CN110256570A - 一种重组融合抗菌肽及应用 - Google Patents
一种重组融合抗菌肽及应用 Download PDFInfo
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- CN110256570A CN110256570A CN201711439057.1A CN201711439057A CN110256570A CN 110256570 A CN110256570 A CN 110256570A CN 201711439057 A CN201711439057 A CN 201711439057A CN 110256570 A CN110256570 A CN 110256570A
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Abstract
本发明公开了一种重组融合抗菌肽及应用。有益效果为:本发明提供的重组融合抗菌肽对革兰氏阴性菌和革兰氏阳性菌均有显著的抑菌效果,其MIC达到1.25~5mmol/L,比单独使用Hepcidin抗菌肽和Moronecidin抗菌肽的抑菌效率提高4~32倍;将抗菌肽用于制备抗菌制剂或抗菌饲料添加剂,所得抗菌制剂具有显著的抑菌效果,该饲料添加剂能够显著降低水产动物的发病率,增强水产动物的抵抗力,提高其成活率。
Description
技术领域
本发明涉及生物工程技术领域,尤其是涉及一种重组融合抗菌肽及应用。
背景技术
在治疗高等动物发生的各种细菌性、病毒性疾病及其他病害中,由于抗生素的大量使用,使病原菌易对药物产生耐药性,因而无法有效的控制疾病的发生,而筛选新的抗生素也极为困难。抗菌肽(antimicrobial peptide)是生物防御外来病菌时,能够迅速诱导合成的一系列的具有抗菌或杀菌活性多肽类物质。当机体遇到病原体入侵时,以活体形式由相应的细胞合成并释放出来,发挥抗菌作用及其它生物学功能。与传统抗生素相比,抗菌肽的抗菌活性还不够理想,有的还存在细胞毒性。因此,对已有抗菌肽进行人工改造和设计新抗菌肽分子已成为抗菌肽开发的一个重要内容。
发明内容
在第一方面,本发明提供了一种氨基酸序列为SEQ ID NO.1的重组融合抗菌肽,该抗菌肽对革兰氏阴性菌和革兰氏阳性菌均有显著的抑菌效果,其MIC达到1.25~5mmol/L,比单独使用Hepcidin抗菌肽和Moronecidin抗菌肽的抑菌效率提高4~32倍。
本发明中重组融合抗菌肽的氨基酸序列SEQ ID NO.1为:FFHHIFRGIVHVGKTIHKLVTGGGGGSGGGGSGCRFCCNCCPNMSGCGVCCRFGGGGSGGGGSRNYIDLSSRNLSSVPGDLPKEAELIDLSRNLIQLLQRGDFWNTPILRFLNISWNCLESIHPEVFLGTPLLQDLDLSHNCLKNLTDQPYLQRAGNLLFLNLAYNKFVTMTLAREFSSLAKLERLTLGGNVIRVGDFGNIADVELRLLSLHLEGKLLYEPGSLKDAYARRLQVELNKVFPLHLINDALSFFAEVELLKLPEGCRELSRQLSQRAEIYTSRLFLTNVSINWSDFTRCVTVALNTTVSHLSVSDVTLHRLPQTDTPMANTSRVKSVTVREVTVKSFLFSQEAVYNFFINMPVESLALTDTAIIRMTCPKSQSPVTHLSFSHCSLSDTIFSRVEGLITIECKTLGNLRTLTLTRNNFKSLQSLSKRMRYMKSLQDLDLSFNRLVYDGQGECYWPQNISILHLSSNSLTSSAFQCLPTGVERLDLQNNQLSAVSSSTLKLTRLLSLNLNANRLLDLPVCDNFPLLQELLLRSNSLHAPSVDRLESCPRLKTLDVSYNALMCICPLRGFIRLGLESEKNRTGVTFLQWPQGYYCSYPEAFKDSNLNNIWISEISCNTHHHHHH。
在第二方面,本发明提供了编码上述抗菌肽的基因,该基因具有SEQ ID NO.2所示的核苷酸序列。具有该核苷酸序列的基因所编码的多肽对革兰氏阴性菌和革兰氏阳性菌均有显著的抑菌效果,对新型抗菌药物的开发具有重要的意义。
基因的核苷酸序列SEQ ID NO.2为:ttctttcaccacattttccgtggaattgttcacgtcggcaagacgatccacaaacttgtgaccgggggtggcggtggaagcggcggtggcggaagcggctgtcgcttttgctgcaattgctgtcctaatatgagcggatgtggtgtctgctgcaggttcggtggcggtggaagcggcggtggcggaagccgcaactatattgacctctcgtccaggaacctctcgtcggtcccgggagaccttccaaaggaagcggagcttatcgacctgtcacgcaacctcatacagctgcttcaacgaggagacttctggaacacccccatcctcagattcctcaacatttcatggaattgtttggaaagtatccacccagaggtgttcctcggcacgccgctgctgcaggatctggacctgtcacacaactgcctgaagaacctcacggatcagccgtacctgcagcgtgctggaaacctcctgtttctgaatttggcctacaataagtttgtcaccatgaccctggccagggagttcagctccctggcgaagctggagagattaacgctgggagggaacgtcatcagagtgggggacttcgggaatattgctgacgtggagctgcgcctgctgagcctccacctggagggcaaactgctctacgaaccaggatctctgaaggacgcgtacgcacggaggctccaagtggaactgaacaaagtattccccctccacctgataaacgacgctctgtccttctttgccgaagtggagctgctgaagttgcccgagggctgtcgagagctgagcaggcagctgagccagagggctgaaatctacacgtctcgcctgtttttgaccaacgtatccatcaactggtccgactttactcggtgtgtcacagtagccctgaacaccaccgttagccacctgagtgtctccgacgtgaccttacacagactacctcaaacagacacgccgatggcaaacacctctcgagtgaagtctgtcacggtgagagaagtaacggtgaagagttttctgttttcgcaggaggcggtctacaacttcttcatcaacatgccggtggagagtttagcgctcactgacactgccatcatacgcatgacctgcccaaagtcccagagtcccgtcacgcatctgagtttctcccactgcagtctgagcgacaccatcttttccagagtggaaggcttgataacgatcgaatgcaagactcttgggaatttgaggacgttgactctgacgagaaacaacttcaagagtctccagtcgctcagcaaacgcatgcgatacatgaaatccctgcaggatctggacctcagcttcaaccggctggtgtatgacggacagggggagtgctactggccgcagaacatcagcatccttcacctgtcttccaatagtttgaccagctctgcgtttcaatgcctccctacgggtgtggagagactggacctccagaacaatcagctttccgctgtttcatcatccacgttgaaactaacgagacttttgtccttgaacctaaacgccaacaggctgctggacctgcccgtgtgtgacaacttccctttgctgcaggagcttctgctcaggtcaaattctctccacgcaccatctgtggacaggctggagagctgccccagactgaaaaccctggacgttagctacaacgccctcatgtgcatctgtcctctgaggggcttcatccgacttggcctcgaatctgagaagaatcgcacaggtgttacgttcttgcagtggccacagggctactactgcagctacccagaggcctttaaggattccaacctcaacaacatctggatctcagagatttcctgtaacactcaccaccaccaccaccactaa。
在第三方面,本发明提供了包含上述基因的载体,该原始载体为pET-28a或pYES2。原始载体在N端没有其它过多的序列,能较好地还原外源蛋白的真实状态,便于后期开展对蛋白功能的研究。
在第四方面,本发明提供了上述载体转化的宿主细胞,该宿主细胞为大肠杆菌Rosetta宿主细胞或酿酒酵母INVSC1宿主细胞。该宿主为内源蛋白酶缺陷型菌株,使获得的外源表达产物更加稳定。
在第五方面,本发明提供了将抗菌肽用于制备抗菌制剂或饲料添加剂中的用途。将抗菌肽用于制备抗菌制剂,所得抗菌制剂具有显著的抑菌效果;将抗菌肽用于制备饲料添加剂,所得饲料添加剂能够显著降低水产动物的发病率,增强水产动物的抵抗力,提高其成活率,同时降低了抗生素和杀虫剂对水产品和养殖环境造成的污染,保证了食品的安全,对人类健康和良好的生态环境具有重要的意义。
与现有技术相比,本发明的优点在于:本发明提供的重组融合抗菌肽对革兰氏阴性菌和革兰氏阳性菌均有显著的抑菌效果,其MIC达到1.25~5mmol/L,比单独使用Hepcidin抗菌肽和Moronecidin抗菌肽的抑菌效率提高4~32倍;将抗菌肽用于制备抗菌制剂或抗菌饲料添加剂,所得抗菌制剂具有显著的抑菌效果,该饲料添加剂能够显著降低水产动物的发病率,增强水产动物的抵抗力,提高其成活率。
附图说明
图1为TLR1基因克隆、重组TLR1-H-M基因及酶切鉴定;
其中,A.河豚TLR1基因胞外区的PCR结果,DNA DGL2000 Marker;Lane 1河豚TLR1基因胞外区PCR结果;B.含TLR1-H-M重组质粒鉴定;DNA 15000 DL Marker;Lane 1~2pET28a以及重组pET28a-TLR1-H-M质粒电泳;C.pET28a-TLR1-H-M酶切鉴定,DNA DGL2000 Marker;Lane 1pET28a-TLR1-H-M质粒酶切结果;
图2为TLR1-H-M重组融合抗菌肽的原核表达图,其中,Protein Marker分别为14.3kDa、20.1kDa、29.0kDa、44.3kDa、66.4kDa、97.2kDa;Lane 1不加IPTG诱导对照全菌蛋白;Lane 2~4为IPTG诱导2h、4h、6h全菌蛋白(箭头所示为预期的目的蛋白);
图3为TLR1-H-M重组融合抗菌肽在酿酒酵母中的表达,其中,A不同浓度半乳糖诱导TLR1-H-M重组融合抗菌肽表达,M为Protein Marker:14.3kDa、20.1kDa、29.0kDa、44.3kDa、66.4kDa、97.2kDa;C为不加半乳糖诱导对照全菌蛋白;Lane1~4分别为0.5、1.0、1.5及2.0%半乳糖诱导TLR1-H-M重组融合抗菌肽表达;B.2.0%半乳糖不同诱导时间TLR1-H-M重组融合抗菌肽表达,M为Protein Marker:14.3kDa、20.1kDa、29.0kDa、44.3kDa、66.4kDa、97.2kDa;C为不加半乳糖诱导对照全菌蛋白;Lane 1~4分别为24h、48h及72h诱导TLR1-H-M重组融合抗菌肽表达。
具体实施方式
下面通过附图和实施例对本发明做进一步说明:
实施例1:一种重组融合抗菌肽,制备过程如下:
1)鱼类TLR1基因的克隆:参考高等物种中Toll-like-1受体(TLR1)基因序列,设计兼并引物,从河豚中克隆鱼类TLR1基因,具体方法为:选择河豚脾脏组织,提取总RNA,按照Takara 3’Full RACE Core Set试剂盒说明,合成第一链cDNA,分别用引物TLR1-F2、TLR1-F3、TLR1-F4、TLR1-F6、TLR1-R1、TLR1-R2和TLR1-R3扩增TnTLR1基因序列的中间片断(表1),然后采用3’RACE和5’RACE的方法扩增基因两端序列,经拼接组装,获得全长cDNA序列,序列分析表明,TnTLR1cDNA序列全长2587bp,其中5’UTR为122bp,3’UTR为74bp,ORF为2391bp;3’UTR中在终止密码子下游43bp处存在一个polyA加尾信号AATAAA,polyA尾巴位于加尾信号下游13个碱基处。然后在阅读开放框两端设计引物,扩增TLR1全长cDNA,克隆入pUcmT载体中,用设计的表达载体构建引物进行PCR扩增,获得了TnTLR1的胞外区序列(图1中A,SEQ IDNO.3),其长度为1680bp。
表1 PCR克隆TnTLR1所用引物
2)重组融合抗菌肽(TLR1-H-M)基因构建:根据步骤1)中获得的TnTLR1基因序列设计特异性引物,用Takara的PyrobestTMDNA Polymerase扩增TnTLR1受体的胞外可溶性片段编码基因序列,并将该序列与hepcidin和moronecidin编码基因通过柔性铰链区G4S序列进行融合。采用改良的重叠延伸PCR方法将hepcidin和moronecidin基因重组到TnTLR1胞外编码序列的5’端,构建含有目的片段的表达质粒,具体方法为:以克隆了TnTLR1全长序列的pUcmT载体为模板,用引物TLREP-F(5’CGGATGTGGTGTCTGCTGCAGGTTCGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCCGCAACTATATTGACCTCTCGTCC 3’,如SEQ ID NO.18所示,含TLR1胞外区部分序列+编码G4S铰链区序列+部分hepcidin序列)和TLREP-R(5’TTTTCCTTTTGCGGCCGCAGTGTTACAGGAAATCTCTGAGATCC 3’,如SEQ ID NO.19所示,TLR1胞外区reverse引物,含NotI酶切位点)经第一轮PCR扩增,得到TnTLR1胞外区部分并引入铰链区及部分hepcidin序列,然后用PLEEP-F引物(5’GGAATTCCATATGGCCATGGGCTGTCGCTTTTGCTGCAATTGCTGTCCTAATATGAGCGGATGTGGTGTCTGCTGCAGG 3’,如SEQ ID NO.20所示,含Hepcidin全长序列+与TLREP-F引物中部分hepcidin配对序列)和TLREP-R引物(5’TTTTCCTTTTGCGGCCGCAGTGTTACAGGAAATCTCTGAGATCC3’,如SEQ ID NO.21所示),经过第二轮PCR扩增,引入Hepcidin全长序列,得到hepcidin-TLR1,在此基础上,再分别用引物H-F(5’GCAAGACGATCCACAAACTTGTGACCGGGGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCTGTCGCTTTTGCTGCAATT 3’,如SEQ ID NO.22所示,与hepcidin配对序列+部分moronecidin序列)和H-M-R(5’TTTTCCTTTTGCGGCCGCTTAGTGGTGGTGGTGGTGGTGAGTGTTACAGGAAATCTC 3’,如SEQ ID NO.23所示,TLREP-R引物序列+his-tag编码序列)以及M-F(5’CGCGGATCCCGCCACCATGGGCTTCTTTCACCACATTTTCCGTGGAATTGTTCACGTCGGCAAGACGATCCACAAACTTGT 3’,如SEQ ID NO.24所示,含moronecidin全长序列+与H-F匹配序列)和M-H-R(5’TTTTCCTTTTGCGGCCGCTTAGTGGTGGTGGTGGTGGTGAGTGTTACAGGAAATCTC 3’,如SEQ IDNO.25所示,TLREP-R引物序列+his-tag编码序列+BamHI和NcoI酶切位点),经过两轮PCR,在hepcidin-TLR1分子中引入moronecidin,得到moronecidin-hepcidin-TLR1重组分子(命名为TLR1-H-M),其核苷酸序列如SEQ ID NO.2所示,其中Hepcidin全长序列如SEQ ID NO.4所示,moronecidin全长序列如SEQ ID NO.5所示;
3)重组融合抗菌肽(TLR1-H-M)原核表达质粒构建与表达:在20μL反应体系中,分别对载体pET28a和TLR1-H-M进行双酶切(NdeI/NotI),37℃反应4h,割胶回收,用DNA纯化回收试剂盒回收片段,连接pET28a和TLR1-H-M双酶切后回收片段,16℃过夜,将连接产物转化大肠杆菌Top10感受态,37℃培养箱过夜培养,直至长出单菌落。挑取单菌落,鉴定阳性克隆,并送Invitrogen(上海英骏生物技术有限公司)进行序列测定。将含有测序正确的pET28a-TLR1-H-M的菌株进行扩培,用碱裂解法提取质粒,将一部分质粒和菌液于-80℃保存,一部分用于转化大肠杆菌Rossette感受态,诱导目的蛋白表达,采用SDS-PAGE检测表达。
对含TLR1-H-M基因片段的重组质粒(pET28a-TLR1-H-M)以及空质粒(pET28a)分别进行电泳比对分析,结果显示重组质粒分子大于空质粒,而且大小如预期(图1中B);同时,对含TLR1-H-M基因片段的重组质粒及其空质粒分别进行双酶切鉴定,结果得到了片段大小与预期相符的质粒片段和TLR1-H-M重组基因片段(图1中C),可知,序列没有发生变化,读码框也没有发生移位,表明成功地构建了hepcidin-moronecidin与TnTLR1胞外区串联的重组表达载体。
利用已经正确构建的重组表达质粒转化表达菌,利用抗性筛选得到重组菌后,通过IPTG来诱导重组蛋白的表达,结果见图2,由图可见,TLR1-H-M能在表达菌内正常表达,且随着诱导时间的延长,总蛋白中的融合蛋白的表达量也逐渐增加,在诱导4~6h后融合蛋白的表达都基本达到高峰。实验表明利用pET28a质粒作为载体,Rossette菌株作为表达菌能实现TLR1-H-M融合蛋白的正常表达;
