WO2021184500A1 - 一种表达ll-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用 - Google Patents

一种表达ll-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用 Download PDF

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WO2021184500A1
WO2021184500A1 PCT/CN2020/088259 CN2020088259W WO2021184500A1 WO 2021184500 A1 WO2021184500 A1 WO 2021184500A1 CN 2020088259 W CN2020088259 W CN 2020088259W WO 2021184500 A1 WO2021184500 A1 WO 2021184500A1
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recombinant
lactococcus lactis
expression vector
recombinant expression
pnz8149
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PCT/CN2020/088259
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French (fr)
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金万洙
张瀚林
蒋笑笑
郑爱华
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中国科学院动物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

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  • the present invention relates to the technical field of genetic engineering, in particular to a recombinant expression vector expressing LL-37 polypeptide, a recombinant Lactococcus lactis, an antiviral drug, and a construction method and application.
  • LL-37 is an antibacterial peptide which also has antiviral activity.
  • the traditional preparation method of LL-37 is a synthetic method, but the synthetic method is expensive, which greatly limits the application of LL-37.
  • the prior art discloses the use of genetic engineering methods to prepare LL-37 by constructing a recombinant expression vector and recombinant bacteria containing the LL-37 encoding gene.
  • the current method still has the technology of low expression efficiency of LL-37 and high production cost. problem.
  • the purpose of the present invention is to provide a recombinant expression vector for expressing LL-37 polypeptide, recombinant Lactococcus lactis, antiviral drugs, and construction methods and applications.
  • the recombinant expression vector of the present invention can be efficiently expressed in Lactococcus lactis to achieve LL
  • the oral delivery of -37 polypeptide can reduce production costs.
  • the present invention provides a recombinant expression vector for expressing LL-37 polypeptide.
  • the backbone plasmid for constructing the recombinant expression vector is PNZ8149; the recombinant expression vector is inserted with a fusion gene; the nucleotide sequence of the fusion gene is as SEQ ID NO: 1 is shown.
  • the fusion gene is inserted between the SphI and XbaI restriction sites of PNZ8149.
  • the present invention provides a recombinant Lactococcus lactis containing the recombinant expression vector described in the above scheme.
  • the original strain of the recombinant Lactococcus lactis includes Lactococcus lactis NZ3900.
  • the present invention provides a method for constructing the recombinant Lactococcus lactis of the above-mentioned scheme, which includes the following steps: transforming competent cells of Lactococcus lactis with the recombinant expression vector of the above-mentioned scheme to obtain recombinant Lactococcus lactis.
  • the present invention provides the application of the recombinant expression vector or the recombinant Lactococcus lactis in the preparation of antibacterial and/or antiviral drugs.
  • the virus includes a coronavirus.
  • the coronavirus includes SARS coronavirus and/or SARS-CoV2 coronavirus.
  • the present invention also provides an antibacterial and/or antiviral drug, which includes the recombinant Lactococcus lactis described in the above-mentioned scheme.
  • the dosage form of the drug includes tablets, granules, pills, capsules or oral liquids.
  • the present invention provides a recombinant expression vector (PNZ8149-7 ⁇ LL-37) for expressing LL-37 polypeptide.
  • the present invention uses PNZ8149 (lactic acid bacteria food-grade expression vector) as the basic vector.
  • PNZ8149 has a fusion gene inserted into it.
  • the fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition sites DDDDK and LL-37 in sequence. Tandem repeat sequence.
  • the present invention organically combines the living biological drug carrier Lactococcus lactis with antiviral polypeptides, not only uses the simple oral route to perfectly solve the cumbersomeness of injection administration and the uncertainty of metabolism in the body, but also effectively avoids a single tandem LL-
  • the 37 sequence expression efficiency is low and it is difficult to detect the problem.
  • the recombinant Lactococcus lactis constructed by the recombinant expression vector of the present invention can be synthesized and synthesized in an orderly manner.
  • the signal peptide sequence SPusp45 and LEISS sequence are digested and separated from the fusion protein.
  • the seven tandem repeats are released as monomeric LL-37 polypeptides, which are compatible with humans.
  • the expression level of LL-37 is extremely high through the detection of specific antibodies, and the blood absorption of LL-37 in the animal body is detected.
  • the LL-37 expressed by the recombinant expression vector of the present invention has good antiviral activity and can inhibit coronaviruses including SARS coronavirus and SARS-CoV2 coronavirus, with a maximum inhibition rate of 85%.
