CN102653779A - 一种新型重组抗菌多肽药物的制备方法 - Google Patents
一种新型重组抗菌多肽药物的制备方法 Download PDFInfo
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Abstract
本发明提供的一种新型重组抗菌多肽药物的制备方法，涉及重组抗生素药物生产技术。包括如下步骤：(1)制备含有重组质粒的大肠杆菌菌种，冷冻保存，(2)采用液体生产培养基中对菌种进行扩大培养，(3)诱导表达重组抗菌多肽，分离纯化重组抗菌多肽；其特征在于，所述液体生产培养基的配方如下：各组分在终溶液中的质量体积比为：磷酸氢二钠0.4％-0.7％，磷酸二氢钾0.1％-0.6％，氯化铵0.05％-0.2％，氯化钙0.0005％-0.001％，硫酸镁0.5％-2.5％，蛋白胨1％-3％，酵母粉0.5％-1％，葡萄糖0.1％-0.5％，氯化钠0.2％--0.8％其余为水。本发明还进一步在扩大培养参数方面对该方法进行了优化，适合于大规模生产抗菌肽且其制备得到的抗菌多肽纯度高。
Description
技术领域
本发明涉及抗生素药物生产工艺，特别是一种新型重组抗菌多肽药物的制备方法。
背景技术
人们一直致力于开发新型的抗菌素，在细菌胞膜上直接形成离子通道而致细菌死亡是比较有前途的抗菌素开发方向之一。自然界中，为数不少的细菌毒素就是以这种机制来杀死细菌的，其模式标本就是大肠杆菌(Escherichia coli)分泌一种毒素蛋白-大肠菌素(colicin)。早在1946年，青霉素的发明者H.Florey(British J.of Experimental Pathology.1946(27)，378～390.)等就已对大肠菌素的抗菌活性以及安全性、毒理等进行了详尽的评价，他们发现大肠菌素抗菌活性极强，提取物被稀释至数百万倍仍有非常有效的抗菌效果。但抗菌谱非常特异，只能针对大肠杆菌和亲缘相近的某些革兰氏阴性菌。1953年Jacob等发现大肠菌素Ia，在pH 6-7时杀菌功能强大。1978年Finkelstein等发现可形成离子通道的大肠菌素K，可以在人工脂质双分子膜上形成电压依赖性离子通道，从而在根本上揭示了这一类细菌毒素的抗菌机制，即在靶细胞膜上形成致死性离子通道。1996年Qiu和Finkelstein等揭示了大肠菌素Ia在人工脂质双分子膜上形成的离子通道开放和关闭时的跨膜立体结构，为在分子水平上设计和制备新型的抗菌素奠定了理论基础。2001年Qiu应用大肠菌素融合金黄色葡萄球菌信息素构建和制备得到抗耐药性金黄色葡萄球菌的抗菌工程多肽药物，体内与体外实验证实该多肽具有杀菌活性和杀菌选择性，依据同样方法，Qiu构建了分别针对耐万古霉素肠球菌和耐青霉素肺炎链球菌的抗菌工程多肽，在体内和体外实验中，这些多肽对这些现有抗菌素难以对付的致病菌都表现出了特异、稳定、快速的杀菌效应，其药效胜过万古霉素、苯唑西林、青霉素等现用抗菌素数十倍至上千倍。相应研究结果以论文形式发表在Nature Biotechnology(21(12)：1480-85，2003)、Antimicrobial Agents and Chemotherapy(49(3)：1184-1189，2005)等国际学术期刊。
在本项目中，我们提出了利用大肠菌素融合致病菌抗原的抗体模拟物构建抗菌工程多肽的全新研究思路及技术路线，并已经成功制备抗菌工程多肽--广谱抗菌信息菌素药物。在抗菌多肽药物领域有独特的思路和理论创新，已申报专利，同时建立了人工构建小型抗体模拟物方法，相关研究结果已发表在Nature biotechnology(25(8)：921-929，2007)。本项目研发的新型高效广谱抗菌信息菌素药物，因其杀菌机制特殊，对耐药菌有良好的杀菌效果，对多重耐药绿脓杆菌(MDRPA)、耐甲氧西林金葡菌(MRSA)、耐万古霉素肠球菌(VRE)等耐药菌都表现出比现有抗生素更强的抗菌效力。该药物的研发和制备，对于解决日益严重的因耐药菌株而导致的一系列治疗问题，保障人民身体健康有重要作用。
