CN111019001B - 一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用 - Google Patents
一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用 Download PDFInfo
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- CN111019001B CN111019001B CN201911406640.1A CN201911406640A CN111019001B CN 111019001 B CN111019001 B CN 111019001B CN 201911406640 A CN201911406640 A CN 201911406640A CN 111019001 B CN111019001 B CN 111019001B
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Abstract
本发明提供一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用，属于生物基因工程技术领域。与现有技术相比，本发明具有如下优势：(1)本发明制备的溶菌酶抗菌肽融合蛋白抗菌活性高，对革兰氏阳性菌金葡球菌、表皮葡萄球菌和革兰氏阴性菌大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、金黄色葡萄球菌、绿脓杆菌和鼠伤寒沙门氏菌均具有明显的抑制作用，抗菌活性明显改善，拓宽了抗菌谱，且无细胞溶血性与动物毒性。(2)本发明采用的纯化制备之所以过程简单是因为采用色谱复性技术，采用本发明的连接肽，能够有效防止复性纯化蛋白的得率和活性明显下降。
Description
技术领域
本发明属于生物基因工程技术领域，具体地说涉及一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用。
背景技术
抗生素的滥用导致了大量耐药菌的出现，对人体造成直接威胁，已成为一个世界性难题。在禽畜及水产养殖方面也存在着抗生素滥用的现象，畜牧业使用抗生素的量，已经远远超过人类使用量的总和。食物、动物中抗生素残留及由此产生的这些耐药菌通过食物或动物与人的接触传播给人类，危害人类健康。长期滥用食品防腐剂也会导致耐药菌株的产生，其中不少还有较强致癌的危险，并对骨骼生长，肾脏、肝脏产生危害，直接威胁着人类的健康。因此，寻找高效、安全、无环境污染的抗有害微生物制剂具有重要现实意义。
溶菌酶、抗菌肽等作为新型抗菌剂，具有效果稳定、无残留、不易产生抗药性，并且安全无毒害。作为防腐杀菌剂，将它们用于医药、保健品、兽药以及作为食品、饮料的防腐保鲜剂、饲料添加剂等领域，其应用前景十分广阔，因而受到人们的关注。
溶菌酶是一种天然、高效、较广谱的抗菌剂，能明显抑制革兰氏阳性细菌生长。以往实际使用的溶菌酶来源于天然产物的提取，如鸡卵清溶菌酶、乳酸链球菌肽等，由于原料及制备成本较高，限制了它们的大规模应用。利用基因工程技术进行表达生产将是重要的发展方向。和蛋清溶菌酶相比，人溶菌酶溶菌活性比蛋清溶菌酶高出3倍，且热稳定性也高，具有较强的应用性。基因工程表达的人菌酶已开始进入临床实验。
重组人溶菌酶对革兰氏阳性菌中的金葡球菌、表皮葡萄球菌具有很好的抗菌活性，尤其对甲氧西林耐药的金葡球菌(MRSA)、表皮葡萄球菌(MRSE)及克拉霉素、罗红霉素等耐药菌均有效，但对革兰氏阴性菌菌中的大肠埃希菌、肺炎克雷伯菌、枸橼酸杆菌、沙雷菌、铜绿假单胞菌、阴沟肠杆菌等的抗菌作用较弱，因而限制了其应用范围，重组溶菌酶的抗菌范围及抗菌活性还有待进一步改进。
抗菌肽是广泛存在于生物体内的具抗微生物活性的小分子多肽，具有热稳定、水溶性好、无残留、不易产生抗药性的特点。大多抗菌肽对大肠杆菌，金黄色葡萄球菌、念珠球菌、绿脓杆菌、肠细菌、沙雷氏菌、变形杆菌、粪链球等革兰氏阴、阳性菌都有抑菌效果，抗菌的广谱性较优于溶菌酶。此外，抗菌肽对人及动物无毒无害，具有很高的安全性。因此，抗菌肽在新型抑菌药物、食品天然防腐剂及动物饲料添加剂等产品开发领域具有广阔的应用前景。
但抗菌肽天然资源有限，化学合成获得抗菌肽成本又较高，由于抗菌肽本身对宿主菌有一定的灭活作用，目前还不能在基因工程菌中直接表达抗菌肽，而是以融合蛋白的形式进行，这无疑给后续加工和使用带来了极大的不方便，并且大大增加了成本。因此，如何利用基因工程技术在微生物中方便、有效及低成本的生产抗菌肽成为其实际应用的关键。
