胱苷肽,20mmol/L Tris-HCl,pH 8.5)、4号复性液(1mol/L脲素,0.5mol/LL型精氨酸,2mmol/L还原型谷胱苷肽,0.5mmol/L氧化型谷胱苷肽,20mmol/L Tris-HCl,pH 8.5)和Tris-HCl缓冲液(100mmol/L NaCl,50mmol/L Tris-HCl,pH 8.5)进行透析。透析完成后收集蛋白溶液,并用超滤管超滤浓缩至约2mg/ml,分装后保存于-70℃以下。同时取样通过SDS-PAGE对其纯度进行检测,结果发现获得的sIFN-λ4的纯度在95%以上。
3.sIFN-λ1、sIFN-λ3重组蛋白的表达与纯化
根据NCBI上公布的猪IFN-λ1(sIFN-λ1)的基因序列(GeneID:100217388)和猪IFN-λ3(sIFN-λ3)的基因序列(GeneID:100310828),对sIFN-λ1和sIFN-λ3的氨基酸序列进行优化,具体是:去掉sIFN-λ1和sIFN-λ3蛋白的信号肽(sIFN-λ1蛋白的信号肽为:MATAWIVVLATVMLDLARA,sIFN-λ3蛋白的信号肽为:MALGGSLVLVLVLMTVAPPR TGA),且在sIFN-λ1的N端引入6个组氨酸,sIFN-λ3的N端有pET-15b载体自带的6个组氨酸。以IPI-2I细胞(购自中国典型培养物保藏中心)的RNA为模板,利用引物sIFN-λ1-F1(如SEQ ID NO:15所示)、sIFN-λ1-R1(如SEQ ID NO:16所示)和sIFN-λ3-F1(如SEQ ID NO:17所示)、sIFN-λ3-R1(如SEQ ID NO:18所示)通过RT-PCR分别扩增优化后的猪IFN-λ1(sIFN-λ1)和猪IFN-λ3(sIFN-λ3)基因片段,优化后的sIFN-λ1和sIFN-λ3基因扩增产物电泳图分别如图1所示。回收PCR产物,将回收的sIFN-λ1片段用NcoI和XhoI酶切,sIFN-λ3片段用NdeI和XhoI酶切,回收目的片段,分别插入pET-15b的相应位点,转化大肠杆菌后小提质粒,经酶切证实重组质粒构建正确(如图2所示),将其分别命名为pET-15b-sIFN-λ1和pET-15b-sIFN-λ3。将获得的质粒分别转化大肠杆菌BL21(DE3)感受态细胞,制备原核表达的猪IFN-λ1重组蛋白和猪IFN-λ3重组蛋白。
猪IFN-λ1重组蛋白和猪IFN-λ3重组蛋白的大量表达与纯化方法与sIFN-λ4重组蛋白的大量表达与纯化方法相同。
实施例2:sIFN-λ4单克隆抗体的制备与鉴定
1.分泌sIFN-λ4单克隆抗体细胞株的建立
将原核表达并纯化的sIFN-λ4重组蛋白与等体积的快速免疫佐剂(购自博奥龙免疫技术有限公司)混合均匀后经后肢肌肉注射6~8周龄雌性BALB/c小鼠,3次免疫后通过间接ELISA检测小鼠血清中的sIFN-λ4抗体,当抗体效价达到1:12800(如图3所示)时,用不加佐剂的sIFN-λ4重组蛋白加强免疫。加强免疫后3~5d,眼眶放血处死小鼠。
无菌取免疫小鼠的脾细胞与骨髓瘤细胞SP 2/0进行融合,经HAT培养基和HT培养基培养后,以原核表达的sIFN-λ4重组蛋白为抗原建立ELISA方法对融合细胞的培养上清进行检测,对阳性孔的细胞进行3轮克隆与筛选,获得1株可以稳定分泌sIFN-λ4单克隆抗体的杂交瘤细胞株,将其命名为杂交瘤细胞株λ4-1A2,将杂交瘤细胞株λ4-1A2保藏于中国典型培养物保藏中心,保藏时间:2022年6月7日,保藏编号:CCTCC NO:C2022132。
2.单克隆抗体λ4-1A2的亚型鉴定
用小鼠单抗亚类鉴定试剂盒(购自博奥龙免疫技术有限公司),对获得的单克隆抗体λ4-1A2进行亚型鉴定,结果显示,该单克隆抗体的重链为IgG1亚型,轻链为κ亚型。
3.单克隆抗体λ4-1A2的特异性验证
