CN114409756B - 一种牙鲆补体成分C3（PoC3）的应用 - Google Patents

一种牙鲆补体成分C3（PoC3）的应用 Download PDF

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CN114409756B
CN114409756B CN202210070638.7A CN202210070638A CN114409756B CN 114409756 B CN114409756 B CN 114409756B CN 202210070638 A CN202210070638 A CN 202210070638A CN 114409756 B CN114409756 B CN 114409756B
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李墨非
吴蒙
贾贝贝
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Abstract

本发明涉及分子生物学领域，具体的说是一种牙鲆补体成分C3(PoC3)的应用。牙鲆补体成分C3(PoC3)在制备杀菌剂中的应用。本发明补体成分C3是来自于牙鲆补体系统的一种天然蛋白。该补体成分C3蛋白可制备为杀菌剂，其制备过程简单，对环境和鱼体均无安全隐患，进而所得蛋白在抗细菌感染中具有应用潜能。

Description

一种牙鲆补体成分C3（PoC3）的应用
技术领域
本发明涉及分子生物学领域，具体的说是一种牙鲆补体成分C3(PoC3) 的应用。
背景技术
补体系统由大约35种蛋白质组成，它们存在于血清、组织液和细胞膜表面。补体系统可以通过三种不同的途径激活，包括经典途径、凝集素途径和旁路途径。C3是补体系统的中心因子，所有三种补体激活途径都需有 C3转化酶的形成。在补体激活的级联反应中，C3转化酶裂解C3产生C3a 和C3b，C3b参与C5转化酶的形成。C5转化酶将C5裂解为C5a和C5b，C5b参与膜攻击复合物(MAC)的组装。MAC在病原体的表面打孔，导致受感染细胞的破坏和外来病原体的死亡。C3在补体系统中起重要作用，广泛参与先天免疫和适应性免疫。补体成分C3已在多种鱼类中发现，但是，在牙鲆中C3功能和应用性研究尚不十分清楚。
发明内容
本发明目的在于提供一种牙鲆补体成分C3(PoC3)的应用。
为实现上述目的，本发明采用的技术方案为：
一种补体成分C3的应用，牙鲆补体成分C3(PoC3)在制备杀菌剂中的应用。
所述补体成分C3(PoC3)为序列表SEQ ID No.1中氨基酸序列所示。
所述补体成分C3在制备哈维氏弧菌(Vibrio harveyi)或海豚链球菌(Streptococcus iniae)的杀菌剂中的应用。
所述补体成分C3为以牙鲆血清为原材料，用沉降剂沉降血清，所得蛋白沉淀用20mM Tris-HCL缓冲液(pH7.4)重悬，将重悬液加入到Mono Q HR5/5阴离子交换色谱柱中，梯度洗脱收集含有C3蛋白的洗脱液进行混合浓缩并重悬在20mM的2-吗啉乙磺酸(MES)(pH5.8)中，然后将重悬液加入到Mono S HR5/5阳离子交换色谱柱中，梯度洗脱收集含有C3蛋白的洗脱液进行透析浓缩后，即为序列表SEQ ID No.1所示的补体成分C3；
所述沉降剂为终浓度的16％的聚乙二醇、20mM EDTA、10mM苄脒、 1mM苯甲基磺酰氟。
所述梯度洗脱时洗脱液为(0-1M)的NaCl溶液。
进一步的说：
1)以阴离子交换色谱柱获得初步纯化的牙鲆补体C3蛋白
取牙鲆血清(5ml)与沉降剂等体积混匀，在4℃下沉降30min，收集蛋白沉淀，用20mM Tris-HCL缓冲液(pH 7.4)重悬，将重悬液加入到Mono Q HR5/5阴离子交换色谱柱中，借助高效液相色谱仪，使用NaCl溶液(0-1M) 进行梯度洗脱，然后将洗脱液进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，并收集含有C3蛋白的洗脱液，即为初步纯化的补体成分C3蛋白；
2)以阳离子交换色谱柱获得完全纯化的牙鲆补体C3蛋白