溶菌酶和抗菌肽之间有较强的协同抗菌作用。天然状态下，溶菌酶、β-防御素、LL-37相互协调，可明显抑制葡萄球菌、大肠杆菌的生长，抑菌的效果均远高于两者单独使用的效果。
溶菌酶和抗菌肽是两种优良的天然抗菌剂，但实际应用还有相当的不足。对于前者，在抑菌活力、抑菌范围、稳定性等方面还有待进一步改善。而抗菌肽在性能上可以弥补溶菌酶抑菌范围、稳定性等方面的不足，但还不能在微生物中直接进行表达应用。
本专利发明旨在提供新的人溶菌酶-抗菌肽融合蛋白的编码基因及其基因工程制备方法，以提供一种新型、高效、无毒、无公害、稳定性好的抗菌制剂，改变当前抗生素和防腐剂滥用状况，为实现其在医疗、食品生产、动物养殖等领域的广泛应用，保障人民的身体健康，减少抗生素等对环境污染，实现社会的可持续发展。
发明内容
发明目的：本发明所要解决的技术问题是针对现有技术的不足，提供一种重组溶菌酶抗菌肽融合蛋白，从而提供新型、高效、无毒、无公害、稳定性好的抗菌制剂，改变当前抗生素和防腐剂滥用状况。
本发明还要解决的问题是提供上述重组溶菌酶抗菌肽融合蛋白的制备方法。
本发明最后要解决的问题是提供上述重组溶菌酶抗菌肽融合蛋白的应用。
为了解决上述技术问题，本发明公开了一种重组溶菌酶抗菌肽融合蛋白，其该蛋白从N端-C端依次由人溶菌酶的氨基酸编码序列、连接肽的氨基酸编码序列和抗菌肽的氨基酸编码序列连接所得。
其中，所述人溶酶菌的氨基酸编码序列如SEQ ID NO.1所示；SEQ ID NO.1：MKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV。
其中，所述连接肽的氨基酸编码序列如SEQ ID NO.2所示；
SEQ ID NO.2：SSSSSGSSSSGSSSSS。
其中，所述抗菌肽的氨基酸编码序列如SEQ ID NO.3所示；
SEQ ID NO.3：AVLKVLISSAASSTWKWKWRSSRFRFRAASSAAKLFK。
其中，所述抗菌肽的基因编码序列如SEQ ID NO.6所示。
其中，所述抗菌肽的分子设计包括如下步骤：根据APD数据库资源，选择若干抗菌肽为蓝本，按照阳离子和两亲性原则、结构简化原则及螺旋论方法，初步设计新型抗菌肽分子；
利用化学合成方法获得若干人工设计的抗菌肽样品，以大肠杆菌(ATCC25922)金黄色葡萄球菌(ATCC25923)为指示菌，微量肉汤稀释法测定抗菌肽样品的最小抑菌浓度(MIC)，获得活性高的抗菌肽，其编码序列如SEQID NO.2所示。
其中，所述重组溶菌酶抗菌肽融合蛋白的氨基酸编码序列如SEQ ID NO.4所示，其包括184个氨基酸；其中，1-131位氨基酸序列为人溶菌酶基因的编码序列，132-147位氨基酸序列为人工设计的连结肽基因的的编码序列，148-184位氨基酸序列为人工设计的抗菌肽基因的的编码序列；
SEQ ID NO.4：
MKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGVSSSSSGSSSSGSSSSSAVLKVLISSAASSTWKWKWRSSRFRFRAASSAAKLFK。
上述的重组溶菌酶抗菌肽融合蛋白的编码基因也在本发明的保护范围之内。
其中，所述编码基因的核苷酸序列如SEQ ID NO.5所示。
其中，所述的多肽氨基酸序列采用大肠杆菌的偏爱密码子将编码上述基因SEQ IDNO.4的密码子进行优化。
上述的重组溶菌酶抗菌肽融合蛋白的制备方法，包括如下步骤：
(1)人工合成编码SEQ ID NO.4基因的核苷酸序列SEQ ID NO.5，将克隆至肠杆菌表达载体pET30a的NdeI和HindIII酶切位点之间，构建重组大肠杆菌表达载体；
(2)将步骤(1)构建的重组大肠杆菌表达载体转化至大肠杆菌宿主细胞BL21(DE3)中，获得重组溶菌酶抗菌肽融合蛋白基因工程菌pET30-RA-BL21(DE3)，IPTG诱导表达后，离心收集菌体，超声波裂解，离心收集包涵体沉淀；
(3)将步骤(2)收集的包涵体沉淀进行纯化，即得到重组溶菌酶抗菌肽融合蛋白。
步骤(3)中，所述的纯化为将包涵体沉淀用洗涤液洗涤，收集二次包涵体沉淀，再进行抽提，离心，收集上清液；利用QAE层析柱、蛋白纯化仪对收集的上清液进行纯化。
具体地，包涵体分别经洗涤溶液(50mmol/L磷酸盐缓冲液，1％Triton 100，2mol/L尿素，pH7.8)溶液洗涤后13000r/min离心20min，收集包涵体沉淀，再以抽提溶液(50mmol/L磷酸盐缓冲液，8mol/L尿素和2mmol/L DTT，pH7.8)25℃搅拌溶解过夜，13000r/min 4℃离心收集上清液。
