CN1291019C - 一种新的天然抗菌肽hkabf及其制备方法和应用 - Google Patents
一种新的天然抗菌肽hkabf及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种海马抗菌肽新基因hkabf及其编码的蛋白HKABF,以及该蛋白在制备抗感染药物中的应用。本发明用RT-PCR的方法,从大海马孵化袋总RNA中筛选得到的抗菌肽新基因hkabf,其DNA序列如序列表中<400>1序列所示。该基因编码的蛋白(重组海马抗菌肽HKABF),其氨基酸序列如序列表中<400>2序列所示。本发明的重组海马抗菌肽HKABF对Gram+菌有明显的抑制作用,可用于制备治疗感染性疾病的药物。
Description
技术领域
本发明涉及一种新的天然抗菌肽HKABF和编码该蛋白的基因hkabf,以及该蛋白在制备治疗感染性疾病药物中的应用。
背景技术
海马(Hippoeampus)是一种奇特的海洋生物,属脊椎动物亚门、鱼纲、海龙目、海龙科(Syngnathidae)。目前,全球已鉴定的海马有35种。在我国,海马主要栖息在东南部海域,常见的共有5种,分别是线纹海马(H.kelloggi)、刺海马(H.histrix)、大海马(H.kuda)、斑海马(H.trimaculatus)以及日本海马(H.japonicus)。
海马是一种名贵的传统中药材,具有补肾壮阳、消肿散结,镇静安神之功效,前述5种海马为中国历版药典所收载。
除了奇特的外形和卓越的药用功能外,海马奇特的雄性孵化繁殖方式也一直是科学家们关注的焦点。作为卵胎生动物,雄海马在臀鳍末端有一个由两层皮膜褶叠而成的“孵化袋”,有些像有袋目动物袋鼠的育儿袋。雌海马在配对后将卵产入雄海马的孵化袋内,由雄海马承担小海马的孵化和保护工作。用通俗的话来说,海马是一种雄性怀孕的动物。雄海马的孵化袋是一个封闭的,具有丰富血管和黏液的皮膜组织,它除了保护受精卵不受物理伤害和捕食者吞噬以外,还有帮助进行气体交换、养分供给、渗透压调节及保护发育中的胚胎免受病原微生物感染等生物功能。
海马具有极高的商业价值,而持续的滥捕滥捞已导致亚洲天然海域的海马处于濒危境地。目前其主要来源是养殖。我国的海马养殖已有四十几年的历史,但要成为一个真正稳定的产业,还有许多疑难问题要解决,其中最大的问题就是幼苗的成活率低,缺乏有效的病害防治措施。由于抗生素的应用日益受到限制,一旦出现病害,目前大多用中药浸泡,但收效甚微。
本研究通过构建大海马孵化袋eDNA文库,筛选出抗菌肽基因hkabf,进而通过重组表达得到有活性的抗菌肽HKABF。天然抗菌肽HKABF的发明,是在海马这一物种的创新性发现,它提供了一个很好的药物后选物,不论是对海马养殖中病害的防治,还是对哺乳动物和人类中感染性疾病的防治。
发明内容
本发明的目的在于提供一种新的天然抗菌肽HKABF和编码该蛋白的基因hkabf。
本发明的另一目的在于提供该蛋白的制备方法。
本发明的第三个目的在于提供该蛋白在制备治疗感染性疾病药物中的应用。
本发明用RT-PCR的方法从大海马孕期孵化袋总RNA中分离得到的海马抗菌肽基因hkabf,其DNA序列如序列表中<400>1序列所示。
本发明所述基因编码的蛋白HKABF,其氨基酸序列如序列表中<400>2序列所示;该蛋白等电点为8.95,分子量为4762道尔顿。
该蛋白还可以通过重组酵母以分泌的形式表达,表达量达到1mg/L。
该重组肽对金黄色葡萄球菌等Gram+菌有明显的抑制作用,且杀菌非常迅速,对人的红细胞溶血性极小。
本发明所选择的海马属于大海马(Hippocampus kuda Bleeker),采自广东陆丰。
雄海马孕期孵化袋eDNA文库的构建:首先匀浆海马孕期孵化袋组织,提取总RNA。反转录为一链eDNA后合成双链eDNA,双链eDNA与peDNA3.0载体连接后转化E.coli并保存每个重组克隆。
本发明通过对以上重组文库克隆进行大规模随机序列测定,从中得到了1个新的编码海马抗菌肽的克隆,命名为hkabf(其DNA序列如序列表中<400>1序列所示)。新基因编码47个氨基酸的成熟肽(其氨基酸序列如序列表中<400>2序列所示),等电点为8.95,分子量为4762道尔顿,具有典型的抗菌肽一级结构、二级结构的特征,即含有较多碱性氨基酸而使分子带正电荷、C末端酰胺化、二级结构为双亲的β-折叠。