将上述步骤1)收集的含有C3蛋白的洗脱液进行混合、浓缩并重悬在 20mM的2-吗啉乙磺酸(MES)(pH5.8)中，然后将重悬液加入到Mono S HR5/5阳离子交换色谱柱中，用NaCl溶液(0-1M)对初步纯化的补体成分C3蛋白进行梯度洗脱，收集洗脱液进行聚丙烯酰胺凝胶电泳 (SDS-PAGE)分析，并进行质谱分析，将质谱结果与序列表SEQ ID No.1所示的牙鲆补体成分C3序列进行比对，确定纯化蛋白为牙鲆C3蛋白，最后将含有牙鲆C3蛋白的洗脱液进行透析浓缩后，即为牙鲆补体C3蛋白。
本发明具有如下优点：
本发明补体成分C3是来自于牙鲆补体系统的一种天然蛋白。该补体成分C3蛋白可制备为杀菌剂，其制备过程简单，对环境和鱼体均无安全隐患，且对哈维氏弧菌和海豚链球菌产生显著的杀菌效应，进而所得蛋白在抗细菌感染中具有应用潜能。
附图说明
图1为本发明实施例提供的牙鲆补体成分C3(PoC3)蛋白对哈维氏弧菌和海豚链球菌的杀菌效应。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例旨在对本发明进行举例描述，而非以任何形式对本发明进行限制。
实施例1
补体成分C3蛋白的纯化
(1)补体成分C3蛋白的纯化
本发明的补体成分C3通过从牙鲆血清中纯化获得，其序列为序列表 SEQ ID No.1中的氨基酸序列。
序列表SEQ ID No.1为：
MGRTQLWLLASLAFASIASLVDGAPLKVMSAPNLLRVGTTENIFVECQD CTGGDIKVDINVMNHPTKVNRLATTSVTLTSGQNFQALGQIRIPAGGFSKDPRLKQYVYLQAQFPDRLLEKVVLVSFQSGFIFIQTDKTIYTPNSKVLYR MFAVTPTMEPVERDEQIQNEASIAIEFVTPEDIVLPLDPVSLKSGIHSGEYKLAEIVSPGLWKVVAKFHSNPQESFSAEFEVKEYVLPSFEVKLTSTSPFFY VESEDLTINIKATYLFGEEVSGTAYVVFGVVLEGEKKSFPSSLQRISIDNGIGLVQLKRQQITQSFQDINALVGNSLFVAVSVLTESGGEMVEAELRGIQIV KSPYTIHFRKTPKYFKPGMTFDIVVEVTNPDETPAQGVAVVVDPGAVKGLTAANGFAKLTVNTMAGVARLPVTAKTNDPLIPLARQAEATMTALPYNTK SNNYIHIGVDTAELELGDNLKVNLNLKKQSNQDTDITYLILSRGQLIQSGRYRTRGQVLISLIIPITKVMLPSFRIVAYYHLDDNEVVSDSVWVDVKDSC MGTLKLESSRPAPSYEPRKMFGLKVTGVPEAMVGLVAVDKGVYVLNNK HRITQKKVWDIIEKYDTGCTPGGGKNSMSVFYDAGLLFESSAASGTPYRQELKCPVPARRKRATTVMNVTTTLVNKYKNELQRECCSEGMKETILSYT CEVRSEYILDGASCVDAFLHCCKEMENLRGEMKQDQLDLARSEEDDSYMDSNEISSRTKFPESWLWSDIQLPACPQQTPNCESTSFMKPVPLQDSITTW QFTGISLSRTHGICVGEPLEVIVRKDFFIDLRLPYSAVRGEQLEIKAILHNY SPDVITVRVDLIEEENVCSVASKHTRYRQEVKVGQQTTRSVPFVIIPMKEGQFNIEVKAAVKDSSLNDGIVKVLRVVPEGVLIKQPQIITLDPSKTGGEQ VEILNSRIPRKDMVPNTPTTTQISVTGRENVGQLVENAISGQSMGTLIYQPSGCGEQNMIHMTLPVIAATYLDKTNQWETVGFQKRNEALQHIKTGYTN ELAYRKKDGSFSVWASHGSSSWLTAYVAKVFAMANSLVAVQNNVICDAV KYLILNAQQPDGVFKEVGRVAHGEMIGDVRGTDSEASMTAFCLIAMQESRTLCAATVNSLPGSVEKSVLYLERRLESLTNPYAVAITSYALANEGRLNKD VLYKFASPELSHWPVPKGRVYTLEATAYALLALVKTKDFEKARPIVRWFSQQQKVGGGYGSTQATIIVYQAVAEYWAAAKEPEYDLNVDILLPGRSKPE