将溶菌酶抗菌肽融合蛋白尿素抽提溶液对Q-Sepharose Fast Flow阴离子交换柱上样，上样结束后用同样缓冲液洗涤后再用溶液IV(50mmol/L磷酸盐缓冲液，2mmol/LDTT，pH7.8)洗涤。用0-1mol/L浓度的NaCl、50mmol/L磷酸盐缓冲液、谷胱甘肽(GSSG/GSH)的溶液进行梯度洗脱，收集目的样品用PBS溶液透析过夜并测定各峰蛋白活性。
上述重组溶菌酶抗菌肽融合蛋白在抑制革兰氏阳性菌和/或革兰氏阴性菌中的应用也在本发明的保护范围之内，具有广谱的对革兰氏阳、阴性菌均有抗菌作用。
有益效果：与现有技术相比，本发明具有如下优势：
(1)本发明制备的溶菌酶抗菌肽融合蛋白抗菌活性高，对革兰氏阳性菌金葡球菌、表皮葡萄球菌和革兰氏阴性菌大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、金黄色葡萄球菌、绿脓杆菌和鼠伤寒沙门氏菌均具有明显的抑制作用，抗菌活性明显改善，拓宽了抗菌谱，且无细胞溶血性与动物毒性。
(2)本发明利用大肠杆菌表达系统和离子交换色谱复性纯化技术成功实现了重组制备，获得了抗菌活性和抗菌谱都有改善的抗菌蛋白制剂，拓宽了抗菌肤的生物资源，在医药、兽药、饲料添加剂、食品保鲜剂的研制与开发方面具有广阔的应用潜力，最终实现该产品的产业化廉价生产具有重要意义。
(3)本发明专利的重组蛋白制备不需要酶制剂的后续加工，能够有效降低成本。
(4)本发明采用的纯化制备之所以过程简单是因为采用色谱复性技术，采用本发明的连接肽，能够有效防止复性纯化蛋白的得率和活性明显下降。
附图说明
图1为不同纯化阶段的目的蛋白SDS-PAGE电泳图谱。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：
委托生物技术公司人工合成编码SEQ ID NO.4基因的核苷酸序列SEQ ID NO.5，将其克隆至大肠杆菌表达载体pET30a的NdeI和HindIII酶切位点之间，获得构建好的重组大肠杆菌表达载体pET30-RA，将该重组载体用CaCl2法转化进转化至大肠杆菌宿主细胞BL21(DE3)中获得重组溶菌酶抗菌肽融合蛋白基因工程菌pET30-RA-BL21(DE3)。挑取鉴定过的pET30-RA-BL21(DE3)甘油冻存菌种接种于5mL LB含100mg/L卡那霉素培养液中，220r/min，37℃培养过夜。取菌液按体积比为1/100的比例加入250mL含100mg/L卡那霉素培养液的摇瓶发酵培养基中，37℃培养至对数生长期，当A600达到0.7时加入IPTG至终浓度1mmoL/L，37℃条件下进行诱导4小时结束发酵。
250mL培养液离心收集菌体，超声波裂解，离心收集包涵体沉淀。包涵体分别经200mL洗涤溶液(50mmol/L磷酸盐缓冲液，1％V/V Triton 100，2mol/L尿素，pH7.8)洗涤后，13000r/min离心20min，收集包涵体沉淀，再加入50mL抽提溶液(50mmol/L磷酸盐缓冲液，8mol/L尿素和2mmo l/L DTT，pH7.8)25℃搅拌溶解过夜，13000r/min4℃离心收集约得0.5mg/L浓度上清液，即溶菌酶抗菌肽融合蛋白8mol/L尿素抽提溶液。
将上述溶菌酶抗菌肽融合蛋白尿素抽提溶液对10mL柱体积的Q-Sepharose FastFlow阴离子交换柱上样，上样结束后用同样缓冲液洗涤3-5个床体积后再用二次洗涤溶液(50mmol/L磷酸盐缓冲液，2mmol/LDTT，pH7.8)洗涤3-5个床体积，用0-1mol/L浓度的NaCl、50mmol/L磷酸盐缓冲液、谷胱甘肽(GSSG/GSH)的溶液进行梯度洗脱，收集目的样品用PBS溶液透析过夜并测定样品蛋白活性。结果见实施例3。
实施例2：
挑取实施例1中鉴定过的pET30-RA-BL21(DE3)甘油冻存菌种接种于50mL LB含100mg/L卡那霉素培养液中，220r/min，37℃培养15小时。按1％的体积比的接种量转接种于5L自动发酵罐中，培养温度37℃，溶氧、pH均自动控制，发酵参数为：37℃，溶氧>25％，pH7.5。利用增加搅拌转速和增加通气量调节溶解氧浓度。发酵开始数小时后当溶解氧浓度快速升高时后，以0.05g/(L·min)葡萄糖的速率进行补料至终浓度2g/L。待葡萄糖耗尽后，开始流加乳糖至10dL进行诱导表达6小时。其中发酵罐培养基含有葡萄糖、少量无机盐(葡萄糖5g/L，MgS04·7H2O 5g/L，pH7.0)的2-YT培养基；补料培养基：葡萄糖100g/L，酵母提取物10g/L，蛋白胨16g/L，NaCl 5g/L，MgS047H2O 5g/L。