4)重组融合抗菌肽(TLR1-H-M)酵母表达质粒构建:利用酿酒酵母(Saccharomycescerevisiae)INVSC1菌株和带有酵母GAL1启动子的E.coli-酵母穿梭表达质粒pYES2进行表达。实验使用的E.coli菌种TOP10和酵母表达质粒pYES2为本实验室保存。具体方法为:以上述构建的TLR1-H-M重组基因为模板,PCR扩增在起始密码子ATG前引入Kozak序列ACC,然后双酶切,重组入pYES2质粒中,转化TOP10,随机挑取单菌落,接种于含Amp(50ug/ml)的LB液体培养基中,37℃培养12~16小时,碱裂解法抽提少量质粒,重组质粒用PCR检测,根据已知的pYES2通用引物基因序列,设计一对引物(pYES2-F:5’AAAACCCCGGATCGGACTAC 3’,如SEQ ID NO.26所示,pYES2-R:5’GGGAGGGCGTGAATGTAAGC 3’,如SEQ ID NO.27所示)进行PCR检测,并设空载体对照,筛选***位点和长度正确的重组子并测序验证。
重组表达质粒以电转化的方式导入酵母感受态细胞。
酵母感受态细胞的制备方法为:挑取YPD培养板上一单克隆INVSC1菌株,接种入2ml YPD液体培养基中,30℃,250~300rpm振荡培养过夜;取少量SC悬浮液涂SC平板,鉴定表型;取200ul接种至100ml含YPD培养基的三角摇瓶中,30℃振荡培养过夜至OD600为1.3~1.5;细胞冰浴15min终止生长;4℃离心收集细胞,用100ml预冷的无菌水重悬并离心洗涤细胞三次,然后用4ml预冷的1M山梨醇重悬并离心洗涤细胞两次,最终将细胞悬浮于100ul预冷的1M山梨醇中,按40ul/管分装,4℃保存一周。
电脉冲转化酿酒酵母细胞的方法为:将40ul酵母悬浮液与5ul质粒DNA混合,置预冷的电转化杯(0.2cm),冰浴5min;脉冲参数为V=1.5kV,25uF,200Ohms,4-5ms;电转化后立刻加1ml预冷的1M山梨醇,涂布SC-U培养基中选择培养,每200ul涂布一块平板;将平板至于30℃培养,直至单菌落出现,然后提取酵母质粒DNA,用PCR法鉴定阳性转化子;
5)重组融合抗菌肽(TLR1-H-M)在酿酒酵母中的表达:挑取步骤4)得到的单菌落重组子,接种至含2%半乳糖的15ml SC-U培养基中,30℃振荡培养过夜;取过夜培养物测OD600值,由此计算在50ml诱导培养基中所需加入的过夜培养物的数量,诱导培养基初始OD值需达到0.4(0.4OD/ml),然后4℃离心收集细胞;用1~2ml诱导培养基重悬细胞,接种至50ml诱导培养基,30℃振荡培养,并在不同时间收集细胞,检测重组蛋白(重组融合抗菌肽)的表达情况。
重组蛋白的提取方法为:用裂解液悬浮酵母细胞,将OD600值调整到50~100,加入500ul裂解液和等体积玻璃珠(Sigma G-8772),振荡30s,冰浴30s,重复4次裂解细胞,然后取部分镜检,观察细胞破碎效果,高速离心10min,收集上清液,用SDS-PAGE电泳分析,如图3所示,利用酿酒酵母INVSC1菌株和pYES2质粒,构建重组转化子,并对重组融合抗菌肽进行诱导表达。经不同浓度半乳糖诱导不同时间,SDS-PAGE检测,表达量可达到总酵母蛋白的20%以上;
实施例2:一种重组融合抗菌肽药效试验:
1)重组融合抗菌肽(TLR1-H-M)的活性测定:重组融合抗菌肽(TLR1-H-M)的活性用最低抑菌浓度(minimum inhibitory concentration,MIC)表示,采用微量稀释法进行测定,在96孔板中加入倍比稀释的不同浓度的重组融合抗菌肽10ul,挑选对数生长期的各种测试菌菌落,用相应测试菌培养基稀释细菌至OD600为0.001(终浓度约105CFU/mL),然后接种90ul至96孔板中,35℃孵育过夜。同时设人工合成的moronecidin抗菌肽(氨基酸序列如SEQ IDNO.5所示)和hepcidin抗菌肽(氨基酸序列如SEQ ID NO.6所示)作为阳性对照,空白PBS液为阴性对照,通过测定OD600吸光值的变化或涂布琼脂平板统计克隆形成单位(cloneforming unit,CFU)判断抗菌肽的抑菌效果,以未添加抗菌物质的OD600为对照,将50%浑浊度的抗菌肽浓度定义为MIC,结果如表2所示,它对所测试的几种鱼类代表性致病菌,包括嗜水气单胞菌(A.hydrophilia)、哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(V.alginolyticus)、灭鲑气单胞菌(A.salmonicida)、迟钝爱德华氏菌(E.tarda)、绿脓杆菌(P.aeruginosa)、副溶血弧菌(V.parahemolyticus)、鳗弧菌(V.anguillarum)等革兰氏阴性菌以及海豚链球菌(S.iniae)、鲑鱼肾杆菌(R.Salmoninarum)、金黄色葡萄(S.aureus)和李斯特菌(L.monocytogenes)等革兰氏阳性菌均有显著的抑菌效果,其MIC达到1.25~5mmol/L,比单独使用Hepcidin和Moronecidin的抑菌效率提高4~32倍,而且对革兰氏阴性菌和革兰氏阳性菌均显示出较高的抑菌效果,表明重组融合抗菌肽实现了三种抗菌肽的功能互补。
表2 TLR1-H-M重组融合抗菌肽的抗菌活性测定
细菌 | 性质 | Hepcidin | Moronecidin | TLR-H-M |
嗜水气单胞菌 | Gram-negative | >20 | >10 | >2.5 |
哈维氏弧菌 | Gram-negative | >10 | >5 | >1.25 |
溶藻弧菌 | Gram-negative | >10 | >5 | >1.25 |
灭鲑气单胞菌 | Gram-negative | >20 | >10 | >2.5 |
迟钝爱德华氏菌 | Gram-negative | >20 | >5 | >1.25 |
绿脓杆菌 | Gram-negative | >20 | >10 | >5 |
副溶血弧菌 | Gram-negative | >10 | >5 | >1.25 |
鳗弧菌 | Gram-negative | >20 | >5 | >1.25 |
海豚链球菌 | Gram-positive | >40 | >5 | >2.5 |
鲑鱼肾杆菌 | Gram-positive | >40 | >5 | >1.25 |
