WO2021073372A1 - 具有强杀菌效果的杂合抗菌蛋白及其应用 - Google Patents

具有强杀菌效果的杂合抗菌蛋白及其应用 Download PDF

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WO2021073372A1
WO2021073372A1 PCT/CN2020/116756 CN2020116756W WO2021073372A1 WO 2021073372 A1 WO2021073372 A1 WO 2021073372A1 CN 2020116756 W CN2020116756 W CN 2020116756W WO 2021073372 A1 WO2021073372 A1 WO 2021073372A1
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hybrid
protein
antibacterial
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陆海荣
黄青山
李国栋
黄晋江
赵晓蔚
陆婉英
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江西缘生生物科技有限公司
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Definitions

  • the present invention relates to hybrid antibacterial proteins.
  • Antibiotics have been widely used in the fields of medical and health, agricultural breeding, and have played an important role in the treatment of infectious diseases and the protection of public health safety.
  • the problem of bacterial resistance to antibiotics has become increasingly serious, which has caused clinical treatment difficulties and increased mortality rates worldwide, which has seriously threatened human health.
  • Multi-drug resistant bacteria mainly include Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Enterococcus and Staphylococcus aureus, among which Gram-negative bacteria (G - bacteria) account for the largest proportion, especially Baumann In recent years, the drug resistance of bacillus has become more and more serious (Mezzatesta ML, D'Andrea MM, Migliavacca R, et al. Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy[J].
  • Peptidoglycan is the main component of bacterial cell wall. It is a three-dimensional network structure cross-linked by sugar chains, peptide chains and peptide bonds. It maintains the mechanical strength of the cell wall and protects bacteria from external factors.
  • Peptidoglycan hydrolases is a type of protein that can specifically lyse bacterial cell wall peptidoglycans (Vollmer W, Joris B, Charlier P, et al. Bacterial peptidoglycan (murein) hydrolases[J]. , 2008, 32(2): 259-286.), mainly including phage lyase, bacteriocin and bacterial autolyase. Such as Staphylococcus aureus phage lyase LysK, bacteriocin lysostaphin that mimics the expression of Staphylococcus, and Streptococcus pneumoniae autolysase LytA.
  • Peptidoglycan hydrolase is generally composed of two domains, namely a catalytic domain (catalytic domain) responsible for hydrolyzing peptidoglycan and a domain (binding domain) responsible for specifically targeting bacterial cell walls.
  • peptidoglycan hydrolase When peptidoglycan hydrolase is added to the bacterial solution, the hydrolase can specifically bind to sensitive bacteria and quickly lyse their cell walls.
  • the antibacterial activity of peptidoglycan hydrolase in vivo and in vitro has been extensively proven (Gerstmans H, Rodr ⁇ guez-Rubio L, Lavigne R, et al.
  • Peptidoglycan hydrolase can quickly kill specific bacteria by directly lysing bacterial peptidoglycan, with a narrow antibacterial spectrum, and its sterilization mechanism is different from traditional antibiotics. It can be used repeatedly for a long time and is not easy to induce drug resistance. During each growth cycle of bacteria, peptidoglycan hydrolase can directly cleave it. The narrow-spectrum peptidoglycan hydrolase hardly affects the normal flora.
  • Wild-type LysK contains two catalytic domains (CHAP and Amidase domains), of which the catalytic activity of the Amidase domain is very low. Removing it from LysK (LysK ⁇ 221-390) does not affect the activity of the protein (Becker S C, Dong S) ,Baker J R, et al. LysK CHAP endopeptidase domain is required for analysis of live staphylococcal cells [J].
  • the CHAP catalytic domain of LysK alone has no antibacterial activity in TSB medium. In a complex environment, the catalytic domain needs the help of the binding domain to take effect. However, the binding domain of LysK itself is not the best, and there is a low affinity and efficiency. The recognition range is too small. Therefore, the construction of a highly active hybrid antibacterial protein is of great significance for promoting the development of new antibacterial drugs, and can provide an effective solution to the problem of bacterial resistance.
  • Gladskin the world's first antibacterial protein product, was launched. It is used for inflammatory skin diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) for intact skin, such as eczema, rosacea and acne.
  • MRSA methicillin-resistant Staphylococcus aureus
  • many antibacterial proteins have entered clinical research, such as Lysostaphin (China, treatment of S. aureus infection), P128 (India, removal of Staphylococcus aureus colonization in nasal mucosa), CF301 (U.S., treatment of sepsis caused by S.
  • the purpose of the present invention is to provide a hybrid antibacterial protein with strong bactericidal effect and its application, so as to overcome the defects of the prior art and meet people's needs.