4L培养液离心收集菌体，经均质机高压裂解，离心收集包涵体沉淀。包涵体分别经4L洗涤溶液(50mmol/L磷酸盐缓冲液，1％W/V Triton 100，2mol/L尿素，pH7.8)洗涤后，13000r/min离心20min，收集包涵体沉淀，再以1L抽提溶液(50mmol/L磷酸盐缓冲液，8mol/L尿素和2mmol/L DTT，pH7.8)25℃搅拌溶解过夜13000r/min 4℃离心收集约得1.5mg/L溶菌酶抗菌肽融合蛋白8mol/L尿素抽提溶液。
将此溶菌酶抗菌肽融合蛋白8mol/L尿素抽提溶液对300mL柱体积的Q-SepharoseFast Flow阴离子交换柱上样，上样结束后用同样缓冲液洗涤3-5个床体积后再用二次洗涤溶液(50mmol/L磷酸盐缓冲液，2mmol/LDTT，pH7.8)洗涤3-5个床体积，用0-1mol/L浓度的NaCl、50mmol/L磷酸盐缓冲液、谷胱甘肽(GSSG/GSH)的溶液进行梯度洗脱，收集目的样品经Sephadex G-25柱脱盐冻干即为纯度大于95％的溶菌酶抗菌肽融合蛋白RA纯品，其抗菌活性见实例3(图1为不同纯化阶段的目的蛋白SDS-PAGE电泳图谱，其中1为菌体；2为层析纯化后的样品；3为包涵体；4为尿素抽提透析上清液)。
实施例3：
以大肠杆菌、沙门氏菌、铜绿假单胞菌、金黄色葡萄球菌、绿脓杆菌为指示菌，微量肉汤稀释法测定最小抑菌浓度(MIC)。将对数生长期的各种指示菌(CFU:1X 106)接种在LB培养基中，再加入10μL不同浓度的抗菌蛋白，(终浓度为2000、1000、500、250、125、62.5、31.25、15.125、7.8125、3.9μg/mL)37℃孵育12h后，在492nm测定各管的吸光度。吸光度无明显变化的抗菌肽的最低浓度为MIC。将大于MIC的各管涂于LB固体培养基平板上，37℃孵育12h后观察，无细菌生长的最低浓度为MBC。同时，以青霉素钠、阿莫西林两种抗生素作为阳性对照，比较纯度大于95％的抗菌肽融合蛋白RA样品与抗生素的相对抑制细菌生长活性，结果如下表。
表1
从表1可知，本发明设计并制备的融合蛋白对革兰氏阴性、阳性菌均有较好的抗菌活性，且抗菌活性由于青霉素和阿莫西林，因此，其作为新的抗菌制剂，在医药、兽药、饲料添加剂、食品保鲜剂方面具有广阔的应用前景。
对比例：对人溶菌酶和抗菌肽的抑菌效果进行检测，检测方法同实施例3。
表2
由表2可知，本发明设计并制备的融合蛋白的抗菌活性优于其内部单独的溶菌酶和抗菌肽的抗菌活性，表明这两个蛋白结构域在抗菌方面发挥了较好的协同作用，也表明本发明设计的连接肽在其间发挥了很好的作用。
本发明提供了一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
序列表
<110> 金陵科技学院
<120> 一种重组溶菌酶抗菌肽融合蛋白及其制备方法与应用
<160> 6
<170> SIPOSequenceListing 1.0
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Met Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu
1 5 10 15
Gly Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu
20 25 30
Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala
35 40 45
Gly Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr
50 55 60
Trp Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu
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Ser Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys
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Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala
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Cys Gly Val
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Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Ser
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Ala Val Leu Lys Val Leu Ile Ser Ser Ala Ala Ser Ser Thr Trp Lys
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Ala Lys Leu Phe Lys
35
<210> 4
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Met Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu
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Gly Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu
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Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala
35 40 45
Gly Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr
50 55 60
Trp Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu
65 70 75 80
Ser Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys
85 90 95
Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala
100 105 110
Trp Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly
115 120 125
Cys Gly Val Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser
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<210> 5
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<212> DNA
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atgaaagttt ttgaacgttg tgaactggct cgtaccttga aacgtctggg tatggatggc 60
tatcgcggta tttccttagc caattggatg tgcttggcca aatgggaatc tggctataac 120
acccgcgcta ctaattacaa cgcaggcgat cgtagcacag attacggtat cttccaaatc 180
aatagtcgtt actggtgtaa cgatggcaaa acgccaggtg ctgttaatgc atgtcattta 240
tcttgctcag cactgttaca ggataacatt gctgatgcag tggcctgcgc aaaacgcgtt 300
gtgcgtgatc cacaaggtat ccgtgcctgg gtcgcctggc gtaatcgttg tcagaaccgt 360
gatgtccgcc aatacgttca gggttgcggc gtgagtagta gtagtagtgg tagtagtagt 420
agtagtggta gtagtagtag tgccgtccta aaggtgctaa tcagtagtgc cgcaagtagt 480
