CN113004386A - 草鱼过敏性毒素C5a重组蛋白及其多克隆抗体 - Google Patents

草鱼过敏性毒素C5a重组蛋白及其多克隆抗体 Download PDF

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CN113004386A
CN113004386A CN202110465468.8A CN202110465468A CN113004386A CN 113004386 A CN113004386 A CN 113004386A CN 202110465468 A CN202110465468 A CN 202110465468A CN 113004386 A CN113004386 A CN 113004386A
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许宝红
刘巧林
肖调义
苏杭
江石峰
蒋海鹏
严海亮
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Abstract

一种草鱼过敏性毒素C5a重组蛋白及其多克隆抗体，是根据草鱼补体5基因蛋白全序列，通过生物信息学分析得到草鱼过敏性毒素C5a蛋白序列，再将C5a蛋白序列全基因克隆至pET‑28a‑SUMO载体上，通过表达、纯化，得到草鱼过敏性毒素C5a重组蛋白，以该重组蛋白为抗原多次免疫动物即得到草鱼过敏性毒素C5a多克隆抗体。通过本方法能得到满足后续实验需求的草鱼过敏性毒素C5a多克隆抗体，提供了研究C5a蛋白的分子工具，为后续有效检测草鱼在不同条件下过敏性毒素C5a的表达调控规律提供了重要实验试剂。

Description

草鱼过敏性毒素C5a重组蛋白及其多克隆抗体
技术领域
本发明涉及分子生物学领域，具体涉及草鱼过敏性毒素C5a重组蛋白，同时涉及利用该草鱼过敏性毒素C5a重组蛋白作为抗原制备的多克隆抗体。
背景技术
补体5（Complement Component 5）是补体系统的重要组成成分。当补体C5被激活后，C5转化酶裂解会形成C5a和C5b大小两种活性分子。C5a产生后进入液相，是一种非常有效的促炎蛋白，是过敏毒素作用最强的介质，可直接作用于肥大细胞，使之释放组胺，引起血管扩张、通透性增加、平滑肌收缩及局部水肿。大量的C5a可以诱导中性粒细胞、嗜酸性粒细胞和单核细胞沿着浓度梯度定向移动，产生趋化作用。高浓度的C5a可以促进溶酶体酶释放，加快氧化代谢进程，甚至可以刺激中性粒细胞粘附，增强其产生超氧化物的能力。除此之外，C5a的活化对机体的免疫应答反应具有明显的增强作用，可诱导炎症因子的大量表达。对炎症和免疫性疾病的治疗而言，将C5a作为靶向进行补体抑制是一种具有发展前景的治疗策略。
草鱼在我国具有悠久的养殖历史，养殖范围广阔，养殖产量位居前列，但其自身抗逆性不高，因此疾病防控在草鱼的养殖过程中十分关键。草鱼出血病作为一种病毒性疾病，传染性强、发病速度快、死亡率高，严重制约草鱼养殖的发展，因此高抗性的草鱼苗种具有巨大的研究价值。然而，补体C5a作为先天免疫中重要的促炎蛋白在草鱼出血病炎症过程中的作用及表达规律尚未得到充分地研究。
发明内容
本发明要解决的技术问题是，针对上述现有技术的不足，提供一种草鱼过敏性毒素C5a重组蛋白及其多克隆抗体，利用本方法从草鱼过敏性毒素C5a序列开始构建，能够得到符合后续检测要求的草鱼过敏性毒素C5a多克隆抗体，为后续有效检测草鱼在不同条件下过敏性毒素C5a的表达调控规律提供了重要实验试剂。
为解决上述技术问题，本发明所采用的技术方案是：一种草鱼过敏毒素C5a重组蛋白，其氨基酸序列如SEQ ID NO.3所示。
本发明同时提供含有上述草鱼过敏毒素C5a重组蛋白的编码基因的重组表达载体，该重组表达载体命名为pET-28a-SUMO-C5a。
本发明还提供一种多克隆抗体，是以上述的草鱼过敏毒素C5a重组蛋白作为抗原免疫动物得到。
具体地，上述多克隆抗体的制备方法如下：
（1）根据登录号为MT150869的草鱼补体5基因蛋白全序列，通过生物信息学分析得到SEQ ID NO.3所示的草鱼过敏性毒素C5a蛋白序列，将该序列全基因合成到pET-28a-SUMO载体上，导入表达大肠杆菌中表达，纯化后获得重组蛋白；
（2）利用该重组蛋白为抗原免疫实验级日本大耳白兔。
上述步骤（1）中的草鱼过敏性毒素C5a蛋白序列是位于SEQ ID NO.1所示的β链和SEQ ID NO.2所示的α链之间的柔性LINK序列RLKR之后的α链N端前71个氨基酸的序列。
