CN104945494A - 一种赤链华游蛇来源抗菌多肽sac及应用 - Google Patents
一种赤链华游蛇来源抗菌多肽sac及应用 Download PDFInfo
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Abstract
本发明属于生物医学技术领域，具体的说是一种赤链华游蛇来源抗菌多肽SAC及应用。赤链华游蛇来源抗菌多肽SAC由29个氨基酸残基组成，为直链多肽，分子量3569.5Da，等电点11.59。本发明所述抗菌多肽SAC具有分子量小、化学合成方法简单、抗菌作用广谱高效、溶血性低的有益特点，具有广泛的应用前景。此外本发明还涉及SAC的编码基因和应用。
Description
技术领域
本发明属于生物医学技术领域，具体的说是一种赤链华游蛇来源抗菌多肽SAC及应用。
背景技术
近年来传统抗生素的大规模滥用导致越来越严重的病原微生物耐药问题，给人类健康带来巨大的威胁。临床上应对耐药微生物感染的措施是使用对耐药微生物尚未使用过的新的或者替代性的抗生素，因此这就需要持续开发新的抗微生物药物。
抗菌肽是生物体基因编码的一种天然小分子多肽，是生物体免疫系统的一种重要分子，对细菌、真菌、病毒甚至原虫均具有直接的杀灭作用。抗菌肽具有分子量小、结构简单、抗菌活性强、杀菌机制独特、毒性低和不易引起耐药性等优点，因此自发现之日起就被认为是具有极大开发潜力的新一代抗生素。到目前为止，已从不同生物体中发现超过1500多种不同的抗菌肽，而且其数目还在增加。
发明内容
本发明的目的在于提供一种赤链华游蛇来源抗菌多肽SAC及其编码基因和应用。
为实现上述目的，本发明采用的技术方案为：
一种赤链华游蛇来源抗菌多肽SAC，赤链华游蛇来源抗菌多肽SAC由29个氨基酸残基组成，为直链多肽，分子量3569.5Da，等电点11.59。
所述抗菌多肽SAC氨基酸序列为SEQ ID NO.1所示。
所述抗菌多肽SAC的前体的基因自5’端至3’端的碱基序列由SEQ IDNO.2所示。
所述SEQ ID NO.2所示碱基序列应用于多肽重组表达，转基因动物、植物、植物部分、动物细胞或植物细胞中。
一种赤链华游蛇来源抗菌多肽SAC的应用，所述赤链华游蛇来源抗菌多肽SAC可用于制备抗菌药物、抑制细菌生长药物、防腐剂、动物饲料或化妆品前体。
所述赤链华游蛇来源抗菌多肽SAC可用于制备抗革兰氏阳性细菌、革兰氏阴性细菌或真菌的药物或组合物。
本发明所具有的优点：
本发明所述赤链华游蛇来源抗菌多肽SAC具有分子量小、化学合成方法简单、抗菌作用广谱高效、溶血活性低的有益特点，具有广泛的应用前景。
具体实施方式
下面用实施例来进一步说明本发明的实质性内容，但本发明的内容并不局限于此。
实施例1
赤链华游蛇来源抗菌多肽SAC编码基因的克隆：
1)赤链华游蛇肺总RNA提取：
①取300mg赤链华游蛇肺组织，放入研钵中加入液氮研磨成粉末，转移到EP管中，加入1m1总RNA提取试剂(Trizol，美国Life tech公司产品)，充分混匀，而后于4℃，12000rpm离心10min。
②离心取上清，加入0.2ml氯仿溶液，剧烈混匀，室温放置10分钟，而后以4℃，12000rpm离心10分钟，弃除沉淀。
③上清加入等体积的异丙醇，室温放置10分钟，以4℃，12000rpm离心10分钟，收集沉淀用75％(V/V)乙醇洗一次，晾干，管底沉淀物即为赤链华游蛇肺总RNA。
2)赤链华游蛇肺cDNA文库构建：采用CLONTECH公司In-Fusion SMARTerTM Directional cDNA Library Construction Kit构建。
(1)cDNA第一链合成(mRNA反转录)：
①经DEPC处理(DEPC处理是在含0.1％(V/V)DEPC的水浸泡过夜，高压灭菌，烘干)的离心管中加入1μl赤链华游蛇肺总RNA、1μl 3’端一链合成引物(3’In-Fusion SMARTer CDS Primer)和2.5μl经DEPC处理(DEPC处理是将含0.1％(V/V)DEPC的水，放置过夜，高压灭菌)的水使总体积达到4.5μl，混匀后短暂离心(2000rpm，30s)，离心后于72℃保温3分钟；保温后再将离心管在42℃孵育2分钟。