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gccgtcctaa aggtgctaat cagtagtgcc gcaagtagta cctggaagtg gaagtggcgt 60
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Claims (5)
1.一种重组溶菌酶抗菌肽融合蛋白，其特征在于，其该蛋白从N端-C端依次由人溶菌酶的氨基酸编码序列、连接肽的氨基酸编码序列和抗菌肽的氨基酸编码序列连接所得；
其中，所述人溶酶菌的氨基酸编码序列如SEQ ID NO.1所示；
其中，所述连接肽的氨基酸编码序列如SEQ ID NO.2所示；
其中，所述抗菌肽的氨基酸编码序列如SEQ ID NO.3所示。
2.权利要求1所述的重组溶菌酶抗菌肽融合蛋白的编码基因，其特征在于，所述编码基因的核苷酸序列如SEQ ID NO.5所示。
3.权利要求1所述的重组溶菌酶抗菌肽融合蛋白的制备方法，其特征在于，包括如下步骤：
(1)将编码重组溶菌酶抗菌肽融合蛋白的碱基序列克隆至大肠杆菌表达载体pET30a的NdeI和HindIII酶切位点之间，构建重组大肠杆菌表达载体；
(2)将步骤(1)构建的重组大肠杆菌表达载体转化至大肠杆菌宿主细胞BL21中，IPTG诱导表达后，离心收集菌体，超声波裂解，离心收集包涵体沉淀；
(3)将步骤(2)收集的包涵体沉淀进行纯化，即得到重组溶菌酶抗菌肽融合蛋白。
4.根据权利要求3所述的制备方法，其特征在于，步骤(3)中，所述的纯化为将包涵体沉淀用洗涤液洗涤，收集二次包涵体沉淀，再进行抽提，离心，收集上清液；利用QAE层析柱、蛋白纯化仪对收集的上清液进行纯化。
5.权利要求1所述的重组溶菌酶抗菌肽融合蛋白在制备抑制大肠杆菌、金黄色葡萄球菌、沙门氏菌、绿脓杆菌或枯草芽孢杆菌抗菌制剂中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649311A (zh) * | 2009-09-15 | 2010-02-17 | 吉林大学 | 人溶菌酶-抗菌肽Catesbeianin-1融合蛋白制备方法及其在防治奶牛乳房炎中的应用 |
| EP2679677A1 (en) * | 2012-06-29 | 2014-01-01 | Lysando Aktiengesellschaft | Composition for use in Mycobacteria therapy |
| CN105061603A (zh) * | 2015-08-11 | 2015-11-18 | 浙江益肽生物科技有限公司 | 抗菌肽-溶菌酶融合蛋白的制备方法及其应用 |
-
2019
- 2019-12-31 CN CN201911406640.1A patent/CN111019001B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649311A (zh) * | 2009-09-15 | 2010-02-17 | 吉林大学 | 人溶菌酶-抗菌肽Catesbeianin-1融合蛋白制备方法及其在防治奶牛乳房炎中的应用 |
| EP2679677A1 (en) * | 2012-06-29 | 2014-01-01 | Lysando Aktiengesellschaft | Composition for use in Mycobacteria therapy |
| CN105061603A (zh) * | 2015-08-11 | 2015-11-18 | 浙江益肽生物科技有限公司 | 抗菌肽-溶菌酶融合蛋白的制备方法及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| Enhanced Expression and Functional Characterization of the Recombinant Putative Lysozyme-PMAP36 Fusion Protein;Rao Z等;《MOLECULES AND CELLS》;20190331;第42卷(第3期);第262-269页 * |
| 人溶菌酶-抗菌肽tachyplesins融合蛋白的原核表达及其抗菌活性;欧阳萍等;《中国生物制品学杂志》;20100831;第23卷(第8期);第829-833页 * |
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