CN113980112B - 一种眼镜王蛇抗菌肽oh-cath30的表达载体和表达产物及其构建制备方法 - Google Patents
一种眼镜王蛇抗菌肽oh-cath30的表达载体和表达产物及其构建制备方法 Download PDFInfo
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Abstract
本发明公开了一种眼镜王蛇抗菌肽OH‑CATH30的表达载体和表达产物及其构建制备方法,涉及一种可高效表达眼镜王蛇抗菌肽OH‑CATH30和OH‑CATH30R的表达载体的构建与表达。眼镜王蛇抗菌肽OH‑CATH30和OH‑CATH30R的表达载体由DAMP4载体蛋白,通过linker分别连接眼镜王蛇抗菌肽OH‑CATH30N端到C端、C端到N端基因构建PET‑28a‑DAMP4‑F‑OH30、PET‑28a‑DAMP4‑F‑OH30R。通过该表达载体在TSsetta(DE3)Chemically Competent Cell表达感受态细胞中诱导表达可溶性产物和高效纯化可溶性表达产物的方法,从而获得有活性的体外抗菌肽OH‑CATH30和OH‑CATH30R表达产物。该方法实现了抗菌肽OH‑CATH30从N‑C和从C—N的原核表达,实验流程简单易行,获得的重组抗菌肽在纯化后不经任何处理即具有明显的抑菌活性,经凝血酶酶切之后抑菌活性有了明显的提高。
Description
技术领域
本发明属于基因工程技术领域,特别是涉及一种爬行纲抗菌肽眼镜王蛇抗菌肽OH-CATH30的表达载体和表达产物及其构建制备方法。
背景技术
抗菌肽(AMPs)作为治疗抗生素耐药病原体的小分子抗菌剂成为一种很有前途的替代品,在过去几十年里引起了广泛的关注。AMPs是一类由10-40个氨基酸残基组成的具有抗菌活性的多肽分子,由宿主基因编码,是宿主自身的免疫防御系统中重要的组成部分。天然AMPs已经在哺乳动物、两栖动物、昆虫和植物等中被发现。AMPs具有广谱的抗菌活性,能够在较短的时间内起到快速和高效的抗微生物作用,对革兰氏阴性菌、革兰氏阳性菌、真菌,以及一些包膜病毒和原生生物都具有较强的杀菌活性。
和传统的抗生素相比,AMPs具有快速且彻底的杀菌作用,从而不易产生细菌耐药性,且对哺乳动物具有极低的毒性。因此,在抗生素耐受严重的今天,AMPs成为了肽类药物的潜在候选药物,在新型抗菌药物研发方面具有广阔的前景。目前,已经报告并鉴定的AMPs有3000多种,并建立了AMPs数据库(http://aps.unmc.edu/AP/main.html),许多AMPs已经进入临床前研究和临床研究或者应用到畜牧业上去。现在已知的AMPs主要由两大家族组成,一类是包含多对二硫键的防御素家族;另一类是Cathelicidin家族。Cathelicidins是一个重要的AMPs家族,是一类具有广谱抗微生物活性,并在动物先天免疫系统中发挥重要作用的多功能抗菌肽。Cathelicidin抗菌肽首先是以无活性的前体蛋白形式表达。前体蛋白的前体由N端信号肽序列(29-30个氨基酸残基),中间保守的Cathelin区域(94-114个氨基酸残基)和C端成熟肽序列(12-100个氨基酸残基)构成。前体蛋白通常情况下是没有活性的,被特异性的蛋白酶切割掉保守的cathelin-like结构域后即可释放出有活性的成熟肽,这可能是由于其N端所带的阴离子的片段抑制C端的阳离子,使生物体避免AMPs伤害自身细胞。迄今为止,在几乎所有种类的脊椎动物体内均发现有Cathelicidin抗菌肽,不同物种来源的成熟肽二级结构存在一定差异。根据Cathelicidin抗菌肽二级结构的差异,可以分为α-螺旋Cathelicindin、含有半胱氨酸的Cathelicindin以及富含脯氨酸或酪氨酸的cathelicindin。与其它家族,如defensins、hepcidins、昆虫AMPs和蛙科皮肤来源AMPs相比,cathelicidins家族表现出更强的抑菌活性。除此之外,Cathelicidins还参与免疫调节、促进细胞增殖以及迁移、抑制组织损伤和促进损伤修复、促进细胞因子和组胺释放、促进血管生成等其他重要的生物学功能。
同已经发现的大多数Cathelicidin相比,来源于眼镜王蛇的cathelicidin(OH-CATH30)具有更强,更广谱的抗菌活性,而且在高剂量下并没有发现明显的溶血活性。这些特点表明蛇来源的Cathelicidin有可能开发为全身性给药的抗细菌感染药物。但是从生物体中直接获取的天然Cathelicidin提取量比较少。
