CN114057866A - 一种具有抑菌和溶血功效的水蛭多肽及其应用 - Google Patents
一种具有抑菌和溶血功效的水蛭多肽及其应用 Download PDFInfo
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- CN114057866A CN114057866A CN202111590989.2A CN202111590989A CN114057866A CN 114057866 A CN114057866 A CN 114057866A CN 202111590989 A CN202111590989 A CN 202111590989A CN 114057866 A CN114057866 A CN 114057866A
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- polypeptide
- leech
- leech polypeptide
- hemolysis
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Abstract
本发明提供了一种具有抑菌和溶血功效的水蛭多肽及其应用,属于功能蛋白技术领域;所述水蛭多肽的氨基酸序列如SEQ ID NO.1所示。本发明的水蛭多肽兼具溶血和抑菌效果。本发明的水蛭多肽的抗菌活性为14.17μg/mL,具有较好的抑菌活性。本发明的水蛭多肽的溶血率能达到44.06%,接近50%,具有较好的溶血率,在抗血栓药物研发方面,具有较好的应用价值。
Description
技术领域
本发明属于功能蛋白技术领域,具体涉及一种具有抑菌和溶血功效的水蛭多肽及其应用。
背景技术
宽体金线蛭(Whitmania pigra Whitman)隶属于无吻蛭目,黄蛭科,金线蛭属,是中药材水蛭的基原动物之一,具有较大的药用价值,在我国分布广泛,常年栖息于水田及与其相同的沟渠中,靠吸食动物的新鲜血液或体液为生,且饱食一次可维持半年甚至长达一年。
现有技术公开了水蛭来源的多种活性多肽。例如,水蛭酸性多肽具有高度特异的抗凝血酶活性,抑制凝血酶结合底物,有抗凝血作用。再比如,中国专利CN107177654A公开了一种水蛭蛋白多肽,具有降脂功效。中国专利CN110437307A公开了一类具抗血栓和脑神经细胞保护作用的水蛭多肽。
目前还没有关于兼具溶血和抑菌效果的水蛭多肽的报道。
发明内容
有鉴于此,本发明的目的在于提供一种具有抑菌和溶血功效的水蛭多肽及其应用,本发明的水蛭多肽兼具溶血和抑菌效果。
本发明提供了一种具有抑菌和溶血功效的水蛭多肽,氨基酸序列如SEQ ID NO.1所示。
本发明还提供了上述方案所述水蛭多肽的编码基因,核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种重组质粒,插入有上述方案所述的编码基因。
优选的,所述重组质粒的原始质粒包括pET-32a。
本发明还提供了一种重组菌,包含上述方案所述的重组质粒。
本发明还提供了上述方案所述的水蛭多肽在抑菌和/或溶血中的应用。
本发明还提供了上述方案所述的水蛭多肽或者所述的编码基因或者所述的重组质粒或者所述的重组菌在制备抑菌和/或溶血产品中的应用。
优选的,所述抑菌包括抑制微球菌。
优选的,所述溶血产品包括抗血栓药物。
本发明提供了一种具有抑菌和溶血功效的水蛭多肽,氨基酸序列如SEQ ID NO.1所示。本发明的水蛭多肽兼具溶血和抑菌效果。本发明的水蛭多肽的抗菌活性为14.17μg/mL,具有较好的抑菌活性。本发明的水蛭多肽的溶血率能达到44.06%,接近50%,具有较好的溶血性,在抗血栓药物研发方面,具有较好的应用价值。
附图说明
图1蛋白表达鉴定SDS-PAGE分析结果,其中,M:marker;a:诱导未破碎;b:未诱导破碎沉淀;c:诱导破碎上清;d:诱导破碎沉淀;e:未诱导破碎上清;
图2为蛋白纯化SDS-PAGE分析结果,其中,M:marker;a:诱导未破碎;b:未诱导破碎沉淀;c:诱导破碎上清;d-i分别为咪唑0、咪唑25、咪唑50、咪唑100、咪唑250、咪唑500;
图3为蛋白浓度标准曲线。
具体实施方式
本发明提供了一种具有抑菌和溶血功效的水蛭多肽,氨基酸序列如SEQ ID NO.1所示,具体为:
MMKSAIYSCFALLTIVLALSEVNSQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI。