金黄色葡萄球菌 | Gram-positive | >40 | >5 | >2.5 |
李斯特菌 | Gram-positive | >40 | >10 | >5 |
2)重组融合抗菌肽(TLR1-H-M)对鲫鱼病害的防治作用:取一龄鲫鱼,背鳍皮下注射TLR-H-M抗菌肽0.1ml(30mg/尾),12小时后人工感染嗜水气单胞菌(1′108CFU/尾),同时再注射TLR-H-M 0.1ml(30mg)/尾,12小时后再注射一次,28℃水温观察2周,统计发病死亡率,对照组感染细菌,同时注射空白PBS液,结果如表3所示,注射TLR-H-M抗菌肽的实验组,其传染性出血性败血症的发病率显著下降,平均成活率为58.56%,而注射空白PBS液的对照组,其平均成活率为23.55%,表明注射TLR-H-M抗菌肽后能显著增强鲫鱼对嗜水气单胞菌感染的抵抗力,平均成活率能提高35.01%。
表3鲫鱼体内注射抗菌肽抗病试验结果
3)重组融合抗菌肽(TLR1-H-M)对甲鱼病害的防治作用:取一龄中华鳖,足部皮下注射TLR-H-M抗菌肽0.1ml(30mg/尾),12小时后人工感染嗜水气单胞菌(1′108CFU/尾),同时再注射TLR-H-M 0.1ml(30mg)/尾,12小时后再注射一次,28℃水温观察2周,统计发病死亡率,对照组感染细菌,同时注射空白PBS液。结果如表4所示显示,注射TLR-H-M抗菌肽的实验组,其穿孔病的发病率显著下降,平均成活率为59%,而注射空白PBS液的对照组,其平均成活率为25%,表明注射TLR-H-M抗菌肽后能显著增强中华鳖对嗜水气单胞菌感染的抵抗力,平均成活率能提高34%。
表4甲鱼体内注射抗菌肽抗病试验结果
4)重组融合抗菌肽(TLR1-H-M)在大黄鱼养殖中的病害防治作用:将养殖大黄鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含鱼类重组融合抗菌肽的饲料(100mg/Kg饲料)投喂,连续投喂4周,统计自然发病死亡率。其中,饲料的制备方法为:取鱼粉10份,发酵花生粕22份,鱼油2份,大豆磷脂油1份,面粉40-45份,氯化胆碱0.1-0.3份混合均匀,再加入0.01份重组融合抗菌肽(TLR1-H-M),搅拌均匀,再加入0.004份N-甲基乙酰胺和0.006份甲基环戊二烯,经过粗粉机和超微粉碎机,分选、制粒、熟化、烘干即得饲料。试验期间不使用其它药物。所用的饲料适口性好,易被消化吸收;加入的N-甲基乙酰胺、甲基环戊二烯与氯化胆碱具有协同作用,促进了细菌膜表面对重组融合抗菌肽(TLR1-H-M)的吸附,使疏水性的C端***膜内疏水区并改变膜的构象,大大破坏了细胞膜的完整性,造成细胞内容物泄露,进而导致了细胞的凋亡。实验结果如表5所示,使用含鱼类重组融合抗菌肽饲料的养殖大黄鱼,平均成活率达到91.03%,而不使用鱼类重组融合抗菌肽饲料的对照组大黄鱼,平均成活率为69.76%,使用鱼类重组融合抗菌肽饲料的大黄鱼平均成活率提高21.27%,表明鱼类重组融合抗菌肽具有明显提高大黄鱼的抗病能力。
表5鱼类重组融合抗菌肽在大黄鱼养殖中的抗病试验
5)饲料添加重组融合抗菌肽(TLR1-H-M)对鲫鱼病害的防治作用:取一龄鲫鱼,连续饲喂添加TLR-H-M重组融合抗菌肽的饲料(每公斤饲料添加表达TLR-H-M酿酒酵母工程菌粉100mg,TLR-H-M蛋白在酵母中的表达量为30%左右,重组融合抗菌肽在饲料中的含量为30mg TLR-H-M/Kg饲料)一周,每天饲喂两次,然后感染嗜水气单胞菌(1′108CFU/尾),感染后再连续饲喂1周,28℃水温观察2周,统计发病死亡率,对照组饲喂不含抗菌肽的正常饲料,结果如表6所示。
表6鲫鱼饲料添加抗菌肽抗病试验结果
实验组鲫鱼的传染性出血性败血症的发病率显著下降,平均成活率为43.01%,而对照组鲫鱼的平均成活率为22.22%,表明以饲喂重组融合抗菌肽的形式也能增强鲫鱼对嗜水气单胞菌感染的抵抗力,平均成活率能提高20.79%。
6)饲料添加重组融合抗菌肽对鲈鱼病害的防治作用:取一龄养殖花鲈,连续饲喂添加TLR-H-M重组融合抗菌肽的饲料(每公斤饲料添加表达TLR-H-M酿酒酵母工程菌粉100mg,TLR-H-M蛋白在酵母中的表达量为30%左右,重组融合抗菌肽在饲料中的含量为30mg TLR-H-M/Kg饲料)一周,每天饲喂两次,然后感染哈维氏弧菌(5′107CFU/尾),感染后再连续饲喂1周,28℃水温观察2周,统计发病死亡率,对照组饲喂不含抗菌肽的正常饲料,结果如表7所示,实验组鲈鱼的出血性皮肤溃疡病发病率显著下降,平均成活率为43.14%,而对照组鲈鱼的平均成活率为19.99%,表明以饲喂重组融合抗菌肽的形式也能增强鲈鱼对哈维氏弧菌感染的抵抗力,平均成活率能提高23.15%。
表7鲈鱼饲喂含抗菌肽饲料的抗病试验结果
7)重组融合抗菌肽(TLR1-H-M)在鲈鱼养殖中的病害防治作用:将养殖鲈鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含鱼类重组融合抗菌肽的饲料(100mg/Kg饲料)投喂,连续投喂4周,统计自然发病死亡率。试验期间不使用其它药物。结果如表8显示,使用含鱼类重组融合抗菌肽饲料的养殖鲈鱼,平均成活率达到91.26%,而不使用鱼类重组融合抗菌肽饲料的对照组鲈鱼,平均成活率为69.16%,使用鱼类重组融合抗菌肽饲料的鲈鱼的平均成活率提高了22.09%,表明重组鱼类重组融合抗菌肽能明显提高鲈鱼抗病能力。
表8鱼类重组融合抗菌肽鲈鱼养殖中的抗病试验
以上实施方式仅用于说明本发明,而并非对本发明的限制,本领域的普通技术人员,在不脱离本发明的精神和范围的情况下,还可以做出各种变化和变型。因此,所有等同的技术方案也属于本发明的范畴,本发明的专利保护范围应由权利要求限定。
序列表
<110> 杭州皇冠农业生物工程技术研究中心有限公司
<120> 一种重组融合抗菌肽及应用
<160> 27
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工合成(Saccharum)
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<213> 人工合成(Saccharum)