  • LL-37 expressed by the recombinant expression vector of the present invention has no other modifications or other protein tags, does not have antigenicity, and has high safety.
  • the present invention also provides a recombinant Lactococcus lactis containing the recombinant expression vector of the above solution.
  • the polypeptide drug LL-37 with anti-bacterial and anti-viral activity is injected into the human body through recombinant Lactococcus lactis, relying on its high-efficiency anti-bacterial and anti-viral effects, convenient route of administration and low production cost. It can be directly and quickly transformed into safe and mature therapeutic drugs, thereby reversing the current serious shortage of anti-new coronavirus drugs and meeting the medical needs of clinical frontline and patients.
  • Figure 1 is the construction of the PNZ8149-7 ⁇ LL-37 expression strain in Example 2;
  • Figure 2-a is the silver staining result of the protein expression level of the PNZ8149-7 ⁇ LL-37 expression strain in Example 3;
  • Figure 2-b shows the protein expression level of the PNZ8149-7 ⁇ LL-37 expression strain in Example 3 and the Western blot result;
  • Figure 3 shows the pharmacokinetic results of the PNZ8149-7 ⁇ LL-37 expression strain in Example 4.
  • Figure 4 shows the detection result of LL-37's inhibition efficiency against SARS simulated coronavirus in Example 5;
  • Fig. 5 shows the detection result of the inhibitory efficiency of LL-37 on SARS-CoV2 simulated coronavirus in Example 6.
  • the present invention provides a recombinant expression vector (PNZ8149-7 ⁇ LL-37) for expressing LL-37 polypeptide.
  • the backbone plasmid for constructing the recombinant expression vector is PNZ8149; the recombinant expression vector is inserted with a fusion gene (SPusp45 -LEISS-DDDDK-7 ⁇ LL-37); the nucleotide sequence of the fusion gene is shown in SEQ ID NO: 1, specifically:
  • nucleotide sequence of SPusp45 is shown in SEQ ID NO: 2, specifically:
  • the nucleotide sequence of the LEISS is shown in SEQ ID NO: 3, specifically: TTGGAAATATCGTCGACTTGTGATGCT; the nucleotide sequence of the DDDDK is shown in SEQ ID NO: 4, specifically: GACGATGACGATAAG; the 7 ⁇ LL The nucleotide sequence of -37 is shown in SEQ ID NO: 5, specifically:
  • the present invention uses PNZ8149 (lactic acid bacteria food-grade expression vector) as the basic vector.
  • PNZ8149 has a fusion gene inserted into it.
  • the fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition sites DDDDK and LL-37 in sequence. Tandem repeat sequence.
  • the recombinant vector of the present invention is a lactic acid bacteria food-grade recombinant expression vector, and the recombinant expression vector can be successfully expressed in Lactococcus lactis.
  • the LEISS is a synthetic short peptide LEIS.STCDA, which is used to enhance the secretion efficiency of foreign proteins in Lactococcus lactis.
  • the present invention organically combines the live biological drug carrier Lactococcus lactis with antiviral polypeptides for the first time, not only uses the simple oral route to perfectly solve the cumbersomeness of injection administration and the uncertainty of metabolism in the body, but also effectively avoids a single tandem LL -37 sequence expression efficiency is low and difficult to detect.
  • the recombinant Lactococcus lactis constructed by using the recombinant expression vector of the present invention can be synthesized in an orderly manner And release the aforementioned fusion protein, and under the action of enterokinase in the intestine, the signal peptide sequence SPusp45 and LEISS sequence are digested and separated from the fusion protein. At the same time, the seven tandem repeats are released into monomeric LL-37 polypeptide, which produces and Human endogenous LL-37 structural polypeptide with the same function.
  • the expression level is extremely high through the detection of specific antibodies, and the blood absorption of LL-37 in the animal body is detected.
  • the LL-37 polypeptide expressed by the recombinant expression vector of the present invention is completely consistent with the human LL-37 polypeptide, so it can also effectively inhibit human acquired immunodeficiency virus (HIV-1), influenza A virus (IAV), and respiratory tract synergy.
  • HIV-1 human acquired immunodeficiency virus
  • IAV influenza A virus
  • Viruses such as cytoplasmic virus (RSV), rhinovirus (HRV), vaccinia virus (VACV), herpes simplex virus (HSV) and hepatitis C virus (HCV), as well as Grams including Bacillus, Enterococcus, Streptococcus, etc.