②在上述离心管中加入以下试剂(均为CLONTECH公司In-FusionSMARTer TM Directional cDNA Library Construction Kit建库试剂盒中配备)，2.0μl 5×第一链缓冲液、0.25μl 100mM DTT、1.0μl 10mM dNTPMix、1.0μl SMARTer V Oligonucleotide、0.25μl RNase Inhibitor和1.0μl SMARTScribe Reverse Transcriptase反转录酶(所用试剂均为CLONTECH公司In-Fusion SMARTer TM Directional cDNA LibraryConstruction Kit建库试剂盒中配备)，混合离心管中试剂并短暂离心(2000rpm，30s)，在42℃保温90min，然后68℃保温10min。保温处理后将离心管置于冰上中止第一链的合成。从离心管取2μl所合成的cDNA第一链备用。
(2)采用长末端聚合酶链式反应(LD-PCR)方法扩增第二链(所用试剂均为CLONTECH公司In-Fusion SMARTer TM Directional cDNA LibraryConstruction Kit建库试剂盒中配备)
①将2μl cDNA第一链(mRNA反转录)、80μl去离子水、10μl 10×Advantage 2PCR缓冲液、2μl 50×dNTP混合物、2μl 5’PCR引物、2μl CDS III/3’PCR引物以及2μl 50×Advantage 2Polymerase Mix(所用试剂均为CLONTECH公司In-Fusion SMARTer TM Directional cDNALibrary Construction Kit建库试剂盒中配备)在95℃预热的PCR管中进行混合。
②在PCR仪中按以下程序扩增：95℃，1min；18个循环：95℃，15sec，65℃，30sec，68℃，6min。循环结束后，将离心管中合成的cDNA双链－80℃保存。
(3)赤链华游蛇来源抗菌多肽SAC编码基因克隆筛选：
根据蛇类抗菌肽编码基因中间保守区，人工设计合成正向引物进行PCR扩增，其序列为5'-GTCATGGRRTGCACAGGCTACT-3'，PCR另一扩增引物为CLONTECH公司In-Fusion SMARTer TM Directional cDNA LibraryConstruction Kit中的3’-PCR引物，其序列为5’-CGGGGTACGATGAGACACCAT-3’。PCR反应在如下条件下进行：94℃5min，94℃30sec，57℃30sec和72℃1min，30个循环。扩增完成后用胶回收试剂盒(天根生物)进行目的片段回收。将回收的目的片段连接到pMD19-T载体(Takara,大连)，转化进CaCl2-MgCl2法制备好的DH5α感受态细胞。涂板并进行氨苄青霉素和蓝白斑双重筛选，挑取单菌落用M13引物PCR检测插入片段大小。挑取阳性菌落，摇菌提取质粒，使用Applied BiosystemsDNA sequencer,model ABI PRISM 377进行核苷酸测序。
测定结果：
SEQ ID NO.2所示的编码赤链华游蛇来源抗菌多肽SAC前体的基因自5’端至3’端序列为：
gtcatggaatgcacaggctactacttctttggggagacgcccccagtgctggtcctcacctgtgaagctgtgggtgaagaggaggaggcggagcagcagcaggaagaagggaacggagaggaggtggagaaggaggaaaaggaggaagacaagaaggatcagcccaggagggtcaagagattcaagaaatttttcaagaagttgaagaagagcgtgaagaaacatgtcaagaaattcttcatcggggtctccatccccttctaaggggggattcggaagggccggcagcctccgatccgaggggaaacggcagagaaaaacgatgcgggatgttttggagtccgtccaatcattccccaaaaagctaccccagcaataaataaatgaataaataaaagaaaaaaaaaaaaaaaaaa
其中编码抗菌肽SAC为第175-261位核苷酸。
赤链华游蛇来源抗菌多肽SAC前体的编码基因核苷酸序列表为：序列长度为416个碱基，序列类型：核酸，链数：单链，拓扑学：直链状，序列种类：cDNA，来源：赤链华游蛇肺。