目前,大多数AMPs都是从生物体中分离得到或者通过化学方法合成的传统AMPs的生产策略,而通过化学方法合成AMPs的成本较高,通过基因工程和发酵工程的方法制备抗菌肽可以有效降低生产成本,生产工艺容易放大;产生的废液处理工艺较为成熟。目前使用原核表达系统制备抗菌肽占所有抗菌肽基因表达系统的80%左右。人们使用原核表达系统表达抗菌肽已经积累了许多经验,对抗菌肽表达质粒构建,生产工艺的中试和放大也有所进展。但是抗菌肽的原核表达依然具有很大技术挑战性,其不仅对宿主菌有毒性,而且由于抗菌肽分子量较少,易被宿主菌各种内源性蛋白酶降解。因此通过微生物平台生产的AMPs经常与载体蛋白结合来进行诱导表达。目前常用的融合标签有硫氧还蛋白(Trx)、类固醇异构酶(KSI)及谷胱甘肽-S-转移酶(GST)等,但在实际应用中,发现上述标签存在标签蛋白过大、促可溶性效果差、表达产物剪切效率低等问题。
2018年Sun等人发现了一个能够耐高温高盐的DAMP4载体蛋白,它是一个四个螺旋结构的小分子蛋白,能够通过非层析的方法实现重组蛋白的分离纯化,节省了繁琐的分离纯化步骤,降低了生产成本。但是由于不同的抗菌肽的生化物理特性不同,并不是所有的抗菌肽都能使用该载体蛋白进行原核表达成功(见实施例五和六)。
CathelicidinOH-CATH30是一条由30个氨基酸残基组成的多肽,具有强效、广谱、不依赖盐的抗菌活性,无明显的溶血活性。因此表明OH-CATH30是一个潜在的药物候选分子。然而,通过大肠杆菌表达系统获得大量且有活性的OH-CATH30是一个巨大的挑战。TongYi Sun利用大肠杆菌对合成的OH-CATH30的密码子进行了优化,并将其亚克隆到载体pET-32a中,使肽以硫氧还蛋白融合蛋白的形式表达,获得的最高蛋白质表达水平为100mg/L的细菌培养物,但是本研究并未重复出该文章中的实验结果,如具体实施例五,且分离和纯化步骤繁琐,生产成本高。
发明内容
本发明的目的旨在通过构建一种含有眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R的表达载体以及在TSsetta(DE3)Chemically Competent Cell表达感受态细胞中诱导表达可溶性产物和高效纯化可溶性表达产物的方法,从而获得有活性的重组体外抗菌肽OH-CATH30和OH-CATH30R表达产物。
一种眼镜王蛇抗菌肽OH-CATH30的的表达载体,是由DAMP4载体蛋白,通过linker分别连接眼镜王蛇抗菌肽OH-CATH30 N端到C端、C端到N端基因构建PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R,具有凝血酶切割位点,其重组质粒诱导表达的重组蛋白为140aa,其载体质粒PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R的图谱分别如图1、图2所示。
本发明所述的眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的原核表达载体PET-28a上连接载体蛋白DAMP4取代PET-28a本身所带的N-His,N-Thrombin标签,偶联N端-C端的OH30和C端-N端的OH30基因(Genbank登录号:EU622894)所构建的PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R表达质粒,具有凝血酶切割位点。其可溶性表达产物DAMP4-F-OH30、DAMP4-F-OH30R通过N端DAMP4载体蛋白耐受高温高盐的性质,90℃高温加热去除杂蛋白,纯化的重组蛋白产物可用凝血酶将融合蛋白中的DAMP4载体蛋白除去,从而获得具有抑菌活性的体外抗菌肽OH-CATH30和OH-CATH30R。通过计算得知,PET-28a-DAMP4-F-OH30重组质粒诱导表达的蛋白为140aa,理论分子量为15.71kDa。PET-28a-DAMP4-F-OH30表达质粒的核苷酸序列如SEQ ID No.1所示。氨基酸序列如SEQ ID No.2所示。PET-28a-DAMP4-F-OH30R重组质粒诱导表达的蛋白为140aa,理论分子量为15.71kDa。PET-28a-DAMP4-F-OH30R表达质粒的核苷酸序列如SEQ ID No.3所示。氨基酸序列如SEQ ID No.4所示。