本发明通过设计了一对引物,将编码海马抗菌肽的新基因hkabf用PCR方法从peDNA3.0载体上扩增出来,克隆到甲醇酵母表达载体pPICZαA上,构建成表达质粒并将其转化毕赤酵母X-33。此表达载体以PAOX1为启动子,采用分泌表达的形式将重组蛋白分泌到发酵上清中。通过对培养基pH值,诱导时间,甲醇浓度等条件的摸索和优化,重组蛋白的表达量可达到1mg/L。
本发明还摸索和优化了重组海马抗菌肽HKABF的纯化条件,发酵上清液经CM sepharoseFF阳离子交换柱纯化,目的蛋白在300mM NaCl的20mM PBS(pH7.0)中被洗脱下来,纯度达95%以上。
本发明获得的重组海马抗菌肽HKABF具有生物活性。该重组海马抗菌肽HKABF对金黄色葡萄球菌等Gram+菌有明显的抑制作用,可通过琼脂弥散法看到明显的透明抑菌圈。因此,本发明的重组或合成海马抗菌肽具有很强的抗菌作用,可用于制备抗感染性疾病的药物。
由本发明的含有hkabf海马抗菌肽成熟肽编码序列的表达质粒pPICZαA经Xho I/NotI双酶切,可得到188bp的片段,即为大海马抗菌肽HKABF成熟肽编码序列。
本发明的表达质粒的复制方法:参照Sambrook(Sambrook,et al.1989,Molecular cloing.Cold Spring Harbor Labroratory Press.USA)方法,按CaCl2法在E.coli DH5α菌株中转化质粒,用含氨苄青霉素(100μg/mL)的LB培养基转化细菌,碱法提取质粒。
附图说明
图1为本发明的海马天然抗菌肽HKABF与其它几个同源抗菌肽的比对结果,其中“*”表示相同;“:”表示差异较小;“.”表示差异明显;空格表示完全不同。
图2为大海马孕期孵化袋总RNA电泳结果。
图3为大海马抗菌肽hkabf基因的RT-PCR电泳结果。1:PCR目的条带;2:空白对照;3:100bp DNAmarker。
图4为含基因hkabf的pPICZαA-hkabf表达质粒酶切鉴定图。1:pPICZαA-hkabf质粒;2:pPICZαA-hkabf Xho I/Not I双酶切;3:1kb DNA marker
图5为重组抗菌肽HKABF经纯化后的SDS-PAGE(Tricine)分析。1:Sigma多肽marker;2:纯化的重组抗菌肽HKABF。
图6为用重组酵母分泌表达覆盖法得到的抗菌肽HKABF抗金黄色葡萄球菌的抑菌圈实验结果。
图7为含基因hkabf的重组表达质粒pPICZαA-hkabf构建图。
具体实施方式
以下实施例将有助于本领域的普通技术人员进一步理解本发明,但不以任何形式限制本发明。
实施例1:海马孵化袋总RNA的提取和RT-PCR扩增
总RNA的提取和cDNA合成:在Trizol中匀浆海马孕期孵化袋组织,采用一步热酚法提取海马总RNA,抽提去除蛋白质。获得总RNA浓度为1.04μg/μl,其A260/A280=1.90,经1%甲醛变性胶电泳检测可见清晰的18s和28s两条带,比值大于1(见图2),表明总RNA完整性良好。取1μg总RNA以SMART IV oligonucleotide(5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3′)和CDSIII/3′PCR引物(5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3′)进行逆转录合成第一链,得到10μl cDNA第一链产物。2μl第一链产物以5’PCR primer(5’-AAGCAGTGGTATCAACGCAGAGT-3’),CDS III /3’PCR primer(5’-ATTCTAGAGGCCGAGGCGGCCGACATGd(T)30N-1N-3’)进行二链PCR扩增。将二链蛋白酶K消化并纯化后进行Sfi I切割,连接入同样Sfi I酶切处理过的pcDNA3.0文库载体上。随机测序获得两个抗菌肽基因hkabf的表达序列标签(EST),经软件分析发现该EST为全长。