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tgtcctaata tgagcggatg tggtgtctgc tgcaggttcg gtggcggtgg aagcggcggt 180
ggcggaagcc gcaactatat tgacctctcg tccaggaacc tctcgtcggt cccgggagac 240
cttccaaagg aagcggagct tatcgacctg tcacgcaacc tcatacagct gcttcaacga 300
ggagacttct ggaacacccc catcctcaga ttcctcaaca tttcatggaa ttgtttggaa 360
agtatccacc cagaggtgtt cctcggcacg ccgctgctgc aggatctgga cctgtcacac 420
aactgcctga agaacctcac ggatcagccg tacctgcagc gtgctggaaa cctcctgttt 480
ctgaatttgg cctacaataa gtttgtcacc atgaccctgg ccagggagtt cagctccctg 540
gcgaagctgg agagattaac gctgggaggg aacgtcatca gagtggggga cttcgggaat 600
attgctgacg tggagctgcg cctgctgagc ctccacctgg agggcaaact gctctacgaa 660
ccaggatctc tgaaggacgc gtacgcacgg aggctccaag tggaactgaa caaagtattc 720
cccctccacc tgataaacga cgctctgtcc ttctttgccg aagtggagct gctgaagttg 780
cccgagggct gtcgagagct gagcaggcag ctgagccaga gggctgaaat ctacacgtct 840
cgcctgtttt tgaccaacgt atccatcaac tggtccgact ttactcggtg tgtcacagta 900
gccctgaaca ccaccgttag ccacctgagt gtctccgacg tgaccttaca cagactacct 960
caaacagaca cgccgatggc aaacacctct cgagtgaagt ctgtcacggt gagagaagta 1020
acggtgaaga gttttctgtt ttcgcaggag gcggtctaca acttcttcat caacatgccg 1080
gtggagagtt tagcgctcac tgacactgcc atcatacgca tgacctgccc aaagtcccag 1140
agtcccgtca cgcatctgag tttctcccac tgcagtctga gcgacaccat cttttccaga 1200
gtggaaggct tgataacgat cgaatgcaag actcttggga atttgaggac gttgactctg 1260
acgagaaaca acttcaagag tctccagtcg ctcagcaaac gcatgcgata catgaaatcc 1320
ctgcaggatc tggacctcag cttcaaccgg ctggtgtatg acggacaggg ggagtgctac 1380
tggccgcaga acatcagcat ccttcacctg tcttccaata gtttgaccag ctctgcgttt 1440
caatgcctcc ctacgggtgt ggagagactg gacctccaga acaatcagct ttccgctgtt 1500
tcatcatcca cgttgaaact aacgagactt ttgtccttga acctaaacgc caacaggctg 1560
ctggacctgc ccgtgtgtga caacttccct ttgctgcagg agcttctgct caggtcaaat 1620
tctctccacg caccatctgt ggacaggctg gagagctgcc ccagactgaa aaccctggac 1680
gttagctaca acgccctcat gtgcatctgt cctctgaggg gcttcatccg acttggcctc 1740
gaatctgaga agaatcgcac aggtgttacg ttcttgcagt ggccacaggg ctactactgc 1800
agctacccag aggcctttaa ggattccaac ctcaacaaca tctggatctc agagatttcc 1860
tgtaacactc accaccacca ccaccactaa 1890
<210> 3
<211> 1680
<212> DNA
<213> 人工合成(Saccharum)
<400> 3
cgcaactata ttgacctctc gtccaggaac ctctcgtcgg tcccgggaga ccttccaaag 60
gaagcggagc ttatcgacct gtcacgcaac ctcatacagc tgcttcaacg aggagacttc 120
tggaacaccc ccatcctcag attcctcaac atttcatgga attgtttgga aagtatccac 180
ccagaggtgt tcctcggcac gccgctgctg caggatctgg acctgtcaca caactgcctg 240
aagaacctca cggatcagcc gtacctgcag cgtgctggaa acctcctgtt tctgaatttg 300
gcctacaata agtttgtcac catgaccctg gccagggagt tcagctccct ggcgaagctg 360
gagagattaa cgctgggagg gaacgtcatc agagtggggg acttcgggaa tattgctgac 420