本发明还保护一种眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的表达菌株,其为含有PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R表达质粒的TSsetta(DE3)ChemicallyCompetent Cell表达菌株。利用IPTG诱导后收集包含重组表达蛋白DAMP4-OH30和DAMP4-OH30R的细菌沉淀。
本发明还保护眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的表达载体和表达产物的构建制备方法,其步骤如下:
S1:重组表达载体PET-28a-DAMP4-F-OH30和PET-28a-DAMP4-F-OH30R的构建
所述的重组表达载体是由DAMP4载体蛋白,通过linker分别连接眼镜王蛇抗菌肽OH-CATH30N端到C端、C端到N端基因构建PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R;
S2:表达菌株的获得:将S1的重组表达载体用热击法转化到大肠杆菌top10感受态细胞,分别得到含有眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的重组PET-28a-DAMP4-F-OH30阳性克隆菌株和PET-28a-DAMP4-F-OH30R阳性克隆菌株,将阳性克隆菌株分别摇菌提取质粒,再用热击法将重组质粒PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R转化至TSsetta(DE3)Chemically Competent Cell表达感受态细胞中,得到含重组质粒PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R的TSsetta(DE3)Chemically Competent Cell表达菌株;
S3:重组表达工程菌的诱导表达:用将上述步骤S2得到的表达菌株进行诱导表达,得到融合表达表物DAMP4-F-OH-CATH30(DAMP4-OH30(ReOH30)和DAMP4-OH-CATH30R(DAMP4-OH30R(ReOH30R);
S4:重组抗菌肽的分离、纯化:将上述S3步骤中表达产物经过离心收集菌体、菌体破碎后再经离心、分离纯化、透析、酶切,即得重组表达抗菌肽OH-CATH30(OH30)和OH-CATH30R(OH30R);
S5:重组抗菌肽抑菌活性的测定
测定重组表达产物酶切反应前和经过凝血酶酶切后得到的产物对大肠杆菌25922菌株的抑菌效果。
进一步地,所述的重组表达载体PET-28a-DAMP4-F-OH30的构建方法如下:重组表达载体PET-28a-OH30由生工生物工程(上海)股份有限公司进行全基因合成,全基因合成DAMP4-F-OH30,从NcoI和HindIII双酶切插入表达载体PET-28a而得到。
进一步,所述的重组表达载体PET-28a-DAMP4-F-OH30R的构建方法如下:
A)根据北京擎科生物有限公司构建的重组载体pET47b-OH30R为模板,合成引物28a-FF和28a-RR,引物序列如SEQ ID No.5和SEQ ID No.6所示,用高保真酶Max DNA Polymerase扩增DMP4-OH30R表达序列,得到PCR产物大小为462bp的带同源臂的目的基因DAMP4-OH30R,PCR产物跑琼脂糖凝胶并对目的片段进行切胶回收;
B)PET-28a空载质粒用限制性核酸内切酶NcoI和Hind III在37℃下双酶切1小时,并对酶切的PET-28a载体进行琼脂糖凝胶电泳和切胶纯化,从而得到线性化PET-28a载体;
C)待插入目的片段DAMP4-OH30R和线性化PET-28a载体按照摩尔比为3:1加入反应体系中,50℃孵育15min,得到重组载体PET-28a-DAMP4-F-OH30R。
进一步地,所述的步骤S4重组抗菌肽分离纯化包括如下步骤:
S41、剩余诱导表达菌液离心收集沉淀,用菌体裂解缓冲液洗涤两次,再用菌体裂解缓冲液重悬菌体,加入蛋白酶抑制剂(PMSF),超声破碎,离心将上清和沉淀分开,跑SDS-PAGE胶,确定目标蛋白是否为可溶性表达;