实施例2:海马抗菌肽基因序列的分析
BLAST同源分析表明,该海马抗菌肽新基因ORF全长为234bp,编码78个氨基酸残基的前体蛋白,其中包括N末端19个氨基酸的信号肽、C末端12个氨基酸的Proregion区域和中间47个氨基酸的成熟肽。其中成熟肽羧基末端的Lys酰胺化。海马抗菌肽前体蛋白的这种组成模式及C末端酰胺化是抗菌肽一级结构的典型特征。成熟肽因含6个碱性氨基酸及C末端酰胺化而使分子带正电荷。预测成熟肽等电点为8.95,分子量为4762道尔顿。该成熟肽,与其他抗菌肽的同源性只有55%,但含有8个保守的Cys残基而使分子内形成4对二硫键,这是该抗菌肽的主要结构特征(见图1)。此外,其疏水性氨基酸占40%,预测二级结构可形成3个双亲的β-折叠。
实施例3:重组海马抗菌肽酵母表达质粒的构建
依据hkabf基因的两端序列合成一对引物,上游引物含Xho I切割位点,下游引物含Not I切割位点。
上游引物(B1):
5’-CCTTAGGCCCCTCGAGaaaaga CTGTCGTGCAGATTTGGACGC-3’;
下游引物(B2):
5’-GCCGAGCTCTGCAGCGGCCGCtca CTTCTTGCCGACACTAACACC-3’。
以含hkabf基因的pcDNA3.0质粒为模板,B1、B2为引物PCR扩增,得到特异扩增的单一条带,产物大小在188bp左右。PCR扩增产物经Xho I/Not I酶切克隆到甲醇酵母表达载体pPICZαA上,得到重组表达载体pPICZαA-hkabf(其构建过程如图7所示)。表达载体经Xho I/Not I双酶切鉴定(酶切结果见图4)正确。表达载体pPICZαA-hkabf中的外源基因经测序鉴定正确。
实施例4:重组海马抗菌肽的表达
将线性化的pPICZαA-hkabf电击转化毕赤酵母菌株X-33,得到重组酵母基因工程菌。该重组酵母菌株经甲醇诱导表达后,其发酵上清液用Tricine-Tris SDS-PAGE电泳及银染分析。结果表明,菌体经诱导后有明显的特异表达产物带,其分子量与预测的理论值5kD相符(图5)。
经过对培养基pH值、甲醇诱导浓度、诱导时间等条件的摸索,确定了培养基pH6.0,甲醇浓度1%,诱导时间3天的表达条件。最终的表达量为1mg重组蛋白/L BMMY培养基(1%yeast extract,2%peptone,100mM potassium phosphate,pH6.0,1.34%YNB,4×10-5% biotin,0.5% methanol)。
实施例5:重组海马抗菌肽的纯化
实施例四所得的含有海马重组抗菌肽的发酵上清液,经Sephadex G-25柱换成平衡缓冲液(20mM PBS,pH6.0)后,上CM sepharose FF阳离子交换柱。收集穿流峰,用前述平衡缓冲液洗柱至平台期。然后,用溶液A(20mM PBS,pH7.0)和溶液B(含有1M NaCl的20mM PBS,pH7.0)的混合液线性梯度洗脱,收集各洗脱峰,用Tricine/Tris SDS-PAGE电泳(银染)分析。结果显示,目的蛋白在含300mM NaCl的20mM PBS(pH7.0)中被洗脱下来。最后可得到纯度在95%以上的重组海马抗菌肽HKABF,得率约为100μg/L培养基(见图5)。
实施例6:溶血实验
用血色素的泄露来评估溶血性大小。用pH7.6的10mM Tris-HCl(含154mM NaCl)缓冲液洗涤人A型红细胞,再用含抗菌肽的前述缓冲液重悬红细胞,孵育0.5小时后离心,去掉完整的红细胞,取上清,稀释后测A540。当HKABF浓度为100μg/ml时,只有不超过3%的溶血性发生。与其它已发表的许多抗菌肽相比,溶血性很小,说明海马抗菌肽对正常细胞的毒性很小。
实施例7:重组海马抗菌肽的抗菌活性实验
重组海马抗菌肽HKABF的生物活性参照Wu和Hancock的方法,分析其对包括枯草杆菌、金黄色葡萄球菌、E.coli K12D31、白色念珠菌等在内的常见敏感菌株及致病菌株的最小抑菌浓度(MIC),同时还进行了抑菌圈实验(见图6)和杀菌活性的时间依赖性(杀菌时间与存活率之间的关系)分析。结果表明,HKABF对大多数菌株的MIC不超过10μg/ml,在2分钟之内就可杀死98%以上的金黄色葡萄球菌,说明其抗菌的高效、快速,而且抗菌谱广。