实施例2
赤链华游蛇来源抗菌多肽SAC的化学合成：
Ⅰ、赤链华游蛇来源抗菌多肽SAC的化学合成方法：根据基因推导的成熟肽氨基酸序列，用自动多肽合成仪(433A,Applied Biosystems)合成其全序列，通过HPLC反相柱层析脱盐。
Ⅱ、分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)。
Ⅲ、纯化的赤链华游蛇来源抗菌多肽SAC用高效液相色谱HPLC方法鉴定其纯度，分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)，等电聚焦电泳测定等电点，用自动氨基酸测序仪测定氨基酸序列结构。
赤链华游蛇来源抗菌多肽SAC是赤链华游蛇来源抗菌多肽SAC编码基因编码的一种直链多肽，含有29个氨基酸残基，分子量3569.5Da，等电点11.59。其SEQ ID NO.1氨基酸序列为：
Lys1Arg2Phe3Lys4Lys5Phe6Phe7Lys8Lys9Leu10Lys11Lys12Ser13Val14Lys15Lys16His17Val18Lys19Lys20Phe21Phe22Ile23Gly24Val25Ser26Ile27Pro28Phe29。
实施例3
赤链华游蛇来源抗菌多肽SAC抗菌活性检测：
(1)分别挑取保存于斜面上的试验菌株均匀涂布于MH固体培养基(购自青岛海博生物技术有限公司)平板上，将经过灭菌的0.5cm直径的滤纸片置于培养基表面，滴加溶解于灭菌去离子水的2mg/ml的抗菌多肽SAC样品溶液10μl，于37℃倒置培养18－20小时，观察抑菌圈形成与否。若样品具有抗菌活性，则会在滤纸片周围形成清晰透明的抑菌圈，抑菌圈越大表明样品抗菌活性越强。
(2)赤链华游蛇来源抗菌多肽SAC最小抑菌浓度(Minimum InhibitoryConcentration)测定(2倍稀释法)：
选择上步实验中具有抑菌圈的菌株进行MIC测定实验。试验菌株接种到MH液体培养基(青岛海博生物技术有限公司)中，37℃振荡培养到对数生长期，而后用新鲜MH液体培养基(购自青岛海博生物技术有限公司)将培养至对数生长期的培养液稀释到2×105cfu/ml待用。
在无菌96孔板各孔中预先加入100μl MH液体培养基，然后在第一孔中加入100μl用MH液体培养基稀释到一定浓度的经0.22mm孔滤膜过滤的的赤链华游蛇来源抗菌多肽SAC样品溶液，混匀后取100μl加入第2孔，依次倍比稀释(参见表1)，自第9孔吸出100μl弃去，第10孔系对照管。
表.1稀释方法
将上述各管混匀后放置37℃缓慢振荡培养18小时，于600nm波长处测定光吸收。最小抑菌浓度为看不见细菌生长的最低样品浓度。结果如表2所示。
由表2可见，赤链华游蛇来源抗菌多肽SAC对革兰氏阳性细菌、革兰氏阴性细菌和真菌均表现出很强的抗菌活性，MIC值处于2.34-75μg/ml的范围。
表2赤链华游蛇来源抗菌多肽SAC抗菌活性
| 试验菌株 | MIC(μg/ml) |
| 大肠杆菌ATCC 25922 | 2.34 |
| 痢疾杆菌 | 2.34 |
| 肺炎克雷伯菌 | 9.38 |
| 产酸克雷伯菌 | 18.75 |
| 奇异变形杆菌 | 18.75 |
| 嗜麦芽窄食单胞菌 | 9.38 |
| 铜绿假单胞菌ATCC27853 | 18.75 |
| 甲型副伤寒沙门氏菌 | 4.69 |
| 金黄色葡萄球菌ATCC25923 | 18.75 |
| 蜡样芽孢杆菌 | 9.38 |
| 枯草芽孢杆菌 | 75 |
| 屎肠球菌 | 37.5 |
| 白色念珠菌 | 4.69 |
| 光滑念珠菌 | 18.75 |
MIC：最小抑菌浓度，以上结果为三次独立重复实验平均值。
实施例4
赤链华游蛇来源抗菌多肽SAC溶血活性测定
将采集的新鲜兔血与阿氏液混合抗凝，生理盐水洗涤2次并重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的赤链华游蛇来源抗菌多肽SAC样品混合，37℃保温30min，再于1000rpm离心5min，上清液于540nm测吸收值。阴性对照使用生理盐水，阳性对照使用Triton X-100，溶血百分比按以下公式计算：溶血百分比H％＝A样品-A阴性对照/A阳性对照×100％。