上述步骤（1）中的提及的表达大肠杆菌为大肠杆茵感受态细胞菌株E. coliRosetta。
本发明能够得到满足后续实验需求的浓缩草草鱼过敏性毒素C5a多克隆抗体，为后续检测草鱼过敏性毒素C5a的表达调控提供了重要实验试剂。
附图说明
图1是本发明确定的草鱼过敏性毒素C5a片段在NCBI网站（https://www.ncbi.nlm.nih.gov/）中的注释结果。
图2是本发明草鱼C5a大肠杆菌表达后破菌纯化PAGE凝胶电泳图；
图中，1：0.4 mg/mL BSA；2：Marker；3：pET-28a-SUMO空载诱导表达；4：上清；5：上清（2M尿素溶解包涵体）6：包涵体2倍稀释（8M尿素溶解包涵体）；7：包涵体10倍稀释（8M尿素溶解包涵体）。
图3是本发明亲和纯化用蛋白检测结果；
图中，1：Marker；2：0.4 mg/mL BSA；3：包涵体2倍稀释（8M尿素溶解包涵体）；4：包涵体10倍稀释（8M尿素溶解包涵体）；
表明：亲和纯化用重组蛋白与破菌纯化后浓度和纯度差异不大，可进行抗原亲和纯化。
图4是本发明抗原WB检测结果；
图中，两只日本大耳兔血液纯化得到的多克隆抗体稀释1000倍后，一只可检测到1ng抗原，另一只可检测到1 ng和500 pg抗原，说明得到的草鱼过敏性毒素C5a多克隆抗体浓度正常。
图5是使用本发明得到的草鱼过敏性毒素C5a多克隆抗体对健康草鱼肝脏进行Western Blot杂交结果图；
图中，β-actin为内参蛋白。
具体实施方式
以下实施是对本发明的进一步说明和解释，而不是对本发明的限制。
实施例1 草鱼过敏性毒素C5a蛋白片段的确定
以NCBI网站（https://www.ncbi.nlm.nih.gov/）GenBank数据库中NCBI中人类C5的蛋白序列（登录号: AAA51925.1）的注释：19~673aa为C5的β链，678~1676aa为C5的α链，682~750aa为C5a。随后运用Clustal w 2.0对人、小鼠和虹鳟的C5序列进行同源性分析发现，C5的α链和β链连接处高度保守，是由4个氨基酸组成的柔性LINK序列连接。C5a是位于α链的N端大约70多个氨基酸的序列，在补体激活过程中在C5转化酶的作用下游离出来。因此C5a的序列为保守LINK序列与C5转换酶作用位点“R”之间的序列，故可通过生物信息学分析草鱼补体5序列（登录号MT150869），得到草鱼C5a在C5上的位置特点，即草鱼过敏性毒素C5a蛋白序列是位于SEQ ID NO.1所示的β链和SEQ ID NO.2所示的α链之间的柔性LINK序列RLKR之后的α链的N端71个氨基酸的序列。将得到的71个氨基酸序列放入NCBI（www.ncbi.nlm.nih.gov/）进行分析，得到了与人类C5a相同的注释，说明确定的草鱼C5a蛋白序列无误（图1）。
实施例2 重组蛋白的获得
将确定的草鱼过敏性毒素C5a蛋白序列所对应的基因片段采用全基因合成的方式，直接克隆至pET-28a-SUMO表达载体（购自华越洋生物（北京）科技有限公司）上，得重组质粒pET-28a-SUMO-C5a，经测序确定连接正确后，导入大肠杆菌感受态细胞菌株E.coliRosetta中，培养至OD600nm为0.5-0.6。随后加入0.8 mM IPTG，37℃诱导4小时以实现破菌，采用SDS-PAGE凝胶电泳对目的蛋白进行纯化，得到蛋白浓度和纯度达到免疫要求的重组蛋白，大小为27kDa，序列如SEQ ID NO.3所示，请结合参见图2，图2表明：PCR扩增得到的草鱼C5a序列在大肠杆菌表达体系中充分表达，表达得到的蛋白质产物大小符合预期。
实施例3 草鱼C5a多克隆抗体的获得
挑选健康的6周大小的实验级日本大耳兔（由武汉爱博泰克生物科技有限公司提供），稳定一周后首次从耳静脉采血10 ml作为阴性对照，然后用上述实施例制得的重组蛋白作为抗原多次免疫两只实验级日本大耳兔：免疫部位为背部5个部位，每个部位注射250μL，第一次免疫采用的免疫剂量为0.3 mg，使用完全弗氏佐剂；12天后进行第二次免疫，使用草鱼过敏性毒素C5a抗原的剂量为0.15 mg，使用不完全弗氏佐剂；第一次免疫26天后进行第三次免疫，使用草鱼过敏性毒素C5a抗原的剂量为0.15 mg，使用不完全弗氏佐剂；第一次免疫40天后进行第四次免疫，使用草鱼过敏性毒素C5a抗原的剂量为0.15 mg，使用不完全弗氏佐剂；第一次免疫54天后进行第五次免疫，使用草鱼过敏性毒素C5a抗原的剂量为0.15 mg，使用不完全弗氏佐剂。第一次免疫后第66天，将日本大耳兔处死，取血，得到抗血清，将抗血清用浓度为3mg/ml的重组蛋白抗原进行抗原亲和纯化（见图3），从两只实验级日本大耳白兔抗血清中分别得到浓度为0.79 mg/ml和1.09 mg/ml的浓缩草鱼过敏性毒素C5a抗体，结合参见图4。