  • the hybrid antibacterial protein with strong bactericidal effect on gram-negative bacteria of the present invention includes the amino acid sequence shown in SEQ ID NO 1, named AB469, and the hybrid protein composed of a sequence similar to the catalytic domain of AB469. Synthetic protein, a hybrid protein composed of a sequence similar to the binding domain of AB469, or a hybrid protein composed of the positions of the catalytic domain and the binding domain of AB469 interchanged;
  • the AB469 contains 285 amino acids, has a molecular weight of 31.8kD, and a theoretical isoelectric point of 9.8. After two steps of purification, the purity can reach more than 95%. SDS-PAGE electrophoresis shows that the molecular weight of AB469 is about 32kD;
  • the nucleic acid sequence encoding the AB469 is as SEQ ID NO: 2, named as ab469;
  • 10 ⁇ g/mL AB469 can cause Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 15442) and Salmonella typhi (CMCC 50094) to decrease by more than 7 Log values after 2 hours, making Klebsiella pneumoniae (ATCC) 700603) and Escherichia coli (ATCC 35218) decrease by more than 2 Log values; 50 ⁇ g/mL AB469 can cause Klebsiella pneumonia (ATCC 700603) and Escherichia coli (ATCC 35218) to decrease by more than 7 Log values; Bacillus (ATCC 700323) and Staphylococcus aureus (ATCC 6538) decreased by about 4 Log values.
  • Acinetobacter baumannii ATCC 19606
  • Pseudomonas aeruginosa ATCC 15442
  • CMCC 50094 Salmonella typhi
  • 50 ⁇ g/mL AB469 can cause Klebsiella
  • Antibacterial protein AB469 can effectively kill clinically isolated Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. At the same time, it is resistant to multiple drug resistance (ceftazidime, ceftriaxone, meropenem, imipe The resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae, which are resistant to South, Tobramycin and Sulbactam, have the same effect.
  • the AB469 also has high activity under complex conditions such as serum and body fluids;
  • hybrid protein composed of the sequence similar to the catalytic domain of AB469 also has high activity, such as SEQ ID NO 3, named AB46M9;
  • the nucleic acid sequence encoding the AB46M9 is as SEQ ID NO: 4, named as ab46m9;
  • the hybrid protein composed of the sequence similar to the binding domain of AB469 also has high activity; such as SEQ ID NO 5, named AB469M;
  • the nucleic acid sequence encoding the AB469M is as SEQ ID NO: 6, named as ab469m;
  • the hybrid protein composed of the positions of the catalytic domain and the binding domain of AB469 is also highly active, such as SEQ ID NO 7, named B946;
  • the nucleic acid sequence encoding the B946 is as SEQ ID NO: 8, named as b946;
  • AB469 Compared with wild-type ABgp46, AB469 has one more binding domain, which can significantly improve the antibacterial activity of ABgp46 in complex environments, including certain ionic strength, culture media, serum and body fluids.
  • ABgp46 (2 ⁇ m, ⁇ 40 ⁇ g/mL) expressed by Oliverira on Pseudomonas aeruginosa (a decrease of 4 log values) is not as good as that of Acinetobacter baumannii (a decrease of more than 5 log values); and this patent Among them, AB469 with a lower protein concentration (0.31 ⁇ m, 10 ⁇ g/mL), under 150mm NaCl conditions, shows a strong bactericidal effect on both Acinetobacter baumannii and Pseudomonas aeruginosa (a decrease of more than 7 log values) .
  • the ABgp46 is a bacteriophage lyase containing only the catalytic domain, which is used in Oliveira H, Boas D V, Mesnage S, et al. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-gram-negactivity [J].Frontiers in microbiology,2016,7. There are detailed reports in the literature;
  • the present invention successfully constructed a new hybrid antibacterial protein, which was expressed by E. coli or yeast, and purified to obtain a high-purity hybrid antibacterial protein; the protein can quickly lyse Bowman Acinetobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Escherichia coli and other Gram-negative bacteria.
  • the antibacterial preparation made from the antibacterial protein can be used to prevent and treat bacterial infections. It is a new, highly effective antibacterial substance that can directly lyse bacteria.
  • the antibacterial protein can be made into antibacterial preparations for the prevention and treatment of bacterial infections, as well as sterilization of medical equipment and medical places.
  • Figure 4 The turbidimetric method detects the biological activity of AB469 against Klebsiella pneumoniae (ATCC 700603).
  • the nucleic acid sequence (SEQ ID NO: 2) that can encode AB469 was artificially designed and synthesized, namely ab469; at its 5'end, a NcoI restriction endonuclease site (italicized part) was added, and at the same time Nucleic acid sequence ct was introduced, together with the g at the end of the restriction site, to form a codon encoding an alanine; a HindIII restriction endonuclease site (in italics) was added to the 3'end.