KIVLNADNSYTTRTSKINDINQDVKVTAKGTGEATVTMVSLYYALPQKKESDCQKFNMSVELIPERIDADESIFKLKIEVLYKDKERDATMSILDIGLLTGFTPNTNDLNLLSKGRARTLAKYEMNTLLSEKGSLIIYLDKVSHTRPEEITF RVHQKMKVGVLQPAAVSVYEYYDQTQCVQFYHPQRKAGQLLRLCRNEECTCAEENCSMQKKGKISNDQRIEKSCETTPTSKIDFVYKVRLEEFTDGG STDIYTMRILEGIKEGSYDVAPQGKLRTFLSYQHCRESLDLGKGKMYLIM GTSKDIHRDDQNESYHYVLGERTWIEYWPTEAECQTEEHRPTCLGLQQMVQQYLLFGCQQ
(a)序列特征：
·长度：1655
·类型：氨基酸序列
·链型：双链
·拓扑结构：线性
(b)分子类型：蛋白质
(c)假设：否
(d)反义：否
(e)最初来源：牙鲆血清
取5ml牙鲆血清，与沉降剂混匀，在4℃下沉降30min，收集蛋白沉淀，用20mM Tris-HCL缓冲液(pH7.4)重悬，将重悬液加入到Mono Q HR5/5 阴离子交换色谱柱(购于美国GEHealthcare公司)中，借助高效液相色谱仪，使用NaCl溶液(0-1M)进行梯度洗脱，然后将洗脱液进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，根据C3蛋白分子量大小分析并收集含有C3蛋白的洗脱液，即为初步纯化的补体成分C3蛋白。将收集的含有C3蛋白的洗脱液进行混合、浓缩并重悬在20mM的2-吗啉乙磺酸(MES)(pH 5.8)中，然后将重悬液加入到Mono S HR5/5阳离子交换色谱柱(购于美国GE Healthcare公司)中，用NaCl溶液(0-1M)再次对PoC3进行梯度洗脱，按洗脱液梯度收集每个浓度的洗脱液进行聚丙烯酰胺凝胶电泳 (SDS-PAGE)分析，并进行质谱分析，将质谱结果与序列表SEQ ID No.1 所示的牙鲆补体成分C3序列进行比对，确定纯化蛋白为牙鲆C3蛋白，最后将含有牙鲆C3蛋白的洗脱液进行透析浓缩后，即为牙鲆补体C3蛋白。
所述沉降剂为终浓度的16％的聚乙二醇、20mM EDTA、10mM苄脒、 1mM苯甲基磺酰氟。
实施例2
牙鲆补体成分C3蛋白(PoC3)对哈维氏弧菌和海豚链球菌的杀菌效应：
步骤1)细菌的培养
将哈维氏弧菌接种于LB培养基，海豚链球菌接种于TSB培养基,将上述细菌28℃培养至OD600约为0.8，收集菌体，用无菌PBS清洗3遍，然后将其重悬于PBS中至终浓度为106CFU/ml。
步骤2)牙鲆补体成分C3蛋白(PoC3)杀菌活性检测
将上述步骤1)中细菌稀释液与浓缩后经PBS配置至终浓度为50μg/ml 的实施例1中纯化的PoC3、相同浓度的牛血清白蛋白(BSA)或PBS(对照组)混合，室温下孵育1h。为计算准确将混合液梯度稀释，均匀涂在固体LB或TSB平板上，放于28℃培养箱中培养12h，对平板进行计数。结果表明，PoC3可以直接杀伤哈维氏弧菌和海豚链球菌，显著的降低细菌的存活率(图1)，具有较强的杀菌效应，而BSA组和对照组处理的细菌存活率没有差异，因此补体成分C3蛋白(PoC3)能够作为一种天然杀菌剂应用于牙鲆细菌性疾病的防治。
序列表
<110> 中国科学院海洋研究所
<120> 一种牙鲆补体成分C3（PoC3）的应用
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His Ile Lys Thr Gly Tyr Thr Asn Glu Leu Ala Tyr Arg Lys Lys Asp
1045 1050 1055
Gly Ser Phe Ser Val Trp Ala Ser His Gly Ser Ser Ser Trp Leu Thr
1060 1065 1070
Ala Tyr Val Ala Lys Val Phe Ala Met Ala Asn Ser Leu Val Ala Val
1075 1080 1085
Gln Asn Asn Val Ile Cys Asp Ala Val Lys Tyr Leu Ile Leu Asn Ala
1090 1095 1100
Gln Gln Pro Asp Gly Val Phe Lys Glu Val Gly Arg Val Ala His Gly
1105 1110 1115 1120