S42、利用高温高盐的非层析方法进行纯化利用载体蛋白DAMP4耐受高温高盐的特点,将获得的上清液在90℃加热30分钟,从而除去大多数杂蛋白,得到纯化后融合蛋白DAMP4-OH-CATH30和DAMP4-OH-CATH30R。所述的融合表达产物DAMP4-OH-CATH30和DAMP4-OH-CATH30R在90℃、1M NaCl的条件下,仍以可溶性的形式存在,且经90℃加热30min后其活性并未受到影响。
S43、分离纯化后目标蛋白的酶切反应
经高温除去大部分杂蛋白的蛋白样品,用透析的方式置换缓冲液为有效的凝血酶酶切缓冲液,BCA定量法测量其蛋白浓度;进行酶切,得到体外抗菌肽OH-CATH30和OH-CATH30R。
优选地,所述步骤S41中PMSF的终浓度为1mM,超声破碎的条件为240W、超声2s、间隔2s、工作总时间为40min。
优选地,所述的菌体裂解缓冲液为25mM Tris、1M NaCl、PH8.0。
优选地,所述的步骤S43中有效酶切缓冲液为20mM Tris、150mM NaCl、PH8.0。
优选地,所述的步骤S43中的酶切条件为常温孵育1小时。
本发明还提供了眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R在抑制大肠杆菌25922生长中的应用。
本发明具有以下有益效果特点:
本发明采用原核表达得载体构建包含眼镜王蛇抗菌肽OH-CATH30的编码基因的重组载体,并且首次使用融合蛋白DAMP4作为载体蛋白,使用Linker(F)将抗菌肽OH-CATH30从N-C、从C—N连接分别建立表达载体并进行原核表达获得的的重组抗菌肽DAMP4-CATH30在纯化后不经任何处理即具有明显的抑菌活性;获得重组抗菌肽DAMP4-CATH30R经酶切处理后,也具有明显的抑菌活性。
本发明首次将抗菌肽OH-CATH30从C—N进行原核表达,得到的重组抗菌肽OH-CATH30R经酶切处理后,也具有明显的抑菌活性,为抗菌肽的重组表达提供了新思路。
本发明方法简单易行,对大肠杆菌25922具有广谱性,应用范围广。这表明本发明提供的重组蛋白可以用于制备成抗菌剂,为当前杀菌消毒领域提供一种新的选择。
附图说明
图1为重组质粒PET-28a-DAMP4-OH30的图谱;
图2为重组质粒PET-28a-DAMP4-OH30R的图谱;
图3为目的片段DAMP4-OH30R PCR扩增片段SDS-PAGE凝胶电泳结果;
图4为含重组质粒PET-28a-DAMP4-F-OH30工程菌在37℃诱导表达、分离纯化的SDS-PAGE结果;
图5、为含重组质粒PET-28a-DAMP4-F-OH30工程菌在37℃诱导表达的WesternBlot结果;
图6、为含重组质粒PET-28a-DAMP4-F-OH30工程菌诱导表达重组蛋白经凝血酶酶切后的SDS-PAGE结果;
图7、为含重组质粒PET-28a-DAMP4-F-OH30工程菌诱导表达重组蛋白经凝血酶酶切后的Western Blot结果;
图8、为含重组质粒PET-28a-DAMP4-F-OH30R工程菌诱导表达SDS-PAGE结果;
图9为含重组质粒PET-28a-DAMP4-F-OH30R工程菌诱导表达后菌体,超声破碎后上清分离纯化的SDS-PAGE结果;
图10为含重组质粒PET-28a-DAMP4-F-OH30R工程菌诱导表达重组蛋白经凝血酶酶切后的SDS-PAGE结果;;
图11为经凝血酶酶切后的重组目的蛋白OH-CATH30的抑菌实验;
图12为经凝血酶酶切后的重组目的蛋白OH-CATH30R的抑菌实验;
图13为含重组质粒PET-28a-DAMP4-F-OH30工程菌诱导表达后菌体,超声破碎后上清分离纯化得到的重组蛋白DAMP4-F-OH30的抑菌实验;
图14为含重组质粒PET-28a-DAMP4-F-OH30工程菌在16℃和25℃诱导表达的Western Blot结果;
图15为含重组质粒Pmal-c5x-OH30工程菌诱导表达SDS-PAGE结果;
图16为含重组质粒PET-32a-OH30工程菌诱导表达SDS-PAGE结果;
图17为含重组质粒PET-28a-DAMP4-F-NA30和重组质粒PET-28a-DAMP4-F-pexigananR工程菌诱导表达的SDS-PAGE结果。
具体实施方式
一、本发明具体实施方式中采用的材料和来源如下:
1、菌株与质粒
TSsetta(DE3)Chemically Competent Cell;DH5α Chemically Competent Cell;PET-28a(+)载体;E.Coil25922均来自本实验室保种或制备。