  • the sequence is as follows:
  • the Cys in the SP10 binding domain was mutated to Ser; the artificially synthesized ab469 gene was digested with NcoI and HindIII and linked to the Nco I of the pET28a(+) vector And Hind III site, the recombinant plasmid pET28a-ab469 was obtained.
  • the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells to obtain the engineered bacteria expressing AB469.
  • the fermentation supernatant was purified by two steps of cation exchange and gel filtration.
  • the purified sample is frozen and stored at -20°C for later use.
  • the size and purity of the recombinant protein samples were determined by 10% SDS-PAGE. It can be seen from the results that the main electrophoresis band of the recombinant hybrid antibacterial protein AB469 is around 32kD (see Figure 1).
  • the mutant AB46M9 (SEQ ID NO: 3) is composed of the catalytic domain AB46M (SEQ ID NO: 10) and the binding domain SP10 (154- 236aa) (SEQ ID NO: 11), with a flexible Linker in the middle.
  • the mutant AB469M (SEQ ID NO: 5) is composed of the catalytic domain AB46 (1-185aa) and the binding domain B9M (SEQ ID NO: 12) which is 82% similar to the amino acid sequence of SP10 (154-236aa), with a section in the middle Flexible Linker.
  • B946 (SEQ ID NO: 7) is composed of binding domain SP10 (154-236aa) and catalytic domain AB46 (1-185aa). In the middle is a flexible linker whose catalytic domain and binding domain are located opposite to AB469.
  • nucleic acid sequences ab46m9 SEQ ID NO 4
  • ab469m SEQ ID NO 6
  • b946 SEQ ID NO 8
  • ab46m9, ab469m and b946, and the specific methods of their expression and purification are the same as ab469.
  • the purified AB46M9, AB469M and B946 samples were frozen and stored at -20°C for later use.
  • the size and purity of the recombinant protein samples were determined by 10% SDS-PAGE (see Figure 1).
  • the overnight cultured Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 15442), and Klebsiella pneumoniae (ATCC 700603) were transferred and grown to mid-log phase (OD 600 approximately 0.5) and centrifuged ( 5,000g, 5 minutes) collect the bacteria.
  • the cells were washed twice with 20mm PBS (pH 7.2), and then resuspended in PBS buffer containing different NaCl concentrations (0-600mM).
  • AB469 can cause the OD value of the bacterial liquid to decrease by lysing the pathogenic bacteria.
  • the rapid decrease of the OD value of the pathogenic bacterial liquid indicates that AB469 is against Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa ( ATCC 15442) and Klebsiella pneumoniae (ATCC 700603) have rapid bactericidal effects.
  • Acinetobacter baumannii ATCC 19606
  • Pseudomonas aeruginosa ATCC 15442
  • Klebsiella pneumoniae ATCC 700603
  • Some non-ionic surfactants such as BriJ98, Tween 20 and Ping Ping, etc.
  • Example 5 The effect of AB469 in killing the standard bacterial strains and clinically isolated strains of pathogenic bacteria in vitro timed and quantitatively
  • the bacteria cultured overnight were transferred and grown to mid-log phase (OD 600 approximately 0.5), and the bacteria were collected by centrifugation (5,000 g, 5 minutes).
  • the cells were diluted with PBS (pH 7.2) to about 1 ⁇ 10 8 cfu/mL.