本项目所研究的广谱抗菌信息菌素药物与目前国内外研究的小肽类抗生素(人穿孔素、家蚕抗菌肽)是完全不同的两类物质。其区别在于(1)该抗菌工程多肽与其原型大肠菌素一样，是以单体(monomer)构象工作的，即一个分子就能组成一个完整的工作单位，而小肽类抗生素是以多体(polymer)构象工作的，即需要数十个分子才能组成一个完整的工作单位；(2)该抗菌工程多肽与其原型大肠菌素一样，可以在血循环和体内工作，而小肽类抗生素不能；(3)该抗菌工程多肽与其原型大肠菌素一样，在靶细胞膜上形成的是电压依赖性离子通道，其杀伤机制和效率远远高于小肽类抗生素在靶细胞膜上形成的孔道。根据检索至2010年本领域文献，在进行动物体内实验时，上述小肽类抗生素几乎都存在着一个致命的缺陷：会被动物体内蛋白酶降解。因此尚没有一个自小肽类抗生素开发出来的药物前体通过了临床测试。而本项目研发和生产的抗菌工程多肽新药的原型本身就是共生在动物和人类消化道细菌所产生的、结构和作用机理与上述小分子多肽完全不同的细菌素，在长达8年的体内外测试中始终表现出强大的杀菌活性；而且在大动物(奶牛、奶山羊等)疾病模型试验中，无论是局部注射和静脉注射均有良好的杀菌效果和治疗作用，因此本抗菌工程多肽新药不存在上述小分子多肽难以在体内使用的局限。
经过文献查阅发现，目前国外对大肠菌素、细菌信号传导多肽、抗体改造等分别进行了相应研究，但尚无任何实验室曾进行过类似本项目研究思路和技术路线的研究，也未见任何类似文章发表。2010年6月经查询美国国家医学图书馆(www.ncbi.nlm.nih.gov)，目前已登录的关于大肠菌素(Colicin)的文献有2600余篇，关于信息素(Pheromone)的文献有7300余篇，关于抗体改造(Antibody reconstitution)的文献有2100余篇，关于免疫毒素(Immunotoxin)的文献有3800余篇，关于抗菌素耐药性(antibiotic resistance)的研究文献94000余篇，在这些信息来源中均未见到任何类似本项目的科学构思、设计理念和研究实践见诸报导。在2010年6月，委托教育部查新工作站(No.1)进行的查新表明，除本项目组人员的报道外，国内外未见利用大肠菌素结合靶细菌信息素或人工设计的靶细菌抗体模拟物，构建针对靶细菌的抗菌工程多肽药物的报道。国内外未见基于本项目的靶向工程抗菌多肽构建方法，生产的抗感染性细菌的人用、兽用药物及农药的报道。
我们已将此广谱抗菌信息菌素药物按照实际需要，分别进行动物药物(治疗奶牛乳房炎的药物)和人用药物(抗菌素)的开发。体内外抗菌测试表明，该信息菌素呈现出很强的抗菌作用，尤其是其体内抗菌效力明显优于体外抗菌效力。2010年5月委托农业部兽药安全监督检验测试中心进行该药物的安全性评价，证明其无毒、无致突变、无致畸作用。已经完成的药物安全评估结果如下：
①大、小鼠急性毒性试验结论为该药物属实际无毒类(报告编号WTPJ20100003)；
②鼠伤寒沙门氏菌回复突变(Ames)试验结果为阴性，表明该药物无致突变性(报告编号WTPJ20100003(2))；
③小鼠骨髓细胞微核试验结果为阴性，无致突变作用(报告编号WTPJ20100003(3))；
④小鼠精子畸形试验结果为阴性，无致小鼠精子畸形作用(报告编号WTPJ20100003(4))。
⑤重组抗菌多肽药物对奶牛乳房炎分离病原菌的体外抑杀试验，试验结果证明：
A重组抗菌多肽药物具广谱抗菌作用，在体外对各种奶牛乳房炎病原菌具有良好的抑杀效应。