Glu Met Ile Gly Asp Val Arg Gly Thr Asp Ser Glu Ala Ser Met Thr
1125 1130 1135
Ala Phe Cys Leu Ile Ala Met Gln Glu Ser Arg Thr Leu Cys Ala Ala
1140 1145 1150
Thr Val Asn Ser Leu Pro Gly Ser Val Glu Lys Ser Val Leu Tyr Leu
1155 1160 1165
Glu Arg Arg Leu Glu Ser Leu Thr Asn Pro Tyr Ala Val Ala Ile Thr
1170 1175 1180
Ser Tyr Ala Leu Ala Asn Glu Gly Arg Leu Asn Lys Asp Val Leu Tyr
1185 1190 1195 1200
Lys Phe Ala Ser Pro Glu Leu Ser His Trp Pro Val Pro Lys Gly Arg
1205 1210 1215
Val Tyr Thr Leu Glu Ala Thr Ala Tyr Ala Leu Leu Ala Leu Val Lys
1220 1225 1230
Thr Lys Asp Phe Glu Lys Ala Arg Pro Ile Val Arg Trp Phe Ser Gln
1235 1240 1245
Gln Gln Lys Val Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Ile Ile
1250 1255 1260
Val Tyr Gln Ala Val Ala Glu Tyr Trp Ala Ala Ala Lys Glu Pro Glu
1265 1270 1275 1280
Tyr Asp Leu Asn Val Asp Ile Leu Leu Pro Gly Arg Ser Lys Pro Glu
1285 1290 1295
Lys Ile Val Leu Asn Ala Asp Asn Ser Tyr Thr Thr Arg Thr Ser Lys
1300 1305 1310
Ile Asn Asp Ile Asn Gln Asp Val Lys Val Thr Ala Lys Gly Thr Gly
1315 1320 1325
Glu Ala Thr Val Thr Met Val Ser Leu Tyr Tyr Ala Leu Pro Gln Lys
1330 1335 1340
Lys Glu Ser Asp Cys Gln Lys Phe Asn Met Ser Val Glu Leu Ile Pro
1345 1350 1355 1360
Glu Arg Ile Asp Ala Asp Glu Ser Ile Phe Lys Leu Lys Ile Glu Val
1365 1370 1375
Leu Tyr Lys Asp Lys Glu Arg Asp Ala Thr Met Ser Ile Leu Asp Ile
1380 1385 1390
Gly Leu Leu Thr Gly Phe Thr Pro Asn Thr Asn Asp Leu Asn Leu Leu
1395 1400 1405
Ser Lys Gly Arg Ala Arg Thr Leu Ala Lys Tyr Glu Met Asn Thr Leu
1410 1415 1420
Leu Ser Glu Lys Gly Ser Leu Ile Ile Tyr Leu Asp Lys Val Ser His
1425 1430 1435 1440
Thr Arg Pro Glu Glu Ile Thr Phe Arg Val His Gln Lys Met Lys Val
1445 1450 1455
Gly Val Leu Gln Pro Ala Ala Val Ser Val Tyr Glu Tyr Tyr Asp Gln
1460 1465 1470
Thr Gln Cys Val Gln Phe Tyr His Pro Gln Arg Lys Ala Gly Gln Leu
1475 1480 1485