  • AB469 final concentration of 10-50 ⁇ g/mL
  • NaCl final concentration of 150mm
  • EDTA final concentration of 0.5mm
  • sodium citrate final concentration of 8mm
  • 10 ⁇ g/mL antibacterial protein AB469 can effectively kill clinically isolated Acinetobacter baumannii and Pseudomonas aeruginosa (Table 4), and has the same effect on multi-drug resistant strains (including ceftazidime, ceftriaxone, merlot). Penem, imipenem, tobramycin, and sulbactam, etc.); 50 ⁇ g/mL antimicrobial protein AB469 can effectively kill clinically isolated Klebsiella pneumoniae, including multi-drug resistant bacteria;

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Abstract

提供了一种具有强杀菌效果的杂合抗菌蛋白及其应用，所述的具有强杀菌效果的杂合抗菌蛋白包括氨基酸序列如SEQ ID NO 1所示的命名为AB469的蛋白，以及由所述的AB469的催化域相似的序列组成的杂合蛋白、由所述的AB469的结合域相似的序列组成的杂合蛋白或由AB469的催化域和结合域位置互换组成的杂合蛋白。

Description

具有强杀菌效果的杂合抗菌蛋白及其应用 技术领域
本发明涉及杂合抗菌蛋白。
技术背景
抗生素已经广泛应用于医疗卫生、农业养殖领域，在治疗感染性疾病以及保障公共卫生安全中，发挥了重要作用。然而，随着抗生素大量使用，细菌对抗生素的耐药性问题日益严重，在世界范围内已经造成临床治疗上的困难及病死率增高，严重威胁人类健康。
多重耐药菌主要包括鲍曼不动杆菌、铜绿假单胞菌、肠杆菌、肠球菌和金黄色葡萄球菌等，其中革兰氏阴性菌(G -菌)占比最大，尤其是鲍曼不动杆菌近几年耐药性越来越严重(Mezzatesta M L,D’Andrea M M,Migliavacca R,et al.Epidemiological characterization and distribution of carbapenem‐resistant Acinetobacter baumannii clinical isolates in Italy[J].Clinical Microbiology and Infection,2012,18(2):160-166.Durante-Mangoni E,Zarrilli R.Global spread of drug-resistant Acinetobacter baumannii:molecular epidemiology and management of antimicrobial resistance[J].Future microbiology,2011,6(4):407-422.)。世界卫生组织(WHO)列出12种重点耐药性细菌中，耐碳青霉烯类抗生素的鲍曼不动杆菌、铜绿假单胞菌、肠杆菌类最为严重，迫切需要开发新型抗菌药物，尤其是与传统抗生素作用模式不同的抗菌药物。
传统抗生素具有广谱抗菌特点，对正常菌群也有同样作用。抗生素大量的使用过程中正常菌群也会产生耐药，耐药基因在正常菌群和致病菌之间相互传播，显著增加了耐药菌的产生的几率。肽聚糖是细菌细胞壁主要成分，是由糖链、肽链和肽键桥交联而成的三维网状结构，它维持着细胞壁的机械强度，保护细菌不受外部因素的影响。肽聚糖水解酶(peptidoglycan hydrolases)，是一类能特异性裂解细菌细胞壁肽聚糖的蛋白质(Vollmer W,Joris B,Charlier P,et al.Bacterial peptidoglycan(murein)hydrolases[J].FEMS microbiology reviews,2008,32(2):259-286.)，主要包括噬菌体裂解酶、细菌素和细菌自溶酶等。比如金黄色葡萄球菌噬菌体裂解酶LysK、模仿葡萄球菌表达的细菌素溶葡萄 球菌酶和肺炎链球菌自溶酶LytA。肽聚糖水解酶一般由两种结构域组成，即负责水解肽聚糖的催化结构域(催化域)和负责特异性靶向细菌细胞壁的结构域(结合域)。
当肽聚糖水解酶加入到细菌溶液中，水解酶能特异性结合敏感菌并快速裂解其细胞壁。肽聚糖水解酶在体内、外的抗菌活性已经被大量证明(Gerstmans H,Rodríguez-Rubio L,Lavigne R,et al.From endolysins to