结果表明SAC浓度为200μg/ml时，溶血百分比为5.54％，说明赤链华游蛇来源抗菌多肽SAC对哺乳动物红细胞具有极低的溶血活性。
最后所应说明的是，以上具体实施方式仅用以说明本发明的技术方案而非限制，尽管参照实例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.一种赤链华游蛇来源抗菌多肽SAC，其特征在于：赤链华游蛇来源抗菌多肽SAC由29个氨基酸残基组成，为直链多肽，分子量3569.5Da，等电点11.59。
2.按权利要求1所述的赤链华游蛇来源抗菌多肽SAC，其特征在于：抗菌多肽SAC氨基酸序列为SEQ ID NO.1所示。
3.按权利要求1所述的赤链华游蛇来源抗菌多肽SAC，其特征在于：抗菌多肽SAC的前体的基因自5’端至3’端的碱基序列由SEQ ID NO.2所示。
4.按权利要求1所述的赤链华游蛇来源抗菌多肽SAC，其特征在于：所述SEQ ID NO.2所示碱基序列应用于多肽重组表达，转基因动物、植物、植物部分、动物细胞或植物细胞中。
5.一种按权利要求1所述的赤链华游蛇来源抗菌多肽SAC的应用，其特征在于：所述赤链华游蛇来源抗菌多肽SAC可用于制备抗菌药物、抑制细菌生长药物、防腐剂、动物饲料或化妆品前体。
6.按权利要求5所述的赤链华游蛇来源抗菌多肽SAC的应用，其特征在于：所述赤链华游蛇来源抗菌多肽SAC可用于制备抗革兰氏阳性细菌、革兰氏阴性细菌或真菌的药物或组合物。
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| CN109512692A (zh) * | 2018-12-29 | 2019-03-26 | 安徽劲诺材料科技有限公司 | 一种湿巾抗菌添加液及其制备方法 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412753A (zh) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | 金环蛇抗菌肽cathelicidin-BF及其基因和应用 |
| CN102311492A (zh) * | 2010-07-09 | 2012-01-11 | 中国科学院昆明动物研究所 | 非天然全D-型蛇毒Cathelicidin抗菌肽及其衍生物、制备方法及应用 |
| CN106146641A (zh) * | 2016-09-13 | 2016-11-23 | 潍坊科技学院 | 泽蛙来源cathelicidin家族抗菌肽及其应用 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412753A (zh) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | 金环蛇抗菌肽cathelicidin-BF及其基因和应用 |
| CN102311492A (zh) * | 2010-07-09 | 2012-01-11 | 中国科学院昆明动物研究所 | 非天然全D-型蛇毒Cathelicidin抗菌肽及其衍生物、制备方法及应用 |
| CN106146641A (zh) * | 2016-09-13 | 2016-11-23 | 潍坊科技学院 | 泽蛙来源cathelicidin家族抗菌肽及其应用 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046122A (zh) * | 2016-06-01 | 2016-10-26 | 潍坊科技学院 | 一种两栖类动物来源缓激肽及其应用 |
| CN106046122B (zh) * | 2016-06-01 | 2019-06-04 | 潍坊科技学院 | 一种两栖类动物来源缓激肽及其应用 |
| CN109512692A (zh) * | 2018-12-29 | 2019-03-26 | 安徽劲诺材料科技有限公司 | 一种湿巾抗菌添加液及其制备方法 |
| CN109512692B (zh) * | 2018-12-29 | 2021-09-07 | 浙江国能科技有限公司 | 一种湿巾抗菌添加液及其制备方法 |
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