高保真酶Max DNA Polymerase、琼脂糖凝胶电泳10*LoadingBuffer、DL2000 DNA Marker、限制性核酸内切酶NcoI和Hind III均为TaKaRa公司产品;Seamless Cloning Kit(无缝克隆试剂盒)买自上海碧云天生物技术有限公司;无内毒素质粒小提中量试剂盒、DNA凝胶回收试剂盒为Tiangen产品;SDS-PAGE配胶试剂盒为雅酶产品;Tricine-SDS-PAGE为北京聚合美产品;SDS-PAGE和Tricine-SDS-PAGE所用Protein Marker为Thermo产品。
二、眼镜王蛇抗菌肽OH-CATH30原核表达方法如下:
1、PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R重组质粒的构建
PET-28a-DAMP4-F-OH30是由上海生工生物有限公司合成,重组质粒PET-28a-DAMP4-F-OH30R通过同源重组的方法构建。以前期北京擎科生物技术有限公司合成的重组质粒pET47b-OH30R为模板,28a-FF和28a-RR,其序列分别如SEQ ID No.5和SEQ ID No.6所示。其中Linker序列为GGTGGCCCAGGTTCCGGT,核酸序列编码的对应氨基酸序列GGPGSG,其序列分别如SEQ ID No.9和SEQ ID No.10所示,用高保真酶Max DNAPolymerase扩增DAMP4-OH30R表达序列,得到PCR产物大小为462bp的带同源臂的目的基因DAMP4-OH30R,PCR产物跑琼脂糖凝胶并对目的片段进行切胶回收;其次将空载质粒PET-28a用限制性核酸快切内切酶Nco I和Hind III在37℃下双酶切1小时,并对线性化的载体进行琼脂糖凝胶电泳和切胶回收,从而得到线性化的PET-28a载体。将线性化后的PET-28a载体和目的基因DAMP4-OH30R按照1:3加入反应体系,50℃孵育15min,得到重组质粒PET-28a-DAMP4-F-OH30R。
2、重组质粒的扩增、鉴定及转化
2.1、大肠杆菌感受态的制备
从-80℃取感受态细胞,涂板(DH5α涂于无抗平板,TSsetta涂于氯霉素抗性平板)。
挑单克隆摇菌,保种。1ml菌液加入到100mlLB培养基中,当OD600=0.4左右(小于0.45)时,置于冰上30min。
在超净工作台中,将菌液倒入预冷并灭菌的收菌瓶中,4℃、3500rpm、5min,弃上清,留沉淀;加入25ul提前灭菌的100mM的CaCl2,轻摇,悬浮。
悬浮后,4℃、3500rpm、5min,弃上清,留沉淀;加入5ml提前灭菌的100mM的CaCl2(内含15%的甘油),冰上轻摇,悬浮。
加入5ml提前灭菌的100mM的CaCl2(内含15%的甘油),冰上轻摇,悬浮;
冰上静置1h,期间每隔10min轻摇2min。
菌液分装,到提前预冷且灭菌的1.5mlEP管中,放于液氮中,﹣80℃冰箱保存。
2.2、重组质粒的扩增
取100μl冰上融化的DH5α感受态细胞,加入5μl重组质粒PET-28a-OH30R,冰上静置30min;42℃水浴热激90s,迅速转移至冰上,静置5min;向离心管中加入200μl无抗性LB培养基,37℃、200rpm摇床培养60min;取200μl涂布到相应抗性的培养基上,37℃培养箱倒置培养过夜。
2.3、重组质粒的鉴定
取平板上的单克隆菌落于5ml含Kana抗生素的LB中,37℃、200rpm摇床培养8小时;取1μl进行菌液PCR,另取1ml送至北京擎科公司测序,将测序结果通过软件Snapgene进行BLAST,测序结果正确的重新进行摇菌,通过质粒小提中量试剂盒进行质粒抽提,用NanoDR2000测量抽提质粒的浓度。
2.4、重组质粒的转化
将100ngPET-28a-DAMP4-F-OH30和PET-28a-DAMP4-F-OH30R质粒加入至100μlTSsetta(DE3)感受态细胞中,轻轻混匀,冰上静置30min;42℃水浴热激90s,迅速转移至冰上,静置5min;向离心管中加入200μl无抗性LB培养基,37℃、200rpm摇床培养60min;取200μl涂布到含Kana的LB培养基上,37℃培养箱倒置培养过夜。
3、重组质粒的诱导表达
挑取单克隆菌落到15ml含Kana的LB培养基中,37℃、180rpm摇床震荡培养12小时。按照1:100的接种比,取10ml菌液至1L含Kana的LB培养基中37℃、200rpm摇床震荡培养至OD600≈0.5-0.8;取1ml作为未诱导,其余加入终浓度为0.5mM的IPTG,含重组质粒PET-28a-DAMP4-F-OH30的表达菌株37℃、110rpm诱导表达6小时;含重组质粒PET-28a-DAMP4-F-OH30R的表达菌株25℃、180rpm诱导表达12小时。
4、诱导表达重组蛋白的鉴定
取1ml诱导表达菌液8000rpm、离心10min,跑SDS-PAGE以及Western Blot确定是否诱导表达成功。剩余诱导表达菌液4000g、离心20min,弃上清留沉淀;用洗涤Buffer洗涤两次,在用裂解Buffer重悬菌体,加入终浓度为1mM蛋白酶抑制剂PMSF,240W、超2s、停2s、工作总时间为40min,4℃、15000rpm离心20min,将上清和沉淀分开,跑SDS-PAGE胶,确定目标蛋白是否为可溶性表达。
5、诱导表达重组蛋白的分离纯化
利用诱导表达蛋白所带重组载体DAMP4耐高温高盐的特征,采用90℃高温30min的方法,去除大部分杂蛋白。
6、目的蛋白的获取和鉴定
经高温除去大部分杂蛋白的蛋白样品,用透析的方式置换Buffer为有效的凝血酶酶切buffer(20mM Tris、150mM NaCl、PH8.0),用BCA定量法测量其蛋白浓度;加入凝血酶,常温孵育1小时。酶切结束后,SDS-PAGE电泳和Western blot鉴定是否酶切成功。
7、液体法抑菌活性的检测
通过微量液体法测定不同药物浓度的杀菌能力,从而得到能够抑制细菌生长的最低药物浓度,即最小抑菌浓度(MIC)。步骤如下:(1)用分光光度计检测新鲜大肠杆菌菌液的OD600,当OD600=0.5时,按照1 OD=1×109CFU/ml,用培养基将菌液浓度调至2×104CFU/ml。(2)在无菌的96孔(除第一孔)加入100μl的新鲜培养基,第一孔加入100μl的凝血酶酶切后的蛋白样品,吹吸混匀,从第一孔取100μl加入第二孔,依次稀释,最后一孔吸出100μl弃之。(3)PET-28a空载质粒诱导表达得到的产物作为阴性对照,固相化学合成的OH-CATH30(终浓度为10ug/ml)作为阳性对照。(4)向每孔加入100μl浓度为2×104CFU/ml的菌液,96孔板混匀仪混匀,置于37℃恒温培养箱孵育16小时,最后用酶标仪检测OD 600的吸光值。每孔做三个独立重复,96孔板四周用200μl培养基水封,防止染菌。
三、抗菌肽OH-CATH30和OH-CATH30R的抑菌活性
1、PCR产物琼脂糖凝胶电泳结果
如图3所示,PCR扩增DAMP4-F-OH30R基因片段大小为462bp,与预期结果相符。琼脂糖凝胶切胶回收测序结果表明PCR产物序列完全正确。
2、SDS-PAGE、Tricine-SDS-PAGE电泳和Western blot免疫印迹结果
含重组质粒PET-28a-DAMP4-F-OH30工程菌经IPTG诱导后和未诱导的菌体沉淀在SDS-PAGE凝胶和Western blot(Western blot所用一抗均为本实验所制备的针对OH-CATH30兔抗,一抗稀释比均为1:5000)上有明显的重组蛋白诱导条带,分子量为15.71kDa,与预期的分子量大小相符(图4、图5),表明DAMP4-OH30(ReOH30)成功表达。分离纯化后的重组蛋白,经凝血酶切割后在SDS-PAGE凝胶和Western blot上均能看到明显的分子量为11.9711kDa和3.739kDa两条带,与预期的分子量相符(图6、图7),表明重组表达蛋白(ReOH30)被凝血酶成功切割其重组目的蛋白的产量为0.5mg/L。含重组质粒PET-28a-DAMP4-F-OH30R工程菌经IPTG诱导后和未诱导的菌体沉淀在SDS-PAGE凝胶上有明显的重组蛋白诱导条带,分子量为15.71kDa,与预期的分子量大小相符(图8),表明DAMP4-OH30R(ReOH30R)成功表达。分离纯化后的重组蛋白,经凝血酶切割后在SDS-PAGE凝胶上能看到明显的分子量为11.971kDa的DAMP4载体蛋白,与预期的分子量相符(图9、图10),表明ReOH30R被凝血酶成功切割。其重组目的蛋白OH-CATH30R(ReOH30R)的产量为1.4mg/L。
3、体外表达抗菌肽OH-CATH30和OH-CATH30R抑菌实验结果