本发明对所述水蛭多肽的来源没有特殊限制,采用本领域常规方法制备或者生物公司合成均可。
本发明还提供了上述方案所述水蛭多肽的编码基因,核苷酸序列如SEQ ID NO.2所示,具体为:
atgatgaagtctgcaatttattcttgtttcgctcttcttacaatcgttttagctcttagcgaggtgaacagtcaaatctcagatccttgtcttcgatgtatttgtaaggaagaagggtgcgagactcaaattggacaatgtaatgatggtacaagtcagagttgcgggccttaccaaattatgagagcatattggattgattgtggaaaacctggaaatgattacgaaacatgcacaaaaactatagactgttctgaggcttgtgtgagagcttacatgaacaggtatggaacctactgcacaggaggaagaactccaacctgccaggactatgccaggatccacaagggtggaccaagtggttgtaatcaatctgaaacttttgtctacgggaaaaaggtgcaggaatgcagtgttattccagctactgaaacaacaactgaaatctag,命名为:wpDestabilase。本发明对所述编码基因的来源没有特殊限制,采用本领域常规方法制备或者生物公司合成均可,在本发明具体实施过程中,所述编码基因的制备方法为:基于PAS(PCR-basedAccurate Synthesis)方法,设计全长拼接引物,合成编码基因wpDestabilase。
本发明通过分析水蛭唾液腺转录组数据获得了所述水蛭多肽的编码基因。
本发明还提供了一种重组质粒pET-32a-wpDestabilase,插入有上述方案所述的编码基因。
在本发明中,所述重组质粒的原始质粒包括pET-32a;所述编码基因的插入位点为NcoI和XhoI之间。
在本发明中,所述重组质粒优选的采用以下构建方法构建得到:将编码基因连入载体pET-32a中;编码基因连入载体pET-32a中的方法优选为酶切连接法。
本发明还提供了一种重组菌,包含上述方案所述的重组质粒。
在本发明中,所述重组菌的原始菌优选为大肠杆菌,更优选为大肠杆菌TOP10克隆菌株。
本发明还提供了上述方案所述的水蛭多肽在抑菌和/或溶血中的应用。
本发明还提供了上述方案所述的水蛭多肽或者所述的编码基因或者所述的重组质粒或者所述的重组菌在制备抑菌和/或溶血产品中的应用。
在本发明中,所述抑菌优选的包括抑制微球菌。
在本发明中,所述溶血产品优选的包括抗血栓药物。
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。
实施例1
一、构建重组质粒
分析水蛭唾液腺转录组数据,找到1条可能与抑菌、溶血功能相关的基因序列,命名为wpDestabilase(如SEQ ID NO.2所示),基于PAS(PCR-based Accurate Synthesis)方法,设计全长拼接引物,合成目的基因wpDestabilase,通过酶切连接法将目的基因连入载体pET-32a中,获得重组质粒pET-32a-wpDestabilase。
二、原核表达
1、将重组质粒1μL加入100μL感受态细菌中,置冰上20min,42℃热激90sec,迅速置冰中5min,加入600μL LB培养液,37℃,220r/min振摇1h,离心后全部涂布于含50μg/mLAmp的LB平板,37℃倒置培养过夜。
2、挑取上述步骤中过夜平板上的单克隆接种于含50μg/mLAmp的3mL LB培养液的试管中,37℃,220r/min振摇过夜;次日按1:100接种于50μg/mL Amp的30mL LB培养液中,37℃,220r/min振摇至菌体OD600为0.6-0.8。
3、取出上述培养物1mL,10000r/min室温离心2min,弃上清,用100μL 1×上样缓冲液重悬菌体沉淀;重悬液进行超声破碎后,分别取上清液与沉淀液加入上样缓冲液重悬。
4、向剩余的培养物中加入IPTG至终浓度为0.5mM,37℃,220r/min振瑶4h,诱导蛋白表达。
5、取出诱导后的培养物1mL,10000r/min室温离心2min,弃上清,用100μL 1×上样缓冲液重悬菌体沉淀;剩余培养物4000r/min,离心10min,弃上清,用PBS重悬菌体沉淀,重悬液进行超声破碎后,分别取上清液与沉淀液加入上样缓冲液重悬。