  • the activity of gram-negative pathogenic bacteria including Achromobacter xylose oxidizing bacteria, Acinetobacter baumannii, Escherichia coli, Brucella, symbiotic actinomycetes, Proteus, etc.
  • the fusion gene is preferably inserted between the SphI and XbaI restriction sites of PNZ8149.
  • the fusion gene was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.
  • the present invention has no special restrictions on the construction method of the recombinant expression vector, and conventional methods in the field may be used.
  • the method for constructing the recombinant expression vector preferably adopts the following steps:
  • the target fragments are recovered for enzyme ligation to obtain the recombinant expression vector PNZ8149-7 ⁇ LL-37.
  • the present invention provides a recombinant Lactococcus lactis containing the recombinant expression vector of the above solution; the original strain of the recombinant Lactococcus lactis includes Lactococcus lactis NZ3900.
  • the present invention provides a method for constructing the recombinant Lactococcus lactis of the above-mentioned scheme, including the following steps: using the recombinant expression vector of the above-mentioned scheme to transform Lactococcus lactis competent cells to obtain recombinant Lactococcus lactis; the method of transformation is preferably electric shock Conversion.
  • the present invention provides the application of the recombinant expression vector or the recombinant Lactococcus lactis in the preparation of antibacterial and/or antiviral drugs;
  • the bacteria preferably include Gram-positive pathogenic bacteria and/or leather Langerhans-negative pathogenic bacteria;
  • the virus includes but is not limited to the coronavirus;
  • the coronavirus preferably includes the SARS coronavirus and/or the SARS-CoV2 coronavirus.
  • the present invention also provides an antibacterial and/or antiviral drug, the drug includes the recombinant Lactococcus lactis of the above-mentioned scheme; the drug preferably also includes a pharmaceutically acceptable excipient;
  • the dosage form preferably includes tablets, granules, pills, capsules or oral liquids.
  • the plasmid was extracted from the cultured transformed bacteria, and then amplified by PCR.
  • the PCR amplification primers were SEQ ID No: 6 (F: cttattgagaaagggaacgacgg) and SEQ ID No. 7 (R: tcaactgctgctttttggcta); the PCR amplification Reaction procedure: 94°C, 3min; ⁇ 94°C, 30s; 58°C, 30s; 72°C, 1min 30s ⁇ 38 cycles; 72°C, 5min.
  • the PCR products were separated and identified by 1% agarose electrophoresis, and the identification results are shown in Figure 1.
  • the PCR product in the rightmost lane is about 1200 bp in size, which is consistent with the target band size of 1257 bp.
  • the plasmid is suspected to be successfully constructed as PNZ8149-7 ⁇ LL-37.
  • the positive plasmid suspected to be PNZ8149-7 ⁇ LL-37 was further identified by sequencing.
  • the sequencing results showed that the PNZ8149-7 ⁇ LL-37 plasmid was successfully constructed without base mutations.
  • Example 5 The anti-simulated coronavirus (SARS) effect of PNZ8149-7 ⁇ LL-37 expression strain
  • the S protein is the main immunogen of coronavirus and the main protein that mediates viral infection;
  • the rVSV-SARS recombinant virus is constructed by replacing the G protein of VSV with the S protein of SARS, and the S protein is displayed on the surface of the VSV virus;
  • the recombinant virus can simulate the infection process of SARS virus;
  • the packaged rVSV-SARS simulated virus was diluted into a virus solution with a concentration of 2000ffu/ml, and LL-37 was diluted with virus solution to 0ng/ml, 10ng/ml, 100ng /ml, 1000ng/ml, 10000ng/ml, 100000ng/ml concentration treatment solution, incubate at room temperature for 30min.