将原核表达的sIFN-λ1重组蛋白、sIFN-λ3重组蛋白和sIFN-λ4重组蛋白从4μg/ml开始进行连续2倍倍比稀释至0.03125μg/ml后,分别包被ELISA板,以杂交瘤细胞株λ4-1A2的培养上清为一抗,羊抗鼠IgG-HRP(购自碧云天生物技术有限公司)为二抗进行间接ELISA检测。
结果显示,杂交瘤细胞株λ4-1A2的培养上清只与sIFN-λ4重组蛋白有特异性反应,与sIFN-λ1重组蛋白和sIFN-λ3重组蛋白无特异性反应,如图4所示,说明杂交瘤细胞株λ4-1A2的培养上清中的单克隆抗体λ4-1A2具有良好的特异性。
4.sIFN-λ4单克隆抗体的大量制备、纯化和效价测定
(1)sIFN-λ4单克隆抗体的大量制备
选取3只10周龄健康雌性BALB/c小鼠,腹腔注射弗氏不完全佐剂,500μl/只,7d后腹腔注射杂交瘤细胞株λ4-1A2,5×105~1×106个细胞/只。10~14d后根据小鼠腹部膨大情况,收集小鼠腹水。将收集的腹水4℃12000r/min离心10min,收集中间黄色清亮层液体进行后续纯化。
(2)sIFN-λ4单克隆抗体的纯化
根据说明书用Protein G柱(购自福因德科技有限公司)对制备的腹水型sIFN-λ4单克隆抗体λ4-1A2进行纯化,将纯化后的单克隆抗体进行SDS-PAGE检测。结果可见轻链和重链两条蛋白带,如图5所示,说明获得了纯度较高的单克隆抗体。
(3)sIFN-λ4单克隆抗体的效价测定
将纯化好的抗体先稀释100倍,然后进行连续2倍倍比稀释,至100×212,以间接ELISA检测抗体水平。结果显示,本实施例制备的腹水型单抗的效价为100×211。
5.单克隆抗体λ4-1A2可变区的扩增与分析
提取杂交瘤细胞株λ4-1A2的总RNA并反转为cDNA,于4℃保存备用。设计11对简并引物用于单克隆抗体λ4-1A2轻链可变区扩增,设计12对简并引物用于单克隆抗体λ4-1A2重链可变区扩增,简并引物序列见表1和表2。
表1扩增轻链可变区的简并引物序列
引物名称 | 引物序列(5’~3’) |
MKV1 | ATGAAGATTGCCTGTTAGGCTGTTGGTGCTG |
MKV2 | ATGGAGWCAGACACACTCCTGYTAYGGGTG |
MKV3 | ATGAGTGTGCTCACTCAGGTCCTGGSGTTG |
MKV4 | ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG |
MKV5 | ATGGATTTWCAGGTGCAGATTWTCAGCTTC |
MKV6 | ATGAGGTKCYYTGYTSAYCTYCTCTGRGG |
MKV7 | ATGGGCWTCAAAGATGGAGTCACAKWYYCWGG |
MKV8 | ATGTGGGGAYCTKTTTYCMMTTTTTCAATG |
MKV9 | ATGGTRTCCWCASCTCAGTTCCTTG |
MKV10 | ATGTATATATGTTTGTTGTCTATTTCT |
MKV11 | ATGGAAGCCCCAGCTCAGCTTCTCTTCC |
MKC | ACTGGATGGTGGGAAGATGG |
表2扩增重链可变区的简并引物序列
重链引物名称 | 引物序列(5’-3’) |
MHV1 | ATGAAATGCAGCTGGGGCATSTTCTTC |
MHV2 | ATGGGATGGAGCTRTATCATSYTCTT |
MHV3 | ATGAAGWTGTGGTTAAACTGGGTTTTT |
MHV4 | ATGRACTTTGGGYTCAGCTTGRTTT |
MHV5 | ATGGGACTCCAGGCTTCAATTTAGTTTTCCTT |
MHV6 | ATGGCTTGTCYTTRGSGCTRCTCTTCTGC |
MHV7 | ATGGRATGGAGCKGGRGTCTTTMTCTT |