6、进行12%SDS-PAGE检测分析,考马斯亮蓝染色显带,结果如图1所示,目的蛋白在诱导破碎后上清和沉淀中均存在。
7、收集破碎后上清和沉淀,过柱纯化,咪唑100洗脱得到目的蛋白,结果如图2所示。
目的蛋白翻译序列如SEQ ID NO.1所示,具体如下:
MMKSAIYSCFALLTIVLALSEVNSQISDPCLRCICKEEGCETQIGQCNDGTSQSCGPYQIMRAYWIDCGKPGNDYETCTKTIDCSEACVRAYMNRYGTYCTGGRTPTCQDYARIHKGGPSGCNQSETFVYGKKVQECSVIPATETTTEI。
三、蛋白浓度标准曲线构建及目的蛋白浓度测定
1、采用酶标板法测定各浓度下的标准蛋白的A562值,按照BCA蛋白浓度测定试剂盒操作说明,选取酶标板上14个孔,分为标准品组和样品组,前12孔加标准品,每个标准品设置1个重复,各孔分别加入5μL相应浓度的标准蛋白质溶液,剩余2孔加入5μL样品稀释液(稀释1倍)。
2、各酶标孔分别放入5μL溶液F,37℃水浴中保温30min。
3、再往每孔中加入200μLBCA工作液,迅速混匀,在37℃水浴中保温30min。
4、冷却至室温后,在酶标仪上测各孔的A562值,以标准品各孔A562平均值为纵坐标,对应蛋白浓度为横坐标,绘制标准曲线(参见图3),通过曲线关系,根据样品稀释液的A562值,计算得到目的蛋白浓度。
结果参见表1:
表1各蛋白浓度下A562值
根据关系方程式,样品稀释液(稀释1倍)A562值为0.126,经计算,原核表达后得到的目的蛋白浓度为1273μg/mL。
实施例2
一、目的蛋白抑菌活性测定
1.1材料
经原核表达得到的目的蛋白,溶菌酶测试盒、微球菌
1.2方法
1.2.1贮备菌液配制
取5mg/支的菌粉1支,倒入匀浆管中,加菌粉溶剂1mL,轻轻缓慢上下旋转研磨3分钟(切勿溅出),即为贮备菌液,将其取出后置于2-8℃冰箱密封保存一周左右。
1.2.2应用菌液的配制
按贮备菌液:菌粉溶剂=1:19进行配制,即取0.5mL贮备菌液溶于9.5mL菌粉溶剂中,现用现配,用时摇匀。
1.2.3标准品贮备液配制
每支标准品准确加双蒸水1.0mL配成2mg/mL的标准品贮备液,置于2-8℃可保存7~10天。
1.2.4标准品应用液的配制
按标准品贮备液:双蒸水=1:799比例稀释成2.5μg/mL(即200U/mL)的标准品应用液,即取0.01mL标准品贮备液溶于7.99mL双蒸水中,现配现用。
1.2.5温浴
将配制好的应用菌液,标准应用液及所需检测的样本均放入37℃水浴箱中预温5分钟以上,使菌液,标准液及检测样本的温度达到37℃。
1.2.6调零
将可见分光光度计于530nm处,1cm光径比色皿,以双蒸水调透光度100%(比色皿准备两只,一只用于调透光度100%,一只用于测定)
1.2.7测定
往相应编号的试管中加入0.2mL待测样本,取2mL应用菌液迅速冲入试管中,立即混匀并计时,设置3个重复。混合液迅速倒入比色皿中,测定530nm处的透光度,20秒时读取透光度值T0,比色皿不要取出,在2分20秒时读取透光度值T2。
标准品应用液测定同上。
1.2.8计算
计算公式:
目的蛋白抗菌活性(μg/mL)=(样本透光度T2-样本透光度T0)/(标准透光度T2-标准透光度T0)×标准品浓度(2.5μg/mL即200U/mL)×样本测试前稀释倍数
1.3结果
取0.2mL稀释1倍的目的蛋白(浓度为636.5μg/mL),与2mL应用菌液混合后于530nm处测定透光度,20秒透光度值T0为30.6(3个重复的平均值),2分20秒透光度值T2为32.3(3个重复的平均值);0.2mL标准品应用液与2mL应用菌液混合后于530nm处测定透光度,20秒透光度值T0为30.1(3个重复的平均值),2分20秒透光度值T2为30.4(3个重复的平均值),按照2.2.8的公式计算得到目的蛋白抗菌活性为14.17μg/mL,具有较好的抑菌活性。
三、目的蛋白溶血实验
3.1材料
经原核表达得到的目的蛋白,四至六周龄、体重20g左右的小白鼠10只,咪唑100
3.2方法
小鼠去眼球取血,滴于2ml离心管中,待血自然凝固,6000r/min,10min离心,弃血清。将血块置于培养皿中,用生理盐水漂洗至液体无色。将血块切至约1g/块,置于培养皿中,放入30℃培养箱,低温烘1h。拿出血块,将其翻面后,重新放入30℃培养箱,1h后取出称重。将称重后的血块分别放入4mL离心管中,设置空白对照组和实验组。实验组加1mL稀释1倍的目的蛋白(浓度为636.5μg/mL),空白对照组加1mL咪唑100,置于37℃、100rpm摇床内反应24h。反应后取出血块,用生理盐水漂洗血块表面杂质后,放入30℃培养箱,低温烘1h。拿出血块,将其翻面后,重新放入30℃培养箱,1h后取出称重。按照“(反应前质量-反应后质量)/反应前质量×100%”计算溶血率。