  • the packaged rVSV-SARS-CoV2 simulated virus was diluted into a virus solution with a concentration of 2000ffu/ml, and LL-37 was diluted to 0ng/ml, 10ng with the virus solution. /ml, 100ng/ml, 1000ng/ml concentration treatment solution, incubate at room temperature for 30min;

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Abstract

提供了一种表达LL-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用，属于基因工程技术领域；所述重组表达载体的构建用骨架质粒为PNZ8149；所述重组表达载体中插入有融合基因；所述融合基因的核苷酸序列如SEQ ID NO：1所示；重组表达载体能够在乳酸乳球菌中高效表达得到的LL-37，能够有效抑制SARS冠状病毒和SARS-CoV2冠状病毒。

Description

一种表达LL-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用 技术领域
本发明涉及基因工程技术领域，尤其涉及一种表达LL-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用。
背景技术
LL-37是一种抗菌肽，也具有抗病毒活性。传统的LL-37制备方法为人工合成的方法，但是人工合成的方法成本高昂，大大限制了LL-37的应用。现有技术公开了利用基因工程手段，通过构建包含LL-37编码基因的重组表达载体、重组菌来制备获得LL-37，但是目前的方法仍然存在LL-37表达效率低、生产成本高的技术问题。
发明内容
本发明的目的在于提供一种表达LL-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用，本发明的重组表达载体能够在乳酸乳球菌中高效表达，从而实现LL-37多肽口服递送，能够降低生产成本。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种表达LL-37多肽的重组表达载体，所述重组表达载体的构建用骨架质粒为PNZ8149；所述重组表达载体中插入有融合基因；所述融合基因的核苷酸序列如SEQ ID NO：1所示。
优选的，所述融合基因插入在PNZ8149的SphI和XbaI限制性酶切位点之间。
本发明提供了一种包含上述方案所述重组表达载体的重组乳酸乳球菌。
优选的，所述重组乳酸乳球菌的原始菌株包括乳酸乳球菌NZ3900。
本发明提供了上述方案所述重组乳酸乳球菌的构建方法，包括以下步骤：利用上述方案所述重组表达载体转化乳酸乳球菌感受态细胞，得到重组乳酸乳球菌。
本发明提供了上述方案所述重组表达载体或者所述重组乳酸乳球菌在制备抗细菌和/或抗病毒的药物中的应用。
优选的，所述病毒包括冠状病毒。
优选的，所述冠状病毒包括SARS冠状病毒和/或SARS-CoV2冠状病毒。
本发明还提供了一种抗细菌和/或抗病毒的药物，所述药物包括上述方案所述重组乳酸乳球菌。
优选的，所述药物的剂型包括片剂、颗粒剂、丸剂、胶囊剂或口服液。
本发明的有益效果：本发明提供了一种表达LL-37多肽的重组表达载体(PNZ8149-7×LL-37)。本发明以PNZ8149(乳酸菌食品级表达载体)为基础载体，PNZ8149上插入有融合基因，所述融合基因包括顺次串联的信号肽SPusp45、益生菌LEISS、肠激酶识别位点DDDDK和LL-37七次串联重复序列。本发明将活体生物药载体乳酸乳球菌与抗病毒多肽有机结合，不仅利用口服的简易途径完美的解决了注射给药的繁琐性和体内代谢的不确定性，而且有效规避了单次串联LL-37序列表达效率低、难以检测的问题，在诱导剂乳酸链球菌素Nisin的严格控制下，实现时空特异性给药；利用本发明的重组表达载体构建得到的重组乳酸乳球菌能够有序合成并释放前述融合蛋白，并且在肠道内肠激酶的作用下将信号肽序列SPusp45、LEISS序列从融合蛋白上酶切分离开来，同时七次串联重复序列释放为单体LL-37多肽，产生与人体内源性LL-37相同功能的结构多肽。经特异性抗体检测LL-37表达量极高，并检测到动物体内LL-37的血液吸收情况。本发明的重组表达载体表达得到的LL-37具有良好的抗病毒活性，能够抑制包括SARS冠状病毒和SARS-CoV2冠状病毒在内的冠状病毒，最大抑制率达到85％。并且本发明的重组表达载体表达得到的LL-37没有其他修饰或其他蛋白标签，不具有抗原性，安全性高。