Figure PCTCN2020116756-appb-000001
s:novel enzyme-based approaches to kill drug-resistant bacteria[J].Biochemical Society Transactions,2016,44(1):123-128.Guo M,Feng C,Ren J,et al.A Novel Antimicrobial Endolysin,LysPA26,against Pseudomonas aeruginosa[J].Frontiers in Microbiology,2017,8.)。肽聚糖水解酶通过直接裂解细菌肽聚糖快速杀死特定细菌，抗菌谱窄，其杀菌机制与传统抗生素不同，能长期反复使用，不易诱导耐药性产生。在细菌的各个生长周期，肽聚糖水解酶都能直接将其裂解。窄谱的肽聚糖水解酶几乎不影响正常菌群。
然而直接应用天然的肽聚糖水解酶会存在一些问题，如抗菌活性低、抗菌谱太窄、表达量低，溶解性差，催化域和结合域并不一定是最佳组合，或者两个结合域中间夹着其它非必要的结构域。野生型LysK含有2个催化域(CHAP和Amidase结构域)，其中Amidase结构域催化活性非常低，将其从LysK中去除后(LysKΔ221-390)并不影响蛋白的活性(Becker S C,Dong S,Baker J R,et al.LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells[J].FEMS microbiology letters,2009,294(1):52-60.)。单独LysK的CHAP催化域在TSB培养基中也没有抗菌活性，在复杂环境下，催化域需要结合域的帮助才能起效，然而LysK自身的结合域效果不是最好的，存在亲和效率低和识别范围太小的问题。因此，构建高活性的杂合抗菌蛋白，对于促进新型抗菌药物的研发具有非常重要的意义，可以为细菌耐药性问题提供一种有效解决方法。
2013年全球第一个抗菌蛋白产品Gladskin上市，用于完整皮肤的耐甲氧西林金黄色葡萄球菌(MRSA)引发的炎症性皮肤病，如湿疹、红斑痤疮和粉刺。目前还有多个抗菌蛋白已经进入临床研究，如Lysostaphin(中国，治疗创面金葡菌感染)、P128(印度，清除鼻腔粘膜金葡菌定植)、CF301(美国，治疗金葡菌引起的败血症)和SAL200(韩国，，治疗金葡菌引起的败血症)等；另外还有一些抗菌蛋白进入临床前研究，如Cpl-1、ClyS、ClyH和PlyV12等，分别治疗金葡菌、肺炎球菌和肠球菌引起的感染。进入临床的抗菌蛋白都集中在治疗G +菌感染，目前还没有治疗革兰氏阴性菌(G -菌)的抗菌蛋白进入临床研究。基于耐药G -菌严峻性和抗菌蛋白药物发展的不平衡，迫切需要研发针对 G -菌的新型抗菌药物。
发明内容
本发明的目的是提供一种具有强杀菌效果的杂合抗菌蛋白及其应用，以克服现有技术存在的缺陷，满足人们的需求。
本发明所述的对革兰氏阴性菌强杀菌效果的杂合抗菌蛋白，包括如SEQ ID NO 1所示氨基酸序列，命名为AB469，以及由所述的AB469的催化域相似的序列组成的杂合蛋白、由所述的AB469的结合域相似的序列组成的杂合蛋白或由AB469的催化域和结合域位置互换组成的杂合蛋白；
所述的AB469含有285个氨基酸，分子量31.8kD，理论等电点9.8，经过2步纯化后纯度能够达到95％以上，SDS-PAGE电泳显示AB469的分子量在32kD左右；
编码所述的AB469的核酸序列如SEQ ID NO:2，命名为ab469；
试验证明，所述的AB469具有较高的溶解性和稳定性；
体外试验证明，所述的AB469具有高活性和广谱性，对鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯、伤寒沙门氏菌和大肠杆菌都有很好的杀菌效果。10μg/mL AB469作用2h能使得鲍曼不动杆菌(ATCC 19606)、铜绿假单胞菌(ATCC 15442)和伤寒沙门氏菌(CMCC 50094)下降大于7个Log值以上，使得肺炎克雷伯菌(ATCC 700603)和大肠杆菌(ATCC 35218)下降大于2个Log值；50μg/mL AB469作用2h能使得肺炎克雷伯菌(ATCC 700603)和大肠杆菌(ATCC 35218)下降大于7个Log值；使得阴沟肠杆菌(ATCC 700323)和金黄色葡萄球菌(ATCC 6538)下降约4个Log值。
抗菌蛋白AB469能有效杀灭临床分离的鲍曼不动杆菌、铜绿假单胞菌和肺炎克雷伯菌，同时，对多重耐药(对头孢他啶、头孢曲松、美洛培南、亚胺培南、妥布霉素和舒巴坦等耐药)的鲍曼不动杆菌、铜绿假单胞菌和肺炎克雷伯菌具有同样效果。
所述的AB469在血清、体液等复杂条件下也同样具有高活性；
由所述的AB469的催化域相似的序列组成的杂合蛋白也具有高活性，如SEQ ID NO 3，命名为AB46M9；