MHV8 | ATGAGAGTGCTGATTCTTTTGTG |
MHV9 | ATGGMTTGGGTGTGGAMCTTGCTTATTCCTG |
MHV10 | ATGGGCAGACTTACCATTCTCATTCCTG |
MHV11 | ATGGATTTTGGGCTGATTTTTTTTATTG |
MHV12 | ATGATGGTGTTAAGTCCTTCTGTACC |
MHCG1 | CAGTGGATAGACAGATGGGGG |
结果显示,有1对引物(MKV2和MKC)可扩增出单克隆抗体λ4-1A2的轻链可变区,有3对引物(MHV5和MHCG1;MHV9和MHCG1;MHV10和MHCG1)可扩增出单克隆抗体λ4-1A2的重链可变区。将获得的扩增片段连接到T载体后测序,分析发现共扩增获得一种抗体轻链可变区序列,三种抗体重链可变区序列,但是扩增获得的三种抗体重链可变区序列中,有两种序列存在终止密码子,属于无效重排基因。因此,本实施例采用简并引物获得一个单克隆抗体λ4-1A2轻链可变区和一个单克隆抗体λ4-1A2重链可变区。
其中,单克隆抗体λ4-1A2的轻链可变区大小为339bp,核苷酸序列如SEQ ID NO:1所示,该轻链可变区的氨基酸序列如SEQ ID NO:2所示,包括氨基酸序列为KSVDSFGKSF的CDR1(如SEQ ID NO:3所示)、氨基酸序列为LVS的CDR2和氨基酸序列为QQNNEDPPT的CDR3(SEQ ID NO:4所示)。该轻链可变区具有完整的抗体可变区结构,抗体V基因片段与MusmusIGKV3-10*01F的同源性为97.25%,抗体J基因片段与Musmus IGKJ1*01F的同源性为100%,因此可以推测单克隆抗体λ4-1A2的轻链可变区是由上述两个胚系基因片段重排形成的。
单克隆抗体λ4-1A2重链可变区大小为330bp,核苷酸序列如SEQ ID NO:5所示,该重链可变区的氨基酸序列如SEQ ID NO:6所示,包括氨基酸序列为GFSLNTYGIG的CDR1(如SEQ ID NO:7所示)、氨基酸序列为IWWNDNK的CDR2(如SEQ ID NO:8所示)和氨基酸序列为ASIKGDY的CDR3(如SEQ ID NO:9所示)。该重链可变区具有完整的抗体可变区结构,抗体V基因片段与Musmus IGHV8-11*01F的同源性为94.50%,抗体J基因片段与Musmus IGHJ4*01F的同源性为81.63%,抗体D基因片段与Musmus IGHD2-1*01F的同源性为100%,因此可以推测单克隆抗体λ4-1A2的重链可变区是由上述三个胚系基因片段重排形成的。
实施例3:单克隆抗体λ4-1A2在检测sIFN-λ4中的应用
1.表达sIFN-λ4的真核表达质粒pCAGGS-Flag-sIFN-λ4的构建
将仙台病毒(SeV)接种LLC-PK1细胞,12h后提取细胞RNA,将其反转录成cDNA后作为模板,以sIFN-λ4-F4(如SEQ ID NO:10所示)和sIFN-λ4-R4(如SEQ ID NO:11所示)为上下游引物进行扩增,获得sIFN-λ4基因的全长片段(如SEQ ID NO:12所示),扩增产物的电泳图如图6所示。回收PCR产物,经EcoR I和Xho I酶切后插入pCAGGS-Flag的相应酶切位点。经酶切鉴定和测序证实重组质粒构建成功(如图7所示),将其命名为pCAGGS-Flag-sIFN-λ4。
2.sIFN-λ4在真核细胞中的表达