本发明还提供了一种包含上述方案所述重组表达载体的重组乳酸乳球菌。本发明通过重组乳酸乳球菌将具有抗细菌和抗病毒活性的多肽药物LL-37以口服的形式输入人体，依靠其高效的抗细菌和抗病毒作用、便捷的给药途径和低廉的生产成本，能够直接、快速地转化为安全、成熟的治疗药 物，从而扭转当前抗新型冠状病毒药物严重匮乏的局面，满足临床一线与患者的用药需求。
附图说明
图1为实施例2中PNZ8149-7×LL-37表达菌株的构建；
图2-a为实施例3中PNZ8149-7×LL-37表达菌株的蛋白表达水平银染结果；
图2-b为实施例3中PNZ8149-7×LL-37表达菌株的蛋白表达水平蛋白质免疫印迹结果；
图3为实施例4中PNZ8149-7×LL-37表达菌株的药代动力学结果；
图4为实施例5中LL-37对SARS模拟冠状病毒抑制效率检测结果；
图5为实施例6中LL-37对SARS-CoV2模拟冠状病毒抑制效率检测结果。
具体实施方式
本发明提供了一种表达LL-37多肽的重组表达载体(PNZ8149-7×LL-37)，所述重组表达载体的构建用骨架质粒为PNZ8149；所述重组表达载体中插入有融合基因(SPusp45-LEISS-DDDDK-7×LL-37)；所述融合基因的核苷酸序列如SEQ ID NO：1所示，具体为：
Figure PCTCN2020088259-appb-000001
Figure PCTCN2020088259-appb-000002
本发明中，所述SPusp45的核苷酸序列如SEQ ID NO：2所示，具体为：
Figure PCTCN2020088259-appb-000003
所述LEISS的核苷酸序列如SEQ ID NO：3所示，具体为：TTGGAAATATCGTCGACTTGTGATGCT；所述DDDDK的核苷酸序列如SEQ ID NO：4所示，具体为：GACGATGACGATAAG；所述的7×LL-37的核苷酸序列如SEQ ID NO：5所示，具体为：
Figure PCTCN2020088259-appb-000004
Figure PCTCN2020088259-appb-000005
本发明以PNZ8149(乳酸菌食品级表达载体)为基础载体，PNZ8149上插入有融合基因，所述融合基因包括顺次串联的信号肽SPusp45、益生菌LEISS、肠激酶识别位点DDDDK和LL-37七次串联重复序列。本发明的重组载体为乳酸菌食品级重组表达载体，所述重组表达载体能够在乳酸乳球菌中成功表达。
本发明中，所述LEISS为人工合成的短肽LEIS.STCDA，用于增强外源蛋白在乳酸乳球菌中的分泌效率。
本发明首次将活体生物药载体乳酸乳球菌与抗病毒多肽有机结合，不仅利用口服的简易途径完美的解决了注射给药的繁琐性和体内代谢的不确定性，而且有效规避了单次串联LL-37序列表达效率低、难以检测的问题，在诱导剂乳酸链球菌素Nisin的严格控制下，实现时空特异性给药；利用本发明的重组表达载体构建得到的重组乳酸乳球菌能够有序合成并释放前述融合蛋白，并且在肠道内肠激酶的作用下将信号肽序列SPusp45、LEISS序列从融合蛋白上酶切分离开来，同时七次串联重复序列释放为单体LL-37多肽，产生与人体内源性LL-37相同功能的结构多肽。经特异性抗体检测表达量极高，并检测到动物体内LL-37的血液吸收情况。
本发明的重组表达载体表达得到的LL-37多肽与人源LL-37多肽完全一致，因此亦能够有效抑制人类获得性免疫缺陷病毒(HIV-1)、甲型流感病毒(IAV)、呼吸道合胞病毒(RSV)、鼻病毒(HRV)、牛痘病毒(VACV)、单纯疱 疹病毒(HSV)和丙型肝炎病毒(HCV)等病毒以及包括芽孢杆菌、肠球菌、链球菌等在内的革兰氏阳性致病菌和包括木糖氧化无色杆菌、鲍氏不动杆菌、大肠杆菌、布鲁氏菌、共生放线菌、变形杆菌等在内的革兰氏阴性致病菌的活性。
本发明中，所述融合基因优选的插入在PNZ8149的SphI和XbaI限制性酶切位点之间。
本发明具体实施过程中，所述融合基因由上海生工生物工程有限公司合成。
本发明对所述重组表达载体的构建方法没有特殊限制，采用本领域常规方法即可。
本发明具体实施过程中，所述重组表达载体的构建方法优选的采用以下步骤进行：
1)全序列化学合成SphI-SPusp45-LEISS-DDDDK-7×LL-37-XbaI，获得pUC57-7×LL-37载体；
2)对PNZ8149和所述pUC57-7×LL-37载体分别进行酶切后，回收目的片段进行酶连，得到重组表达载体PNZ8149-7×LL-37。