编码所述的AB46M9的核酸序列如SEQ ID NO:4，命名为ab46m9；
由所述的AB469的结合域相似的序列组成的杂合蛋白也具有高活性；如SEQ ID NO 5，命名为AB469M；
编码所述的AB469M的核酸序列如SEQ ID NO:6，命名为ab469m；
由AB469的催化域和结合域位置互换组成的杂合蛋白也具有高活性，如SEQ ID NO 7，命名为B946；
编码所述的B946的核酸序列如SEQ ID NO:8，命名为b946；
AB469与野生型ABgp46相比，多了一个结合域，能显著提高ABgp46在复杂环境下的抗菌活性，复杂环境包括在一定离子强度、培养基、血清和体液等。
试验表明，当在ABgp46的杀菌活性检测过程中加上一定离子强度时(150mm NaCl)，ABgp46的活性会显著下降(表1)，Log下降值从5.58减少至1.54，这和很多只有单独催化域的蛋白的结果是一致的(Osipovitch D C,Griswold K E.Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent[J].FEMS microbiology letters,2015,362(2):1-7.)。从表1可以看到，离子强度对ABgp46的影响显著大于对AB469的影响。Oliverira表达的ABgp46(2μm，～40μg/mL)对铜绿假单胞菌的杀菌效果(下降4个log值)不如对鲍曼不动杆菌的杀菌效果(下降大于5个log值)；而本专利中，更低蛋白浓度的AB469(0.31μm，10μg/mL)，在150mm NaCl条件下，对鲍曼不动杆菌和铜绿假单胞菌都显示出强大的杀菌效果(下降大于7个log值)。
所述的ABgp46为一种只含有催化域的噬菌体裂解酶，在Oliveira H,Boas D V,Mesnage S,et al.Structural and enzymatic characterization of ABgp46,a novel phage endolysin with broad anti-gram-negative bacterial activity[J].Frontiers in microbiology,2016,7.文献中，有详细的报道；
所述的具有强杀菌效果的杂合抗菌蛋白，可以用于制备抗菌制剂，所述的抗菌制剂包括抗菌有效量的所述的革兰氏阴性菌强杀菌效果的杂合抗菌蛋白和药物载体，所述的药物载体包括赋形剂，如水、羟丙基甲基纤维素等；
优选的，所述的抗菌制剂中，所述的对革兰氏阴性菌强杀菌效果的杂合抗菌蛋白的重量百分比含量为0.0001～0.05％；
所述的抗菌制剂可以通过喷涂方式施加于需要的人体或物体表面。
本发明的有益效果是：
本发明在大量研究的基础上，成功的构建了一种新的杂合抗菌蛋白，利用大肠杆菌或者酵母菌等进行表达，纯化后获得高纯度的杂合抗菌蛋白；该蛋白能快速裂解鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、沙门氏菌、大肠杆菌等革兰氏阴性菌。由该抗 菌蛋白制成的抗菌制剂可用于预防和治疗细菌感染。它是一种新的、高效的、能直接裂解细菌的抗菌物质。该抗菌蛋白可制成抗菌制剂用于预防和治疗细菌感染，以及医疗器械和医疗场所的杀菌等。
附图说明
图1 AB469、AB46M9、AB469M和B946的SDS-PAGE图。
图2比浊法检测AB469对鲍曼不动杆菌(ATCC 19606)的生物活性。
图3比浊法检测AB469对铜绿假单胞菌(ATCC 15442)的生物活性。
图4比浊法检测AB469对肺炎克雷伯菌(ATCC 700603)的生物活性。
图5比浊法检测AB469、AB46M9、AB469M和B946对鲍曼不动杆菌(ATCC 19606)的生物活性。
具体实施方式
下面结合具体实施例对本发明做进一步的说明，但本发明的保护范围并不局限于此；
本发明通过对抗菌蛋白结构域分析，人工设计了针对革兰氏阴性菌具有高抗菌活性的杂合蛋白，氨基酸序列为SEQ ID NO 1；采用分子生物学方法，实现该蛋白在大肠杆菌中外分泌表达，经纯化后获得高纯度蛋白用于活性检测。
实施例1、AB469的工程菌构建、表达和纯化
1、AB469的工程菌构建
根据大肠杆菌密码子偏爱，人工设计并合成能编码AB469的核酸序列(SEQ ID NO:2)，即ab469；在其5’端加上NcoI限制性核酸内切酶位点(斜体部分)，同时多引入了核酸序列ct，与酶切位点末尾的g一起形成编码一个丙氨酸的密码子；在3’端加上HindIII限制性核酸内切酶位点(斜体部分)。序列如下：
Figure PCTCN2020116756-appb-000002
Figure PCTCN2020116756-appb-000003
为了便于在大肠杆菌中表达，形成可溶的目的蛋白，将SP10结合域中的Cys突变成Ser；人工合成的ab469基因通过NcoI和HindIII双酶切,连入pET28a(+)载体的Nco I和Hind III位点之间，得到重组质粒pET28a-ab469。重组质粒转化大肠杆菌BL21(DE3)感受态细胞，获得表达AB469的工程菌。