Leu Arg Leu Cys Arg Asn Glu Glu Cys Thr Cys Ala Glu Glu Asn Cys
1490 1495 1500
Ser Met Gln Lys Lys Gly Lys Ile Ser Asn Asp Gln Arg Ile Glu Lys
1505 1510 1515 1520
Ser Cys Glu Thr Thr Pro Thr Ser Lys Ile Asp Phe Val Tyr Lys Val
1525 1530 1535
Arg Leu Glu Glu Phe Thr Asp Gly Gly Ser Thr Asp Ile Tyr Thr Met
1540 1545 1550
Arg Ile Leu Glu Gly Ile Lys Glu Gly Ser Tyr Asp Val Ala Pro Gln
1555 1560 1565
Gly Lys Leu Arg Thr Phe Leu Ser Tyr Gln His Cys Arg Glu Ser Leu
1570 1575 1580
Asp Leu Gly Lys Gly Lys Met Tyr Leu Ile Met Gly Thr Ser Lys Asp
1585 1590 1595 1600
Ile His Arg Asp Asp Gln Asn Glu Ser Tyr His Tyr Val Leu Gly Glu
1605 1610 1615
Arg Thr Trp Ile Glu Tyr Trp Pro Thr Glu Ala Glu Cys Gln Thr Glu
1620 1625 1630
Glu His Arg Pro Thr Cys Leu Gly Leu Gln Gln Met Val Gln Gln Tyr
1635 1640 1645
Leu Leu Phe Gly Cys Gln Gln
1650 1655

Claims (5)

1.一种牙鲆补体成分C3的应用，其特征在于：牙鲆补体成分C3（PoC3）在制备哈维氏弧菌（Vibrio harveyi）或海豚链球菌（Streptococcus iniae）的杀菌剂中的应用。
2.按权利要求1所述的补体成分C3的应用，其特征在于：所述补体成分C3（PoC3）为序列表SEQ ID No.1中氨基酸序列所示。
3. 按权利要求1-2任意一项所述的补体成分C3的应用，其特征在于：所述补体成分C3为以牙鲆血清为原材料，用沉降剂沉降血清，所得蛋白沉淀用20mM pH 7.4的Tris-HCL缓冲液重悬，将重悬液加入到Mono Q HR5/5阴离子交换色谱柱中，梯度洗脱收集含有C3蛋白的洗脱液进行混合浓缩并重悬在20mM pH 5.8的2-吗啉乙磺酸中，然后将重悬液加入到MonoS HR5/5阳离子交换色谱柱中，梯度洗脱收集含有C3蛋白的洗脱液进行透析浓缩后，即为序列表SEQ ID No.1所示的补体成分C3；
所述沉降剂为终浓度为16%的聚乙二醇、20mM EDTA、10mM苄脒、1mM苯甲基磺酰氟的组合。
4.按权利要求3所述的补体成分C3的应用，其特征在于：所述梯度洗脱时洗脱液为0-1M的NaCl溶液。
5.按权利要求3所述的补体成分C3的应用，其特征在于：
1) 以阴离子交换色谱柱获得初步纯化的补体成分C3蛋白：
取牙鲆血清与沉降剂等体积混匀，在4℃下沉降30min，收集蛋白沉淀，用20mM pH7.4Tris-HCL缓冲液重悬，将重悬液加入到Mono Q HR5/5阴离子交换色谱柱中，借助高效液相色谱仪，使用0-1M的 NaCl溶液进行梯度洗脱，然后将洗脱液进行聚丙烯酰胺凝胶电泳分析，并收集含有C3蛋白的洗脱液，即为初步纯化的补体成分C3蛋白；
2) 以阳离子交换色谱柱获得纯化完全的补体成分C3蛋白
将上述步骤1) 收集的含有C3蛋白的洗脱液进行混合、浓缩并重悬在20mM pH5.8的2-吗啉乙磺酸中，然后将重悬液加入到Mono S HR5/5阳离子交换色谱柱中，用0-1M 的NaCl溶液再次对初步纯化的补体成分C3蛋白进行梯度洗脱，收集洗脱液进行聚丙烯酰胺凝胶电泳分析，并进行质谱分析，将质谱结果与序列表SEQ ID No.1所示的牙鲆补体成分C3序列进行比对，确定纯化蛋白为牙鲆C3蛋白，最后将含有牙鲆C3蛋白的洗脱液进行透析浓缩后，即为牙鲆补体C3蛋白。
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