gtggagctgc gcctgctgag cctccacctg gagggcaaac tgctctacga accaggatct 480
ctgaaggacg cgtacgcacg gaggctccaa gtggaactga acaaagtatt ccccctccac 540
ctgataaacg acgctctgtc cttctttgcc gaagtggagc tgctgaagtt gcccgagggc 600
tgtcgagagc tgagcaggca gctgagccag agggctgaaa tctacacgtc tcgcctgttt 660
ttgaccaacg tatccatcaa ctggtccgac tttactcggt gtgtcacagt agccctgaac 720
accaccgtta gccacctgag tgtctccgac gtgaccttac acagactacc tcaaacagac 780
acgccgatgg caaacacctc tcgagtgaag tctgtcacgg tgagagaagt aacggtgaag 840
agttttctgt tttcgcagga ggcggtctac aacttcttca tcaacatgcc ggtggagagt 900
ttagcgctca ctgacactgc catcatacgc atgacctgcc caaagtccca gagtcccgtc 960
acgcatctga gtttctccca ctgcagtctg agcgacacca tcttttccag agtggaaggc 1020
ttgataacga tcgaatgcaa gactcttggg aatttgagga cgttgactct gacgagaaac 1080
aacttcaaga gtctccagtc gctcagcaaa cgcatgcgat acatgaaatc cctgcaggat 1140
ctggacctca gcttcaaccg gctggtgtat gacggacagg gggagtgcta ctggccgcag 1200
aacatcagca tccttcacct gtcttccaat agtttgacca gctctgcgtt tcaatgcctc 1260
cctacgggtg tggagagact ggacctccag aacaatcagc tttccgctgt ttcatcatcc 1320
acgttgaaac taacgagact tttgtccttg aacctaaacg ccaacaggct gctggacctg 1380
cccgtgtgtg acaacttccc tttgctgcag gagcttctgc tcaggtcaaa ttctctccac 1440
gcaccatctg tggacaggct ggagagctgc cccagactga aaaccctgga cgttagctac 1500
aacgccctca tgtgcatctg tcctctgagg ggcttcatcc gacttggcct cgaatctgag 1560
aagaatcgca caggtgttac gttcttgcag tggccacagg gctactactg cagctaccca 1620
gaggccttta aggattccaa cctcaacaac atctggatct cagagatttc ctgtaacact 1680
<210> 4
<211> 63
<212> DNA
<213> 人工合成(Saccharum)
<400> 4
ggctgtcgct tttgctgcaa ttgctgtcct aatatgagcg gatgtggtgt ctgctgcagg 60
ttc 63
<210> 5
<211> 66
<212> DNA
<213> 人工合成(Saccharum)
<400> 5
ttctttcacc acattttccg tggaattgtt cacgtcggca agacgatcca caaacttgtg 60
accggg 66
<210> 6
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 6
gtgccgtcac ttcgatattc c 21
<210> 7
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 7
tttcacgcct ttgtgtctta c 21
<210> 8
<211> 20
<212> DNA
<213> 人工合成(Saccharum)
<400> 8
cttggaatgg cctcaggaca 20
<210> 9
<211> 22
<212> DNA
<213> 人工合成(Saccharum)
<400> 9
acctcaaaca gacacgccga tg 22
<210> 10
<211> 22
<212> DNA
<213> 人工合成(Saccharum)
<400> 10
tggctctgtg tttggctcgt gt 22
<210> 11
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 11
cccatcctca gattcctcaa c 21
<210> 12
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 12
agtgggagaa actcagatgc g 21
<210> 13
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 13
gtgccgtcac ttcgatattc c 21
<210> 14
<211> 21
<212> DNA
<213> 人工合成(Saccharum)
<400> 14
tttcacgcct ttgtgtctta c 21
<210> 15
<211> 22
<212> DNA
<213> 人工合成(Saccharum)
<400> 15
ctgatctaga ggtaccggat cc 22
<210> 16
<211> 23