2、AB469的表达和纯化
工程菌挑单克隆接种于LB培养液(含30mg/L卡那霉素)，30℃振荡培养过夜，按1％接种到相同的LB培养液中，30℃振荡培养至光密度值(波长600nm)≈0.6时，加入终浓度为0.05mM的IPTG诱导蛋白表达，继续30℃振荡培养，诱导4h左右，离心收集发酵上清。
发酵上清经阳离子交换和凝胶过滤两步纯化。纯化后的样品置于-20℃冷冻保存备用。用10％SDS-PAGE测定重组蛋白样品的大小和纯度。从结果可见，重组杂合抗菌蛋白AB469的电泳主带在32kD附近(见图1)。
实施例2、AB469突变体AB46M9、AB469M和B946的工程菌构建、表达和纯化
1、突变体的工程菌构建
突变体AB46M9(SEQ ID NO:3)是由与AB46(1-185aa)氨基酸序列(SEQ ID NO:9)具有82％相似的催化域AB46M(SEQ ID NO:10)和结合域SP10(154-236aa)(SEQ ID NO:11)组成，中间为一段柔性的Linker。
突变体AB469M(SEQ ID NO:5)是由催化域AB46(1-185aa)和与SP10(154-236aa)氨基酸序列具有82％相似的结合域B9M(SEQ ID NO:12)组成，中间为一段柔性的Linker。
B946(SEQ ID NO:7)是由结合域SP10(154-236aa)和催化域AB46(1-185aa)组成，中间为一段柔性的Linker，其催化域和结合域的位置与AB469正好相反。
根据大肠杆菌密码子偏爱，人工设计并合成能编码AB46M9、AB469M和B946的核酸序列ab46m9(SEQ ID NO 4)、ab469m(SEQ ID NO 6)和b946(SEQ ID NO 8)；
ab46m9、ab469m和b946的表达载体、工程菌构建，以及它们表达和纯化的具体方法同ab469。纯化后的AB46M9、AB469M和B946样品置于-20℃冷冻保存备用。用10％ SDS-PAGE测定重组蛋白样品的大小和纯度(见图1)。
实施例3、比浊法检测AB469对鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌的杀菌活性
将过夜培养的鲍曼不动杆菌(ATCC 19606)、铜绿假单胞菌(ATCC 15442)、肺炎克雷伯菌(ATCC 700603)转接后生长到对数中期(OD 600大约0.5)，离心(5,000g,5分钟)收集菌体。菌体用20mm PBS(pH 7.2)清洗2次，然后重悬于含不同NaCl浓度(0-600mM)的PBS缓冲液中。在96孔板中加入样品(终浓度2μg/mL)或者PBS对照、10μl 200mm EDTA(终浓度0.5mm)和50μl的菌悬液，补加PBS至总反应体系200μl，反应温度37℃，在600nm波长下读数(间隔1分钟，读数10次)。
比浊检测结果见图2-图4，AB469能通过裂解病原菌导致菌液OD值下降，病原菌菌液的OD值快速下降表明AB469对鲍曼不动杆菌(ATCC 19606)、铜绿假单胞菌(ATCC 15442)、肺炎克雷伯菌(ATCC 700603)具有快速杀菌效果，随着盐浓度的提高，AB469依然保持较高的杀菌活性。一些非离子表面活性剂(如BriJ98、吐温20和平平加等)能显著增强AB469的杀菌活性。
实施例4、比浊法检测AB46M9、AB469M和B946对鲍曼不动杆菌的杀菌活性
比浊法检测同实施例3，比浊检测结果见图5。与AB469相比，突变体AB46M9、AB469M和B946对鲍曼不动杆菌的杀菌活性没有明显下降。
实施例5、AB469体外定时定量杀灭病原菌标准菌株及临床分离菌株的效果
将过夜培养的细菌转接后生长到对数中期(OD 600大约0.5)，离心(5,000g,5分钟)收集菌体。菌体用PBS(pH 7.2)稀释至约1×10 8cfu/mL。在250μl菌液中加入1mL样品含AB469(终浓度为10～50μg/mL)、NaCl(终浓度为150mm)和EDTA(终浓度为0.5mm)或者柠檬酸钠(终浓度为8mm)。室温作用120min，取0.5mL样液进行系列稀释，再各取0.5mL样液进行活菌计数培养。以40μg/mL AB46和EDTA(终浓度为0.5mm)室温作用2h为对照，AB46的制备方法同AB469。
体外杀菌实验结果显示，杂合抗菌蛋白AB469较单独AB46具有更强杀灭鲍曼不动杆菌(ATCC 19606)的活性(表1)；且对离子强度更加耐受。
表1、ABgp46和AB469对鲍曼不动杆菌(ATCC 19606)的杀菌活性比较
Figure PCTCN2020116756-appb-000004
Figure PCTCN2020116756-appb-000005