将HEK293T细胞接种24孔细胞培养板,待细胞长满单层后,转染0.8μgpCAGGS-Flag-sIFN-λ4或pCAGGS-Flag,24h后用RIPA裂解液收集细胞,加入5×SDS-PAGE LoadingBuffer,100℃沸水浴10min,冰浴10min,12000r/min离心10min,吸取上清,进行SDS-PAGE电泳。电泳结束后,将蛋白转印至PVDF膜。用含10%脱脂奶粉的TBST封闭后,加入用TBST稀释的Flag标签的抗体(购自Proteintech),室温孵育3h,洗膜3次。加入用TBST稀释的羊抗鼠IgG-HRP(购自碧云天生物技术有限公司),室温孵育1h,洗膜3次。
结果如图8所示,转染pCAGGS-Flag-sIFN-λ4的细胞样品有特异性反应条带出现,大小约20kDa,与预期相符,而转染pCAGGS-Flag的细胞样品无特异性反应条带,说明sIFN-λ4重组蛋白在真核细胞中获得了正确表达。
3.单克隆抗体λ4-1A2应用于IFA检测sIFN-λ4
将HEK293T细胞接种24孔细胞培养板,待细胞长满单层后,转染0.8μgpCAGGS-Flag-sIFN-λ4或pCAGGS-Flag,24h后吸弃细胞培养液,每孔加入1ml组织固定液,固定15min,加入-20℃预冷的甲醇,静置15min,用PBS洗涤3次,每次5min。加入含5%BSA的PBS,37℃封闭1h,PBS洗涤3次,每次5min。加入实施例2制备的单克隆抗体λ4-1A2(用PBS进行2000倍稀释),每孔250μl,37℃孵育1h。用PBS洗涤3次,每次5min。每孔加入250μl异硫氰酸荧光素(FITC)标记的羊抗鼠IgG(购自碧云天生物技术有限公司)(用PBS做2000倍稀释),37℃孵育1h,全程避光操作。每孔加入250μl DAPI(购自碧云天生物技术有限公司)(用PBS进行5000倍稀释),37℃孵育15min。吸弃荧光二抗与DAPI稀释液,PBS洗涤3次,最后在每孔中加入200μl PBS,置于倒置荧光显微镜下观察并拍照。
结果如图9所示,实施例2制备的单克隆抗体λ4-1A2与转染pCAGGS-Flag-sIFN-λ4的细胞有特异性荧光反应,而与转染pCAGGS-Flag的细胞无荧光反应,说明实施例2制备的单抗λ4-1A2可应用于IFA检测真核表达的sIFN-λ4重组蛋白。
4.单克隆抗体λ4-1A2应用于Western-Blot检测sIFN-λ4
将HEK293T细胞接种24孔细胞培养板,待细胞长满单层后,转染0.8μgpCAGGS-Flag-sIFN-λ4或pCAGGS-Flag,24h后使用RIPA裂解液收集细胞。将细胞样品按照体积比例加入5×SDS-PAGE Loading Buffer,100℃沸水浴10min,冰浴10min,12000r/min离心10min,吸取上清,进行SDS-PAGE电泳,电泳结束后,将蛋白转印至PVDF膜。用含10%脱脂奶粉的TBST封闭后,加入用TBST1:1000稀释的单克隆抗体λ4-1A2,室温孵育3h,洗膜3次。加入用TBST稀释的羊抗小鼠IgG-HRP,室温孵育1h,洗膜3次。
结果如图10所示,实施例2制备的单克隆抗体λ4-1A2与pCAGGS-Flag-sIFN-λ4转染的细胞样品有特异性反应条带,大小约20kDa,与预期大小相符,而与转染pCAGGS-Flag的细胞样品无特异性反应条带,说明实施例2制备的单抗λ4-1A2可应用于Western-Blot检测真核表达的sIFN-λ4重组蛋白。
5.单克隆抗体λ4-1A2应用于ELISA检测sIFN-λ4
将原核表达并纯化的sIFN-λ4重组蛋白的浓度调整为1μg/ml,再进行连续的2倍倍比稀释,直到将sIFN-λ4重组蛋白调整至0.49ng/ml,将不同稀释度的sIFN-λ4重组蛋白分别包被酶标板,同时包被等比稀释的pET-15b空载体转化菌表达蛋白作为对照,4℃包被12h。以单克隆抗体λ4-1A2为一抗、羊抗鼠IgG-HRP(购自碧云天生物技术有限公司)为二抗,进行间接ELISA检测。若待测孔OD630nm:阴性孔OD630nm值≥2.1,即判定为阳性。