实施例4 抗体的检测验证
为了验证得到的草鱼过敏性毒素C5a多克隆抗体是否能够用于后续Western Blot实验，本实验利用得到的草鱼过敏性毒素C5a多克隆抗体检测草鱼过敏性毒素C5a在健康草鱼肝脏中的表达情况。每组实验采集了三条草鱼样本。首先从各检测器官或组织分别剪取0.1g组织，加入到装有1 ml RIPA裂解液、10μl蛋白酶抑制剂和2颗无菌钢珠的匀浆管中，使用冷冻混合球磨仪进行研磨，均质速度55 Hz，1650r/s，研磨结束后置于裂解10分钟，然后4℃、12000 rpm离心15分钟，将离心后的上清分装转移至1.5 ml离心管中用于后续实验。根据每个样品总蛋白量，按照上样量20 μg点样至分离胶浓度为15%的PAGE胶中100V电泳至溴酚蓝到胶底部止。然后将草鱼过敏性毒素C5a蛋白进行转膜。用1×PBST缓冲液清洗3次，每次脱色摇床摇10 min。加适量封闭液，在脱色摇床上封闭15 min。将膜加到多槽盒内，加入草鱼过敏性毒素C5a多克隆抗体稀释液（用封闭液按1：1000的比例稀释），将膜与抗体一起孵育，4℃过夜。孵育结束后，用1PBST缓冲液洗涤3次，每次洗涤10 min。将HRP标记的二抗稀释液（用封闭液按1：2000的比例稀释），将稀释后的二抗与膜共同孵育60 min，然后用1×PBST缓冲液洗涤3次，每次洗涤10 min。最后将显色A液与B液（Thermo）等体积混合，避光，每张膜加200μL，放置1-2 min。将膜放置到荧光成像仪中，观察并拍照。结果显示，健康草鱼肝脏组织C5a表达结果如图5所示。以上结果验证了通过本发明描述的方法能够得到用于草鱼过敏性毒素C5a表达检测的浓缩草鱼C5a多克隆抗体。
序列表
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Glu Phe Thr Phe Glu Thr Glu Asp Pro Arg Trp Ser Gln Ala Pro Leu
420 425 430
Lys Leu Lys Ala Glu Ser Tyr Val Ser Val Asn His Arg Tyr Leu Tyr
435 440 445
Ile Asp Leu His Pro Glu Ser Ser Val Phe Glu Val Gly Arg Tyr Ile
450 455 460
Asn Ile Lys Val Phe Phe His Tyr Arg Asp Tyr Leu Ser Leu Glu Thr
465 470 475 480
Phe Ser Tyr Gln Ile Ile Ser Arg Gly Lys Ile Val Gln Phe Ala Thr
485 490 495
Val Lys Arg Val Ser Glu Lys Ser Gln Ser Leu Asn Ile Lys Ile Thr
500 505 510
Pro Asp Met Val Pro Ser Ala Arg Leu Leu Val Tyr Tyr Val Leu Tyr
515 520 525
Gly Glu Glu Lys Ala Glu Leu Val Ala Asp Ser Ala Trp Ile Asp Val
530 535 540
Lys Ala Lys Cys Val Gln Asn Leu Asn Met Asp Ile Ser Thr Leu Asn
545 550 555 560
Ser Gln Tyr Lys Pro Lys Asp Lys Leu Glu Leu Arg Val Ser Thr Lys
565 570 575
Thr Lys Glu Glu Ser Leu Val Ala Phe Ser Ala Val Asp Thr Ala Leu
580 585 590
Tyr Asn Leu Lys Ser Asn Asp Lys Asp Pro Leu Lys Lys Val Leu His
595 600 605
His Val Glu Arg Ser Asp Leu Gly Cys Gly Arg Gly Gly Gly Lys Asn
610 615 620
Asn Ala Asp Val Phe Asp Arg Ala Gly Leu Met Phe Ile Thr Asn Ala
625 630 635 640
Met Ala Ser Ser Ala Glu Gly Thr Cys Ser Asp Leu Val