体外杀菌实验结果显示(表2)，10μg/mL抗菌蛋白AB469作用2h能有效杀灭鲍曼不动杆菌(ATCC 19606)、铜绿假单胞菌(ATCC 15442)和伤寒沙门氏菌(CMCC 50094)，下降均大于7个Log值以上；使得肺炎克雷伯菌(ATCC 700603)和大肠杆菌(ATCC 35218)下降大于2个Log值；使得阴沟肠杆菌(ATCC 700323)和金黄色葡萄球菌(ATCC 6538)下降约1个Log值。
表3的结果显示，50μg/mL抗菌蛋白AB469作用2h能有效杀灭肺炎克雷伯菌(ATCC700603)和大肠杆菌(ATCC 35218)；使得阴沟肠杆菌(ATCC 700323)和金黄色葡萄球菌(ATCC 6538)下降约4个Log值。
表2、AB469(10μg/mL)/EDTA在150mm NaCl条件下对不同病原菌的杀菌活性
Figure PCTCN2020116756-appb-000006
表3、AB469(50μg/mL)/EDTA在150mm NaCl条件下对不同病原菌的杀菌活性
Figure PCTCN2020116756-appb-000007
10μg/mL抗菌蛋白AB469作用2h能有效杀灭临床分离的鲍曼不动杆菌和铜绿假单胞菌(表4)，对多重耐药菌株具有同样效果(包括对头孢他啶、头孢曲松、美洛培南、亚胺培南、妥布霉素和舒巴坦等)；50μg/mL抗菌蛋白AB469作用2h能有效杀灭临床分离的肺炎克雷伯菌，包括多重耐药菌；
表4、AB469/EDTA在150mm NaCl条件下对不同临床分离病原菌的杀菌活性
Figure PCTCN2020116756-appb-000008
Figure PCTCN2020116756-appb-000009
以上结果表明，杂合后的AB469对革兰氏阴性菌具有非常强的杀菌效果，且对耐药菌具有同样的效果；AB469对金黄色葡萄球菌(革兰氏阳性菌)也有较强的杀菌效果。图1。
实施例6
制造100ml含AB469的生物抗菌制剂。
处方：
Figure PCTCN2020116756-appb-000010
制备方法：
a)按照处方和总配制液体积计算辅料需求量，精确称取至洁净容器中。
b)在配料器皿中加入总配制液70％的水，先溶解NaCl、乙二胺四乙酸二钠、磷酸氢二钠和磷酸二氢钾，充分溶解后再加入甘油，混匀后加入AB469，最后用水定容并且充分混匀。
c)除菌过滤：将配制好的消毒剂通过除菌过滤器，流出的消毒剂接入无菌容器。
d)灌装：将无菌消毒剂灌装于塑料或玻璃瓶。
该抗菌制剂可用于伤口或创面的杀菌消毒，每日1-2次。
Figure PCTCN2020116756-appb-000011
Figure PCTCN2020116756-appb-000012
Figure PCTCN2020116756-appb-000013
Figure PCTCN2020116756-appb-000014
Figure PCTCN2020116756-appb-000015
Figure PCTCN2020116756-appb-000016
Figure PCTCN2020116756-appb-000017
Figure PCTCN2020116756-appb-000018
Figure PCTCN2020116756-appb-000019
Figure PCTCN2020116756-appb-000020

Claims (10)

  1. 具有强杀菌效果的杂合抗菌蛋白，其特征在于，包括氨基酸序列如SEQ ID NO 1所示，命名为AB469，以及由所述的AB469的催化域相似的序列组成的杂合蛋白、由所述的AB469的结合域相似的序列组成的杂合蛋白或由AB469的催化域和结合域位置互换组成的杂合蛋白。
  2. 编码所述的AB469的核酸序列如SEQ ID NO:2，命名为ab469。
  3. 根据权利要求1所述的具有强杀菌效果的杂合抗菌蛋白，其特征在于，由所述的AB469的催化域相似的序列组成的杂合蛋白，如SEQ ID NO 3，命名为AB46M9。
  4. 编码权利要求3所述的AB46M9的核酸序列如SEQ ID NO:4，命名为ab46m9。
  5. 根据权利要求1所述的具有强杀菌效果的杂合抗菌蛋白，其特征在于，由所述的AB469的结合域相似的序列组成的杂合蛋白，如SEQ ID NO 5，命名为AB469M。
  6. 编码权利要求5所述的AB469M的核酸序列如SEQ ID NO:6，命名为ab469m。
  7. 根据权利要求1所述的具有强杀菌效果的杂合抗菌蛋白，其特征在于，由AB469的催化域和结合域位置互换组成的杂合蛋白，如SEQ ID NO 7，命名为B946。
  8. 编码权利要求7所述的B946的核酸序列如SEQ ID NO:8，命名为b946。
  9. 权利要求1、3、5或7所述的具有强杀菌效果的杂合抗菌蛋白的应用，其特征在于，用于制备抗菌制剂。
  10. 一种抗菌制剂，其特征在于，包括抗菌有效量的权利要求1、3、5或7所述的具有强杀菌效果的杂合抗菌蛋白和药物载体。
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