B重组抗菌多肽药物(发明申请名称：一种新型抗生素及其核苷酸序列、制备方法与应用，申请号200910157564.5)对葡萄球菌(金黄色葡萄球菌、表皮葡萄球菌、腐生葡萄球菌、松鼠葡萄球菌)的抑杀作用最好，MIC50可达0.125μg/mL，MBC50可达0.25μg/mL。与对照之头孢噻吩(2ug/ml)、苯唑西林(4ug/ml)、青霉素、氨苄青霉素、林可霉素、庆大霉素(均为8ug/ml以上)有极显著差异。按药物分子量进行标化，多肽(MW 70,000)对所试葡萄球菌的抑杀作用是头孢噻吩(MW 523)作用的2,100倍；是苯唑西林(MW 423)作用的5,300倍。
C重组抗菌多肽药物对链球菌、化脓隐秘杆菌、大肠杆菌等肠杆菌科细菌的抑杀作用基本相当，对链球菌(无乳链球菌、停乳链球菌、乳房链球菌、牛链球菌)的MIC50为1.0μg/mL，MBC50为2.0μg/mL；对化脓隐秘杆菌的MIC50为0.25μg/mL，MBC50为1.0μg/mL；对大肠杆菌等肠杆菌科细菌的MIC50为1.0μg/mL，MBC50为1.0μg/mL。
D重组抗菌多肽药物对细菌敏感株和耐药株的抑杀作用无明显差异，能高效抑杀具各种耐药表型的葡萄球菌、链球菌和大肠杆菌。
在前期的大动物治疗试验(奶牛乳房炎实验性治疗试验)中，治愈112头，治愈率达到95％。经过国家兽药安全评价中心检测，本药物属实际无毒类药物，不会对动物产生毒性和不良影响，同时该类药物属可降解信息菌素物质，避免了常规抗菌素在畜禽疾病治疗后产生药物残留的弊病，经北京市乳品质量监督检验站检测，应用该药物治疗后的奶牛所产牛奶中均无抗生素残留(检测报告编号A2009-249)。
但是上述重组抗菌素的生产，如发明人前期的申请号为200910157564.5，名称为“一种新型抗生素及其核苷酸序列、制备方法与应用”发明申请说明书实施例1中公开了该抗菌肽的完整制备工艺，在获得重组制粒并转化感受态细胞之后的增菌诱导表达重组蛋白的步骤中，采用了较常规的增菌工艺，由于在实验阶段，对生产效率和制备量没有高要求，但随着这些重组抗生素经临床试验、动物试验及安全评估，证明这些重组抗菌肽医用价值之后，采用上述发明申请中的制备工艺，成本太高，且很难获得大量的高纯度重组蛋白表达物，因此，如何进行有效的规模化开发和生产成了上述药物走向实际应用过程中一个必须解决的问题。
发明内容
本发明针对上述领域的空白及急切需求，提供上述重组抗菌多肽的制备方法。
一种新型重组抗菌多肽药物的制备方法，包括如下步骤：
(1)制备含有重组质粒的大肠杆菌菌种，冷冻保存，
(2)采用液体生产培养基对菌种进行扩大培养，
(3)诱导菌种表达重组抗菌多肽；
其特征在于，所述液体生产培养基的配方如下：各组分在终溶液中的质量体积比为：磷酸氢二钠0.4％-0.7％，磷酸二氢钾0.1％-0.6％，氯化铵0.05％-0.2％，氯化钙0.0005％-0.001％，硫酸镁0.5％-2.5％，蛋白胨1％--3％，酵母粉0.5％-1％，葡萄糖0.1％-0.5％，氯化钠0.2％--0.8％其余为水
所述液体生产培养基的配方如下：各组分在终溶液中的质量体积比为：磷酸氢二钠0.68％，磷酸二氢钾0.3％，氯化铵0.1％，氯化钙0.001％，硫酸镁0.02％，蛋白胨2.5％，酵母粉0.75％，葡萄糖0.2％，氯化钠0.6％，其余为水。
所述对菌种进行扩大培养分为三级扩大培养，各级扩大培养的参数为：220rpm、37℃、3-8小时。
所述诱导表达重组抗菌多肽指对步骤(2)得到的菌液做如下处理：搅拌速度220rpm，最大通氧量，30℃，2～4小时；42℃，0.5小时；37℃，1～2小时，达到42℃时加入终浓度为0.5mM的IPTG。
所述步骤(2)之前，菌种进行如下处理：
(1)菌种4℃解冻，在加有终浓度为50μg/ml AMP的LB液体培养基中，220rpm、37℃培养过夜复苏菌种，然后涂布于LB固体培养基上培育单菌落，