通过测定重组表达OH-CATH30和OH-CATH30R的MIC值,可知重组蛋白DAMP4-F-OH30和DAMP4-F-OH30R酶切后(图10、图11)均表现出明显的抑菌活性,其中体外表达抗菌肽OH-CATH30的MIC值为3.4ug/ul,OH-CATH30R的MIC值为3.0ug/ul。重组蛋白DAMP4-F-OH30不经酶切处理(图12)也具有一定的酶切活性,而未经IPTG诱导的PET-28a-DAMP4-F-OH30和PET-28a-DAMP4-F-OH30R产物无抑菌活性。上述结果表明本发明重组OH-CATH30具有较好的体外抑菌效果。
四、不同表达温度的筛选
重组质粒PET-28a-DAMP4-F-OH30在诱导表达时,尝试使用不同的温度条件,分别在16℃、25℃和37℃进行表达,看其是否表达。如图5和图14的Western blot结果表明,表达质粒PET-28a-DAMP4-F-OH30仅在37℃有表达,在16和25℃下不表达。
五、使用不同的融合子(载体蛋白)的表达载体以及原核表达OH-CATH30结果对比
如图15、图16所示,将OH-CATH30的序列插入Pmal-c5x和PET-32a多克隆位点中,以Pmal-c5x质粒上的MBP标签和PET-32a质粒上的TrxA标签作为载体蛋白偶联OH-CATH30,IPTG诱导表达均诱导表达成功,但Pmal-c5x-OH30经直链淀粉柱纯化后并不能被凝血酶切割,未切割的重组Pmal-c5x-OH30并未有抑菌活性,可能是由于MBP载体蛋白将酶切位点包裹,导致凝血酶无法识别酶切位点;PET-32a-OH30超声破碎后,目的蛋白以包涵体的形式存在,包涵体变性复性后,也未能得到有活性的目的蛋白。
六、利用上述方法表达眼镜蛇来源抗菌肽NA30和抗菌肽pexiganan
NA-CATH30(KFFKKLKNSVKKRAKKFFKKPKVIGVTFPF)序列号如SEQ ID No.7所示,同OH-CATH30相比仅差3个氨基酸,抗菌肽pexiganan(GIGKFLKKAKKFGKAFVKILKK)序列号如SEQID No.8所示,使用本发明相同的方法来表达。
结果如图17所示,SDS-PAGE结果表明,使用相同的方法表达抗菌肽NA30,表达不成功。除此之外,将抗菌肽pexiganan从C-N端表达,也并未成功表达。
以上SDS-PAGE和Western blot结果均表明,不是所有的抗菌肽都是可以用DAMP4载体蛋白进行重组都能表达,并且能得到具有抗菌活性的抗菌肽的;并且也不是所有抗菌肽从C-N端进行原核表达都可以成功表达且有活性。
SEQUENCE LISTING
<110> 中国科学院昆明动物研究所
<120> 一种眼镜王蛇抗菌肽OH-CATH30的表达载体和表达产物及其构建制备方法
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Claims (6)
1.一种眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,包括以下步骤:
S1:重组表达载体PET-28a-DAMP4-F-OH30和PET-28a-DAMP4-F-OH30R的构建
所述的重组表达载体是由DAMP4载体蛋白,通过linker分别连接眼镜王蛇抗菌肽OH-CATH30 N端到C端、C端到N端基因构建PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R,具有凝血酶切割位点,重组表达载体诱导表达的重组蛋白均为140aa,重组蛋白DAMP4-F-OH-CATH30的编码核苷酸序列和氨基酸序列分别如SEQ ID No.1和SEQ ID No.2所示,重组蛋白DAMP4-OH-CATH30R的编码核苷酸序列和氨基酸序列分别如SEQ ID No.3和SEQ ID No.4所示;
S2: 表达菌株的获得:将S1的重组表达载体用热击法转化到大肠杆菌top10感受态细胞,分别得到含有眼镜王蛇抗菌肽OH-CATH30和OH-CATH30R的PET-28a-DAMP4-F-OH30阳性克隆菌株和PET-28a-DAMP4-F-OH30R阳性克隆菌株,将阳性克隆菌株分别摇菌提取质粒,再用热击法将重组表达载体PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R分别转化至TSsetta (DE3) Chemically Competent Cell表达感受态细胞中,得到分别含重组表达载体PET-28a-DAMP4-F-OH30、PET-28a-DAMP4-F-OH30R的TSsetta (DE3) ChemicallyCompetent Cell表达菌株;