结果显示,单克隆抗体λ4-1A2可应用于ELISA检测原核表达的sIFN-λ4重组蛋白,且最低可检测到6.25ng的sIFN-λ4原核表达蛋白。
表3单克隆抗体λ4-1A2用于间接ELISA检测原核表达的sIFN-λ4的结果
总的来说,上述单克隆抗体sIFN-λ4效价高、特异性好,能够特异性识别原核表达的sIFN-λ4重组蛋白和真核表达的sIFN-λ4重组蛋白。上述单克隆抗体λ4-1A2可以用于IFA中,检测真核表达的sIFN-λ4重组蛋白。还可以用于Western-Blot中,检测真核表达的sIFN-λ4重组蛋白,又可以用于间接ELISA方法中,检测原核表达的sIFN-λ4重组蛋白。
以上所述之实施例,只是本发明的较佳实施例而已,并非限制本发明的实施范围,故凡依本发明专利范围所述的构造、特征及原理所做的等效变化或修饰,均应包括于本发明申请专利范围内。
SEQUENCE LISTING
<110> 华中农业大学
<120> 分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用
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Claims (10)
1.分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株,其特征在于,所述杂交瘤细胞株为杂交瘤细胞株λ4-1A2,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO: C2022132。
2.权利要求1中所述的杂交瘤细胞株分泌的单克隆抗体。
3.根据权利要求2所述的单克隆抗体,其特征在于,所述单克隆抗体的轻链可变区包括SEQ ID NO:3所示的CDR1、氨基酸序列为LVS的CDR2和SEQ ID NO:4所示的CDR3;所述单克隆抗体的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3。
4.根据权利要求2所述的单克隆抗体,其特征在于,所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示,重链可变区的氨基酸序列如SEQ ID NO:6所示。
5.一种基因工程抗体,其特征在于,所述基因工程抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示,重链可变区的氨基酸序列如SEQ ID NO:6所示。
6.编码权利要求4所述的单克隆抗体的DNA,其特征在于,编码所述单克隆抗体的轻链可变区的DNA序列如SEQ ID NO:1所示,编码所述单克隆抗体的重链可变区的DNA序列如SEQID NO:5所示。
7.一种重组表达载体,其特征在于,所述重组表达载体包含权利要求6所述的DNA。
8.一种宿主细胞,其特征在于,所述宿主细胞包含权利要求7所述的重组表达载体。
9.一种猪IFN-λ4检测试剂盒,其特征在于,所述试剂盒包含权利要求2~4任一项所述的单克隆抗体。
10.权利要求2~4任一项所述的单克隆抗体在检测猪IFN-λ4中的应用,所述应用以非疾病诊断为目的。
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CN104560885A (zh) * | 2014-11-20 | 2015-04-29 | 中国农业科学院哈尔滨兽医研究所 | 一种抗天然牛γ-干扰素的单克隆抗体,分泌该抗体的杂交瘤细胞株及应用 |
CN105602908A (zh) * | 2016-01-26 | 2016-05-25 | 中国农业科学院特产研究所 | 水貂γ-干扰素单克隆抗体及其在检测水貂γ-干扰素中的应用 |
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