(2)挑取单菌落于1.5mlLB液体培养基中，220rpm、37℃培养5-8小时；然后转至100mlLB液体培养基中，220rpm、37℃培养5-8小时；
所述LB培养基的组分及各组分质量体积比为氯化钠1％，蛋白胨1％g，酵母0.5％，琼脂0.8～1％，其余为水。
所述大肠杆菌指含有重组质粒pBHC-SA1、pBHC-SA2、pBHC-SA3、pBHC-SA4、pBHC-SE或pBHC-PA的BL-21工程菌。
基于发明人前期获得的一系列重组抗菌多肽，本发明提供一种制备重组抗菌素的方法，专门用于大量制备高纯度的重组抗菌肽。现有方法不适宜大规模生产，在大规模生产情况下纯度或产量不理想等缺陷，是将发明人前期获得的重组抗菌多肽推向临床应用过程中必须克服的难题。本发明通过对培养基成分的选择和优化组合，提供了一种最适合大肠杆菌表达外源基因的培养基配方，通过选择最适合的扩大培养参数，使整个制备过程环环相扣，兼顾大规模生产的情况下制备重组抗菌多肽的纯度与产量的矛盾。为发明人前期获得重组抗菌多肽的规模化、工业化生产奠定了基础。
附图说明
图1本发明的方法制备的抗菌肽与AMP的对比结果
从左到右：CON：3小时开始浑浊；AMP：4小时开始浑浊；实施例1制备得到的样品：194批次蛋白与198批次蛋白第27个小时澄清。
图2不同批次抗菌工程多肽纯度测试
条带1为对照Marker，条带2为120批次，条带3为122批次，条带4为126批次，条带5为246批次，条带6为247批次，条带7为248批次，条带8为250批次，条带9为252批次。其中120，122，126批次的生产方法为现有方法，246，247，249，250，252批次为本发明的新方法生产。
图3.不同生产规模下的差率比较
从左到右为：摇瓶、42L发酵罐、200L发酵罐连续生产10批次的产率。
具体实施方式
以下以一些具体的重组抗菌肽说明本发明的制备工艺，但本发明不限于制备以下重组抗菌肽。
实施例1.抗大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、绿脓杆菌抗菌肽的制备工艺
本发明用到的培养基：
(1)LB液体培养基
100ml氯化钠1g，蛋白胨1g，酵母0.5g，将各种试剂称好后放入250ml烧瓶中，加入100ml自来水，使其溶解，在高压灭菌锅内120℃8min灭菌
(2)LB固体培养基
100ml氯化钠0.5～1.5g，蛋白胨0.5～2g，酵母0.3～1g，琼脂0.8～3g，LB固体培养基用于菌种复苏后划平板培养单克隆菌落用。将各种试剂称好后放入250ml烧瓶中，加入100ml自来水，使其溶解，在高压灭菌锅内120℃8min灭菌
(3)生产专用培养基(700ml 20L 100L 200L)
各成分在终溶液中的质量体积比为：磷酸氢二钠0.4％-0.7％，磷酸二氢钾0.1％-0.6％，氯化铵0.05％-0.2％，氯化钙0.0005％-0.001％，硫酸镁0.5％-2.5％，蛋白胨1％--3％，酵母粉0.5％-1％，葡萄糖0.1％-0.5％，氯化钠0.2％--0.8％其余为水。
本发明优选配方如表1：
培养基加入相应量的自来水后，高压灭菌，121℃8min灭菌。
(4)硼酸缓冲液：硼酸0.04M，NaCl0.01M，四硼酸钠0.04M，EDTA 2mM
配制方法：硼酸缓冲液5L＝A液1L+B液4L
A液(1L)：硼酸12.368g(0.2M)，NaCl2.925g(0.05M)
B液(4L)：四硼酸钠76.27g(0.05M)，
A液1L+B液4L混合，加入EDTA2.9225g终浓度为2mM
步骤1.制备重组突变质粒
参照申请号为“200910157564.5”，名称为“一种新型抗生素及其核苷酸序列、制备方法与应用”的发明申请说明书中实施例1的记载，制备得到重组突变质粒pBHC-SAl、pBHC-SA2、pBHC-SA3pBHC-SA4、pBHC-SE、pBHC-PA。