S3:重组表达工程菌的诱导表达:用将上述步骤S2得到的表达菌株进行诱导表达,得到表达产物DAMP4-F-OH-CATH30和DAMP4-OH-CATH30R;所述的诱导表达条件为:含重组表达载体PET-28a-DAMP4-F-OH30的表达菌株37℃、110rpm诱导表达6小时;含重组表达载体PET-28a-DAMP4-F-OH30R的表达菌株25℃、180rpm诱导表达12小时;
S4:重组抗菌肽的分离、纯化:将上述S3步骤中表达产物经过离心收集菌体、菌体破碎后再经离心、分离纯化、透析、凝血酶酶切,即得重组表达抗菌肽OH-CATH30和OH-CATH30R;
S5:重组抗菌肽抑菌活性的测定
测定重组表达产物凝血酶酶切反应前和经过凝血酶酶切后得到的产物对大肠杆菌25922菌株的抑菌效果。
2.根据权利要求1所述的眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,其特征在于:所述的重组表达载体PET-28a-DAMP4-F-OH30的构建方法如下:全基因合成DAMP4-F-OH-CATH30的编码核苷酸序列,从NcoI和Hind III双酶切插入表达载体PET-28a而得到重组表达载体PET-28a-DAMP4-F-OH30。
3.根据权利要求1所述的眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,其特征在于:所述的重组表达载体PET-28a-DAMP4-F-OH30R的构建方法如下:
A)以SEQ ID No.3所示的重组蛋白DAMP4-OH-CATH30R 编码核苷酸序列为模板,合成引物28a-FF和28a-RR,引物序列如SEQ ID No.5和SEQ ID No.6所示,用高保真酶PrimeSTAR®Max DNA Polymerase扩增DAMP4-F-OH-CATH30R表达序列,得到PCR产物大小为462bp的带同源臂的目的基因DAMP4-F-OH-CATH30R,PCR产物跑琼脂糖凝胶并对目的片段进行切胶回收;
B)PET-28a空载质粒用限制性核酸内切酶NcoI和Hind III在37℃下双酶切1小时,并对酶切的PET-28a载体进行琼脂糖凝胶电泳和切胶纯化,从而得到线性化的PET-28a载体;
C)待插入目的片段DAMP4-F-OH-CATH30R和线性化PET-28a载体按照摩尔比为3:1加入反应体系中,50ºC 孵育15 min,得到重组表达载体PET-28a-DAMP4-F-OH30R。
4.根据权利要求1所述的眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,其特征在于:所述的步骤S4重组抗菌肽分离纯化包括如下步骤:
S41、剩余诱导表达菌液离心收集沉淀,用菌体裂解缓冲液洗涤两次,再用菌体裂解缓冲液重悬菌体,加入蛋白酶抑制剂PMSF,超声破碎,离心将上清和沉淀分开,跑SDS-PAGE胶,确定目标蛋白是否为可溶性表达;
S42、利用高温高盐的非层析方法进行纯化,所述高温的条件为采用90℃、30 min;
S43、分离纯化后目标蛋白的酶切反应,经S42步骤除去大部分杂蛋白的蛋白样品,用透析的方式置换缓冲液为有效的凝血酶酶切缓冲液,用BCA定量法测量其蛋白浓度;进行凝血酶酶切,所述的酶切条件为加入凝血酶,常温孵育1小时,得到重组抗菌肽OH-CATH30和OH-CATH30R。
5.根据权利要求4所述的眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,其特征在于:所述步骤S41中PMSF的终浓度为1 mM,超声破碎的条件为240 W、超声2s、间隔2s、工作总时间为40 min,所述的菌体裂解缓冲液为25 mM Tris、1M NaCl、PH8.0。
6.根据权利要求4所述的眼镜王蛇抗菌肽OH-CATH30及OH-CATH30R重组抗菌肽的制备方法,其特征在于:所述的步骤S43中有效酶切缓冲液为20 mM Tris、150 mM NaCl、PH8.0。
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