645 650
<210> 2
<211> 1011
<212> PRT
<213> 合成()
<400> 2
Ser Pro Asp Leu Gly Gln Lys Phe Glu Ser Lys Ala Lys Met Tyr Gly
1 5 10 15
Pro Ser Arg Asn Cys Cys Leu Ser Gly Thr Arg Ser Ile Pro Thr Leu
20 25 30
Glu Thr Cys Gly Asp Arg Ala Lys Arg Leu Pro Phe Ser Glu Lys Thr
35 40 45
Thr Asp Tyr Trp Lys Lys Arg Cys Gln Arg Ala Phe Leu Glu Cys Cys
50 55 60
Glu Phe Ala Ile Lys Leu Arg Lys Asp Ser Val Asp Lys Ile Ile Leu
65 70 75 80
Ser Arg Gly Ala Ile Asp Phe Leu Leu Asp Ala Met Pro Ser Gln Ile
85 90 95
Arg Ser Tyr Phe Pro Glu Ser Trp Leu Trp Glu Glu His Thr Ser Lys
100 105 110
Ser Gly Ser Val Ser Ile Thr Lys Asn Leu Pro Asp Ser Leu Thr Thr
115 120 125
Trp Glu Leu Lys Ala Val Gly Val Phe Ser Glu Gly Ile Cys Val Ser
130 135 140
Glu Glu Lys Val Gln Val Ser Gln Asp Ile Ser Val Asp Val Pro Leu
145 150 155 160
Pro Tyr Ser Met Val Arg Gly Glu Gln Ile Glu Leu Arg Gly Ser Val
165 170 175
Tyr Asn Gln Arg Phe Ser Lys Thr Trp Phe Arg Val Thr Leu Thr Ala
180 185 190
Thr Asp Gly Val Cys Val Phe His Gly Thr Gln Gln Gly Lys His Gly
195 200 205
Lys Pro Asn Glu Ile Lys Gly Gln Ile Asp Gly Arg Ser Val Ala Leu
210 215 220
Val Thr Phe Tyr Ile Met Ala Leu Glu Ala Gly Thr His Thr Leu Thr
225 230 235 240
Phe Thr Leu Thr Thr Lys Trp Gly Gly Glu Thr Val Val Lys Lys Leu
245 250 255
Lys Val Val Pro Glu Gly Ile Arg Lys Glu Ile Gln Ser Gly Gly Arg
260 265 270
Ile Asp Pro Asn Gly Val Phe Gly Thr Ser Met Arg Lys Leu Glu Leu
275 280 285
Lys His Ala Ile Pro Pro Asn Ile Ala Pro Lys Ser Thr Val Asp Arg
290 295 300
Ile Leu Thr Ile Asn Gly Glu Val Leu Gly Glu Leu Leu Ser Ile Ile
305 310 315 320
Asn Asn Pro Glu Gly Leu Lys Gln Leu Thr Asn Leu Pro Arg Gly Asn
325 330 335
Ala Glu Met Glu Leu Met Gly Leu Leu Pro Ile Tyr Tyr Val Tyr His
340 345 350
Tyr Leu Glu Lys Thr Glu Lys Trp Asp Ile Leu Gly Lys Glu Ser Ala
355 360 365
Ala Ser Glu Arg Glu Leu Lys Arg Lys Met Gln Ala Gly Ile Thr Ser
370 375 380
Ile Met Ser Tyr Lys Leu Arg Asn Glu Tyr Ala Phe Ser Met Trp Ser
385 390 395 400