步骤2.转化感受态细胞
将6种重组突变质粒100ng分别与制备的BL-21工程菌感受态细胞40ul冰孵5分钟，热冲击42℃，30秒，再置入冰中2分钟，加入SOC培养基160ul，220rpm，37℃摇菌1小时后铺板(LB培养基加1％琼脂，加50ug/ml氨苄青霉素，37℃过夜)，挑取单克隆菌落增菌，获得菌种，低温保存菌种；
步骤3.菌种复苏
1.菌种复苏
取出保存菌种4℃解冻，取1.5ml倒入10ml LB培养基中(含AMP 50μg/ml)，220rpm、37℃培养5-8小时。
2.接种单克隆菌
复苏后的菌液稀释104或105倍，取10ul稀释后的菌液加入到制备好的LB固体培养基(AMP 50μg/ml)平板上，涂平板。放湿盒中，37℃恒温培养箱培养10-12小时，培养基表面长出圆形单菌落。
3.挑菌和扩菌
(1)用已灭菌牙签或接菌环从长好的平板上挑选规则圆形，边缘光滑的单菌落，放入1.5mlLB培养基中，振荡培养：220rpm、37℃培养5-8小时。
(2)1.5mlLB菌液翻至100ml LB培养基中，振荡培养：220rpm、37℃培养5-8小时。
(3)一级扩大培养：将上步的100ml菌液加入到700ml生产专用培养基中培养，振荡培养：220rpm、37℃培养5-8小时。
(4)二级扩大培养：将上步700ml菌液分别加入到6×700ml生产专用培养基中，振荡培养：220rpm、37℃培养5-8小时。
(5)三级扩大培养：将上步6×700ml菌液加入到20L生产专用培养基中，在发酵罐中培养：搅拌速度220rpm、最大通氧量，37℃培养3-5小时。
(6)工程菌发酵和诱导表达蛋白：将上步20L菌液加入到200L生产专用培养基中，在发酵罐中培养和诱导表达蛋白：搅拌速度220rpm，最大通氧量，30℃，2～4小时；42℃，0.5小时；37℃，1～2小时，注：达到42℃时加入终浓度为0.5mM的IPTG
4、离心收菌
培养液6000g，4℃离心20min。收取离心后的沉淀，放入50mM的硼酸缓冲液(pH9.0)中，使菌体重悬浮于硼酸缓冲液中，注：硼酸缓冲液中加入2mM的PMSF(中文名：苯甲基磺酰氟丝氨酸蛋白酶抑制剂，自菌体重悬浮之后的操作须在4℃进行。
5、破碎菌体
待菌体完全悬浮于pH9.0硼酸缓冲液后，用高压匀质机500～600bar高压破碎菌体，反复破碎7次，每次破菌间隔3～5分钟。
6、沉淀菌体DNA
将破碎后的菌液，55000g，4℃离心40min。取上清液，加入硫酸链霉素(每200ml液体加入16瓶100万单位的硫酸链霉素)，在磁力搅拌器上搅拌1h。
7、透析
将上步的菌液55000g，4℃离心20min，取上清液，装入透析袋中，置硼酸缓冲液中透析8h～12小时，每4h换透析液一次。
8、蛋白药物纯化和得到抗菌工程多肽药物
将透析后的菌液55000g，4℃离心20min，取上清液放入烧杯中，采用离子交换法纯化蛋白。取上清上样于CM离子交换柱，充分冲洗后，用含0.3M NaCl的50mM硼酸缓冲液洗脱即可得到新型抗菌工程多肽。
实施例2.多肽药物的抑菌测试
1、在100ml锥形瓶中装入10ml BM培养基(未灭菌)，高压蒸汽灭菌锅121℃，8min灭菌。
2、超净工作台提前用酒精擦干净，开紫外灯灭菌30min。
3、在每个100ml锥形瓶中加入3μl过夜培养的金黄色葡萄球菌。
4、在100ml锥形瓶中分别加入10μl无菌生理盐水、1μl 1mg/ml的AMP、以及本发明实施例1的方法制备得到的0.8mg/ml的pBHC-SA1多肽样品125μl，作好标记。
5、放入振动培养箱中，37℃、220rmp培养。
6、在3h、6h、9h、12h、24h时观察混浊情况。