序列表
<110>中山大学
<120>一种新的天然抗菌肽HKABF及其制备方法和应用
<160>2
<210>1
<211>141
<212>DNA
<213>大海马(Hippocampus kuda Bleeker)
<220>
<221>mat peptide
<222>(1)…(141)
<400>1
ctg tcg tgc aga ttt gga cgc atc gcg tgc att gga tcg tgt cag gtc 48
Leu Ser Cys Arg Phe Gly Arg Ile Ala Cys Ile Gly Ser Cys Gln Val
1 5 10 15
cag aac tgc ggg acc ggc tac tgc cgc ggg gac acg tgt gtc tgt tcg 96
Gln Asn Cys Gly Thr Gly Tyr Cys Arg Gly Asp Thr Cys Val Cys Ser
20 25 30
cgc tgc ggc acc gga ggc atc gtc ggt gtt agt gtc ggc aag aag 141
Arg Cys Gly Thr Gly Gly Ile Val Gly Val Ser Val Gly Lys Lys
35 40 45
<210>2
<211>47
<212>PRT
<213>大海马(Hippocampus kuda Bleeker)
<220>
<221>mat peptide
<222>(1)…(47)
<400>2
Leu Ser Cys Arg Phe Gly Arg Ile Ala Cys Ile Gly Ser Cys Gln Val
1 5 10 15
Gln Asn Cys Gly Thr Gly Tyr Cys Arg Gly Asp Thr Cys Val Cys Ser
20 25 30
Arg Cys Gly Thr Gly Gly Ile Val Gly Val Ser Val Gly Lys Lys
35 40 45
Claims (5)
1.从大海马孕期孵化袋总RNA中分离得到的海马抗菌肽基因hkabf,其DNA序列为:ctg tcgtgc aga ttt gga cgc atc gcg tgc att gga tcg tgt cag gtc cag aac tgc ggg acc ggc tactgc cgc ggg gac acg tgt gtc tgt tcg cgc tgc ggc acc gga ggc atc gtc ggt gtt agt gtcggc aag aag 。
2.权利要求1所述基因编码的蛋白HKABF,其氨基酸序列为:Leu Ser Cys Arg Phe Gly ArgIle Ala Cys Ile Gly Ser Cys Gln Val Gln Asn Cys Gly Thr Gly Tyr Cys Arg Gly Asp Thr CysVal Cys Ser Arg Cys Gly Thr Gly Gly Ile Val Gly Val Ser Val Gly Lys Lys。
3.权利要求2所述的蛋白HKABF的生产方法,按照以下步骤进行:
(1)将权利要求1所述的基因hkabf克隆到甲醇酵母表达载体pPICZαA上,得到重组表达载体pPICZαA-hkabf;
(2)将pPICZαA-hkabf电击转化毕赤酵母菌株X-33,得到重组酵母基因工程菌;
(3)工程菌的培养表达;
(4)发酵上清液经CM sepharose FF阳离子交换柱纯化,可得到纯度在95%以上的重组海马抗菌肽HKABF。
4.按权利要求3所述的蛋白HKABF的生产方法,其特征在于:步骤(3)的工程菌的培养表达是在pH6.0的培养基,以浓度为1%的甲醇,对上述的重组酵母基因工程菌进行诱导表达3天。
5.权利要求2所述的蛋白HKABF在制备治疗感染性疾病药物中的应用。
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