Asn Lys Asp Lys Ser Ala Ser Thr Trp Val Thr Ala Leu Ile Ala Lys
405 410 415
Thr Leu Ala Asp Met His Glu Tyr Val Ser Leu Glu Ser Glu Val Leu
420 425 430
Thr Asn Thr Ile Tyr Trp Leu Ile Lys Asn Arg Gln Asn Asp Asp Gly
435 440 445
Ser Phe Tyr Glu Lys Ser Gln Thr Asn Pro Ile Lys Leu Met Gly Ala
450 455 460
Gly Ala Asp Val Thr Glu Lys Thr Val Phe Leu Thr Ser Phe Val Met
465 470 475 480
Ile Gly Ile Lys Asn Ala Leu Lys Val Pro Asn Ile Asn Ile Gln Ala
485 490 495
Phe Lys Asp Ala Leu Asp His Ala Thr Gln Tyr Leu Cys Ser Lys Ser
500 505 510
Lys Lys Ile Glu Ser Leu Tyr Val Arg Ala Ile Ala Ser Tyr Ala Leu
515 520 525
Thr Leu Val Asp Thr His Ser Met Pro Ala Val Asp Leu Tyr Glu Lys
530 535 540
Ile Lys Gln Gln Ala Gln Val Lys Gly Asn Pro Ala Thr Ile Arg Phe
545 550 555 560
Trp Glu Asp Ser Lys Pro Lys Lys Asp Ser Leu Lys Pro Ser Asp Val
565 570 575
Thr Ala Lys Thr Val Glu Thr Thr Val Tyr Ile Leu Leu Asn Thr Leu
580 585 590
Val Arg Gly Asp Ser Thr Tyr Ala Lys Pro Ile Leu Asn Trp Leu Thr
595 600 605
Gln Asp Gln Arg Tyr Gly Gly Gly Phe His Ser Thr Gln Asp Ser Ile
610 615 620
Leu Thr Leu Glu Ala Leu Thr Lys Tyr Ser Ile Leu Ala Ser Gln Ala
625 630 635 640
Thr Leu Asp Met Val Val Asp Ile Glu Tyr Lys Thr Lys Gly Asp Ile
645 650 655
Ser Arg Ile Lys Leu Thr Gln Gln Lys Pro Val Ala Lys Pro Ile Glu
660 665 670
Val Thr Lys Asn Asp Asp Ile Ile Ile Lys Thr Ala Met Ser Ser Gly
675 680 685
Ile Ser Phe Ala Lys Leu Arg Thr Val Tyr Tyr Glu Met Thr Glu Ser
690 695 700
Asn Glu Asn Cys Gln Phe Glu Met Ser Ile Asp Ile His Asp Ser Val
705 710 715 720
Pro Phe Ser Asp Asp Pro Met Leu Leu Ser Gln Arg Ile Val Ala Cys
725 730 735
Ala Lys Tyr Lys Pro His Glu Asn Thr Phe Glu Thr Glu Ser Gly Leu
740 745 750
Thr Val Met Glu Ile Tyr Leu Pro Thr Gly Val Ile Pro Val Gln Glu
755 760 765
Asp Leu Asp Met Tyr Gln Asn Gly Leu Glu Ser Gln Ile Ser Asn Tyr
770 775 780
Glu Ile Met Gly Asp Gln Val Ile Leu Gln Ile Asp Ser Ile Pro Ser
785 790 795 800
Lys Asp Phe Tyr Cys Val Gly Phe Arg Ile Gln Glu Glu Phe Glu Thr