空白对照和AMP在3h时混浊，样品在9h未混浊，说明制备的样品能够有效抗金黄色葡萄球菌，如图1。从左到右：CON：3小时开始浑浊；AMP：4小时开始浑浊；实施例1制备得到的样品：194批次蛋白与198批次蛋白至第27个小时澄清。
实施例2本发明的制备方法与现有方法制备的抗菌肽比较
将本发明实施例1的方法与原有方法(200910157564.5中公开的方法)以相等的生产规模制备得到的不同批次的抗菌肽pBHC-SA1在聚丙烯酰胺凝胶中电泳、染色，以比较产物的纯度，结果显示，抗菌工程多肽的分子量在75000左右，实施例1的方法生产的多肽所在的泳道5、6、7，8的条带相对单一，而泳道2、3、4内含有大量较小分子量的杂带，即本发明的方法生产的多肽在纯度上得到了明显的提高。
实施例3.生产工艺流程的改进
经过不断的改进和优化生产工艺，生产规模也在不断地扩大，由试验性摇瓶生产(8.4L)，到42L发酵罐生产(25L)，再到现在的200L发酵罐生产(100L)，且随着生产规模的扩大，差率并没有受到影响，见图3，为抗菌多肽的规模化、工业化生产奠定了基础。摇瓶连续生产10批次、42L发酵罐连续生产10批次与200L发酵罐连续生产10批次产量和产率的对比情况，见表2、图3
表2.产量和差率
| 摇瓶 | 42L发酵罐 | 200L发酵罐 | |
| 产量 | 1893.72 | 6654.82 | 31940.00 |
| 产率(mg/L) | 22.54 | 26.61 | 31.94 |
Claims (6)
1.一种新型重组抗菌多肽药物的制备方法，包括如下步骤：
(1)制备含有重组质粒的大肠杆菌菌种，冷冻保存，
(2)采用液体生产培养基中对菌种进行扩大培养，
(3)诱导表达重组抗菌多肽，分离纯化重组抗菌多肽；
其特征在于，所述液体生产培养基的配方如下：各组分在终溶液中的质量体积比为：磷酸氢二钠0.4％-0.7％，磷酸二氢钾0.1％-0.6％，氯化铵0.05％-0.2％，氯化钙0.0005％-0.001％，硫酸镁0.5％-2.5％，蛋白胨1％-3％，酵母粉0.5％-1％，葡萄糖0.1％-0.5％，氯化钠0.2％--0.8％其余为水。
2.根据权利要求1所述的制备方法，所述液体生产培养基的配方如下：各组分在终溶液中的质量体积比为：磷酸氢二钠0.68％，磷酸二氢钾0.3％，氯化铵0.1％，氯化钙0.001％，硫酸镁0.02％，蛋白胨2.5％，酵母粉0.75％，葡萄糖0.2％，氯化钠0.6％，其余为水。
3.根据权利要求1所述的制备方法，所述对菌种进行扩大培养分为三级扩大培养，各级扩大培养的参数为：220rpm、37℃、3-8小时。
4.根据权利要求1所述的制备方法，所述诱导表达重组多肽指对步骤(2)得到的菌液做如下处理：搅拌速度220rpm，最大通氧量，30℃2～4小时；42℃0.5小时；37℃1～2小时，达到42℃时加入终浓度为0.5mM的IPTG。
5.根据权利要求1所述的制备方法，所述步骤(2)扩大培养前，对冷冻保存的菌种进行如下处理：
(1)菌种4℃解冻，在加有终浓度为50μg/ml AMP的LB液体培养基中，220rpm、37℃培养过夜复苏菌种，然后涂布于LB固体培养基上培育单菌落，
(2)挑取单菌落于1.5mlLB液体培养基中，220rpm、37℃培养5-8小时；然后转至100mlLB液体培养基中，220rpm、37℃培养5-8小时；
所述LB固体培养基的组分及各组分质量体积比为氯化钠1％，蛋白胨1％g，酵母0.5％，琼脂0.8～1％，其余为水。
6.根据权利要求1所述的制备方法，所述大肠杆菌指含有重组质粒pBHC-SA1、pBHC-SA2、pBHC-SA3、pBHC-SA4、pBHC-SE或pBHC-PA的BL-21工程菌。