3.3结果参见表2
表2溶血率
从表2可以看出,该目的蛋白溶血率能达到44.06%,接近50%,具有较好的溶血率,在抗血栓药物研发方面,具有较好的应用价值。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
序列表
<110> 贵州省畜牧兽医研究所
<120> 一种具有抑菌和溶血功效的水蛭多肽及其应用
<160> 2
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Cys Ile Cys Lys Glu Glu Gly Cys Glu Thr Gln Ile Gly Gln Cys Asn
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Asp Gly Thr Ser Gln Ser Cys Gly Pro Tyr Gln Ile Met Arg Ala Tyr
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Gly Thr Tyr Cys Thr Gly Gly Arg Thr Pro Thr Cys Gln Asp Tyr Ala
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Arg Ile His Lys Gly Gly Pro Ser Gly Cys Asn Gln Ser Glu Thr Phe
115 120 125
Val Tyr Gly Lys Lys Val Gln Glu Cys Ser Val Ile Pro Ala Thr Glu
130 135 140
Thr Thr Thr Glu Ile
145
<210> 2
<211> 450
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgatgaagt ctgcaattta ttcttgtttc gctcttctta caatcgtttt agctcttagc 60
gaggtgaaca gtcaaatctc agatccttgt cttcgatgta tttgtaagga agaagggtgc 120
gagactcaaa ttggacaatg taatgatggt acaagtcaga gttgcgggcc ttaccaaatt 180
atgagagcat attggattga ttgtggaaaa cctggaaatg attacgaaac atgcacaaaa 240
actatagact gttctgaggc ttgtgtgaga gcttacatga acaggtatgg aacctactgc 300
acaggaggaa gaactccaac ctgccaggac tatgccagga tccacaaggg tggaccaagt 360
ggttgtaatc aatctgaaac ttttgtctac gggaaaaagg tgcaggaatg cagtgttatt 420
ccagctactg aaacaacaac tgaaatctag 450
Claims (9)
1.一种具有抑菌和溶血功效的水蛭多肽,氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述水蛭多肽的编码基因,核苷酸序列如SEQ ID NO.2所示。
3.一种重组质粒,插入有权利要求2所述的编码基因。
4.根据权利要求3所述的重组质粒,其特征在于,所述重组质粒的原始质粒包括pET-32a。
5.一种重组菌,包含权利要求3或4所述的重组质粒。
6.权利要求1所述的水蛭多肽在抑菌和/或溶血中的应用。
7.权利要求1所述的水蛭多肽或者权利要求2所述的编码基因或者权利要求3或4所述的重组质粒或者权利要求5所述的重组菌在制备抑菌和/或溶血产品中的应用。
8.根据权利要求6或7所述的应用,其特征在于,所述抑菌包括抑制微球菌。
9.根据权利要求7所述的应用,其特征在于,所述溶血产品包括抗血栓药物。
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