<212> DNA
<213> 人工合成(Saccharum)
<400> 16
ttcctccttt tccattttgc tga 23
<210> 17
<211> 23
<212> DNA
<213> 人工合成(Saccharum)
<400> 17
tgaggttctt caggcagttg tgt 23
<210> 18
<211> 79
<212> DNA
<213> 人工合成(Saccharum)
<400> 18
cggatgtggt gtctgctgca ggttcggtgg cggtggaagc ggcggtggcg gaagccgcaa 60
ctatattgac ctctcgtcc 79
<210> 19
<211> 44
<212> DNA
<213> 人工合成(Saccharum)
<400> 19
ttttcctttt gcggccgcag tgttacagga aatctctgag atcc 44
<210> 20
<211> 79
<212> DNA
<213> 人工合成(Saccharum)
<400> 20
ggaattccat atggccatgg gctgtcgctt ttgctgcaat tgctgtccta atatgagcgg 60
atgtggtgtc tgctgcagg 79
<210> 21
<211> 44
<212> DNA
<213> 人工合成(Saccharum)
<400> 21
ttttcctttt gcggccgcag tgttacagga aatctctgag atcc 44
<210> 22
<211> 81
<212> DNA
<213> 人工合成(Saccharum)
<400> 22
gcaagacgat ccacaaactt gtgaccgggg gtggcggtgg aagcggcggt ggcggaagcg 60
gctgtcgctt ttgctgcaat t 81
<210> 23
<211> 57
<212> DNA
<213> 人工合成(Saccharum)
<400> 23
ttttcctttt gcggccgctt agtggtggtg gtggtggtga gtgttacagg aaatctc 57
<210> 24
<211> 81
<212> DNA
<213> 人工合成(Saccharum)
<400> 24
cgcggatccc gccaccatgg gcttctttca ccacattttc cgtggaattg ttcacgtcgg 60
caagacgatc cacaaacttg t 81
<210> 25
<211> 57
<212> DNA
<213> 人工合成(Saccharum)
<400> 25
ttttcctttt gcggccgctt agtggtggtg gtggtggtga gtgttacagg aaatctc 57
<210> 26
<211> 20
<212> DNA
<213> 人工合成(Saccharum)
<400> 26
aaaaccccgg atcggactac 20
<210> 27
<211> 20
<212> DNA
<213> 人工合成(Saccharum)
<400> 27
gggagggcgt gaatgtaagc 20
Claims (8)
1.一种重组融合抗菌肽,其特征在于:所述抗菌肽的氨基酸序列为SEQ ID NO.1。
2.编码权利要求1所述抗菌肽的基因。
3. 根据权利要求2所述的基因,其特征在于:所述基因具有SEQ ID NO.2所示的核苷酸序列。
4.包含权利要求2所述基因的载体。
5.根据权利按要求4所述的载体,其特征在于:所述原始载体为pET-28a或pYES2。
6.权利要求4所述载体转化的宿主细胞。
7.根据权利要求6所述的宿主细胞,其特征在于:所述宿主细胞为大肠杆菌Rosetta宿主细胞或酿酒酵母INVSC1宿主细胞。
8.权利要求1所述抗菌肽的应用,其特征在于将抗菌肽用于制备抗菌制剂或抗菌饲料添加剂中的用途。
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Cited By (3)
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CN111662370A (zh) * | 2020-07-03 | 2020-09-15 | 上海海洋大学 | 南极鱼hepcidin抗菌肽及其制备方法和应用 |
CN112798671A (zh) * | 2020-12-29 | 2021-05-14 | 中国科学院海洋研究所 | 一种温和裂解细胞的裂解剂及其应用 |
CN114933644A (zh) * | 2022-06-15 | 2022-08-23 | 河南师范大学 | 一种泥鳅抗菌肽Ma-sHep及其应用 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111662370A (zh) * | 2020-07-03 | 2020-09-15 | 上海海洋大学 | 南极鱼hepcidin抗菌肽及其制备方法和应用 |
CN111662370B (zh) * | 2020-07-03 | 2022-07-26 | 上海海洋大学 | 南极鱼hepcidin抗菌肽及其制备方法和应用 |
CN112798671A (zh) * | 2020-12-29 | 2021-05-14 | 中国科学院海洋研究所 | 一种温和裂解细胞的裂解剂及其应用 |
CN112798671B (zh) * | 2020-12-29 | 2023-09-05 | 中国科学院海洋研究所 | 一种温和裂解细胞的裂解剂及其应用 |
CN114933644A (zh) * | 2022-06-15 | 2022-08-23 | 河南师范大学 | 一种泥鳅抗菌肽Ma-sHep及其应用 |
CN114933644B (zh) * | 2022-06-15 | 2023-08-22 | 河南师范大学 | 一种泥鳅抗菌肽Ma-sHep及其应用 |
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