805 810 815
Gly Leu Thr Arg Ala Ser Val Phe Arg Val Tyr Glu Phe Tyr Asp Gln
820 825 830
Glu Asn Lys Cys Met Lys Phe Tyr His Lys Gln Thr Arg Lys Leu Leu
835 840 845
Arg Leu Cys Glu Gly Asp Gln Cys Gln Cys Met Ala Ala Glu Cys Cys
850 855 860
Asn Phe Lys Ser Thr Ile Asp Thr Thr Ile Thr Ala Glu Gln Arg Arg
865 870 875 880
Asn Tyr Leu Cys Lys Glu Ser Met Lys Tyr Ala Phe Lys Val Leu Ile
885 890 895
Glu Ser Ser Glu Ala Val Gly Asp Phe Val Thr Tyr Lys Ala Lys Val
900 905 910
Lys Thr Val Leu Lys Lys Glu Gln Thr Asp Asp Leu Lys Thr Asp Ser
915 920 925
Glu Ile Asp Phe Val Lys Lys Ala Thr Cys Thr Glu Thr Asn Phe Glu
930 935 940
Val Gly Lys Gln Tyr Leu Ile Met Thr Ser Glu Ser Ile Lys Ile Lys
945 950 955 960
Val Asp His Ala Tyr Lys Tyr Lys Phe Pro Leu Asp Ser His Ala Trp
965 970 975
Val Asp Trp Trp Pro Leu Glu Ser Asp Cys Arg Asn Asp Ala Cys Lys
980 985 990
Gln Tyr Thr Gly Val Leu Asp Asp Phe Gly Leu Asp Phe Leu Leu Ser
995 1000 1005
Gly Cys Asp
1010
<210> 3
<211> 71
<212> PRT
<213> 合成()
<400> 3
Ser Pro Asp Leu Gly Gln Lys Phe Glu Ser Lys Ala Lys Met Tyr Gly
1 5 10 15
Pro Ser Arg Asn Cys Cys Leu Ser Gly Thr Arg Ser Ile Pro Thr Leu
20 25 30
Glu Thr Cys Gly Asp Arg Ala Lys Arg Leu Pro Phe Ser Glu Lys Thr
35 40 45
Thr Asp Tyr Trp Lys Lys Arg Cys Gln Arg Ala Phe Leu Glu Cys Cys
50 55 60
Glu Phe Ala Ile Lys Leu Arg
65 70

Claims (9)

1.一种草鱼过敏毒素C5a重组蛋白，其氨基酸序列如SEQ ID NO.3所示。
2.含有权利要求1所述草鱼过敏毒素C5a重组蛋白的编码基因的重组表达载体。
3.如权利要求2所述的重组表达载体，其特征在于，该重组表达载体命名为pET-28a-SUMO-C5a。
4.一种多克隆抗体，其特征在于，该抗体是以权利要求1所述的草鱼过敏毒素C5a重组蛋白作为抗原免疫动物得到。
5.如权利要求4所述的多克隆抗体，其特征在于，所述草鱼过敏毒素C5a重组蛋白是根据登录号为MT150869的草鱼补体5基因蛋白全序列，通过生物信息学分析得到SEQ ID NO.3所示的草鱼过敏性毒素C5a蛋白序列，将该蛋白序列全基因克隆到pET-28a-SUMO载体上，导入表达大肠杆菌中表达，纯化后得到。
6.如权利要求5所述的多克隆抗体，其特征在于，所述草鱼过敏性毒素C5a蛋白序列是位于SEQ ID NO.1所示的β链和SEQ ID NO.2所示的α链之间的柔性LINK序列RLKR之后的α链N端前71个氨基酸的序列。
7.如权利要求5所述的多克隆抗体，其特征在于，所述表达大肠杆菌为大肠杆茵感受态细胞菌株E. coli Rosetta。
8.如权利要求4所述的多克隆抗体，其特征在于，所述动物为实验级日本大耳兔。
9.权利要求4所述的多克隆抗体在检测草鱼过敏性毒素C5a的表达调控中的应用。
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