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| HK12110533.9A HK1169837A1 (zh) | 2011-03-04 | 2012-10-23 | 種新型重組抗菌多肽藥物的製備方法 |
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| CN104328080A (zh) * | 2014-08-04 | 2015-02-04 | 湖北文理学院 | 大肠埃希菌atcc25922增殖促进剂及其应用方法 |
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| US9611299B2 (en) | 2011-11-25 | 2017-04-04 | Protein Design Lab, Ltd. | Method for highly expressing recombinant protein of engineering bacteria and use thereof |
| CN104328080A (zh) * | 2014-08-04 | 2015-02-04 | 湖北文理学院 | 大肠埃希菌atcc25922增殖促进剂及其应用方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| BR112013022333A2 (pt) | 2016-12-06 |
| JP2014508766A (ja) | 2014-04-10 |
| EA201300884A1 (ru) | 2014-04-30 |
| CL2013002534A1 (es) | 2014-07-11 |
| KR20130138822A (ko) | 2013-12-19 |
| US9765378B2 (en) | 2017-09-19 |
| CA2828998A1 (en) | 2012-09-13 |
| SG193000A1 (en) | 2013-10-30 |
| US20160304927A1 (en) | 2016-10-20 |
| WO2012119524A1 (zh) | 2012-09-13 |
| ZA201306575B (en) | 2016-03-30 |
| EP2682474A1 (en) | 2014-01-08 |
| HK1169837A1 (zh) | 2013-02-08 |
| MY169503A (en) | 2019-04-15 |
| EP2682474B1 (en) | 2017-05-10 |
| JP5916769B2 (ja) | 2016-05-11 |
| KR101566331B1 (ko) | 2015-11-05 |
| EP2682474A4 (en) | 2014-12-10 |
| EA201300884A8 (ru) | 2018-06-29 |
| TWI541351B (zh) | 2016-07-11 |
| AP3486A (en) | 2015-12-31 |
| TW201239092A (en) | 2012-10-01 |
| CO6771436A2 (es) | 2013-10-15 |
| EA030794B1 (ru) | 2018-09-28 |
| AP2013007193A0 (en) | 2013-10-31 |
| AU2012225088B2 (en) | 2015-04-09 |
| CN102653779B (zh) | 2014-02-19 |
| NZ614593A (en) | 2015-07-31 |
| AU2012225088A1 (en) | 2013-09-12 |
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