本发明提供了一种包含上述方案所述重组表达载体的重组乳酸乳球菌；所述重组乳酸乳球菌的原始菌株包括乳酸乳球菌NZ3900。
本发明提供了上述方案所述重组乳酸乳球菌的构建方法，包括以下步骤：利用上述方案所述重组表达载体转化乳酸乳球菌感受态细胞，得到重组乳酸乳球菌；所述转化的方法优选为电击转化。
本发明提供了上述方案所述重组表达载体或者所述重组乳酸乳球菌在制备抗细菌和/或抗病毒的药物中的应用；所述细菌优选的包括革兰氏阳性致病菌和/或革兰氏阴性致病菌；所述病毒包括但不限于冠状病毒；所述冠状病毒优选的包括SARS冠状病毒和/或SARS-CoV2冠状病毒。
本发明还提供了一种抗细菌和/或抗病毒的药物，所述药物包括上述方案所述重组乳酸乳球菌；所述药物优选的还包括药学上可接受的赋形剂；所述药物的剂型优选的包括片剂、颗粒剂、丸剂、胶囊剂或口服液。
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。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 PNZ8149-7×LL-37表达菌株的构建
1、SphI-SPusp45-LEISS-DDDDK-7×LL-37-XbaI全序列化学合成，获得pUC57-7×LL-37载体；
2、构建PNZ8149-7×LL-37载体
1)采用天根质粒小提试剂盒，提取PNZ8149，pUC57-7×LL-37质粒；
2)酶切：PNZ8149，pUC57-7×LL-37酶切体系如表1所示，总体系为50μl，37℃酶切3h；
表1 PNZ8149，pUC57-7×LL-37酶切体系
Figure PCTCN2020088259-appb-000006
3)配制1％琼脂糖凝胶电泳，120V电压下电泳30min后切胶回收；
4)酶连：酶连体系如表2，总体系20μl，插入片段：载体＝7：1，16℃过夜；
表2 酶连体系
Figure PCTCN2020088259-appb-000007
5)连接产物纯化回收：制备好的连接产物中加入2.5倍体积的无水乙醇，1/10体积的2.5mol/L乙酸钠，混勻后于20℃放置lh，12000r/min离心5min，弃上清；用1ml 75％乙醇沉淀一次，12000r/min离心5min，弃上清，室温干燥20min，用去离子水溶解沉淀。
实施例2 PNZ8149-7×LL-37电转乳酸乳球菌NZ3900
1、乳酸乳球菌NZ3900感受态制备
1)取-80℃冻存的乳酸乳球菌MG1363接种于5ml含有5％葡萄糖的M17液体培养基中，30℃过夜培养；
2)将获得菌液按照1％接种于含有2.5％Gly和5％葡萄糖的M17液体培养基中，30℃静置培养至菌体OD 600值为0.3～0.4，收集备用；
3)将以上收集的菌体培养物冰浴10min，4℃离心5000rpm，5min；收集菌体；
4)沉淀用冰冷的10％蔗糖和10％甘油混合溶液1/10体积清洗两次，4℃离心8000rpm，5min，收集沉淀；
5)最后沉淀重悬于1/100体积10％蔗糖和10％甘油混合溶液中，冰浴10min后使用；
2、乳酸乳球菌NZ3900电转化
1)吸取40μl新鲜制备的感受态细菌悬液于冰冷的无菌EP管中，冰浴5min。
2)吸取1μl纯化后的连接产物加入感受态细菌，混匀后，置于冰上5min。
3)将细菌悬液和质粒DNA混合液加入冰冷的电转杯中，放入电击仪，设置条件为2500V，200Ω，25μF；
4)电击完毕后，迅速加入1ml冰冷的GM17-MC恢复培养基；混匀后转入EP管中冰浴5～10min；30℃培养2h。
5)取100μl恢复培养的菌液接种于Elliker选择培养基，30℃过夜培养，挑取黄色菌落进行鉴定。
3、转化菌的验证
从培养的转化菌体中提取质粒，之后用PCR扩增，所述PCR扩增引物为SEQ ID No：6(F:cttattgagaaagggaaacgacgg)和SEQ ID No.7(R：tcaactgctgctttttggcta)；所述PCR扩增的反应程序：94℃，3min；{94℃，30s；58℃，30s；72℃，1min 30s}38个循环；72℃，5min。将上述PCR产物用1％浓度的琼脂糖电泳分离鉴定，鉴定结果参见图1。由图1可以看出，最右 侧泳道的PCR产物约为1200bp大小，与目的条带大小1257bp相符，该质粒疑似构建成功的PNZ8149-7×LL-37。
疑似为PNZ8149-7×LL-37的阳性质粒经进一步测序鉴定，测序结果显示PNZ8149-7×LL-37质粒构建成功，不存在碱基突变。
实施例3 银染与Western检测PNZ8149-7×LL-37表达菌株的蛋白表达能力
1、PNZ8149-7×LL-37在乳酸乳球菌中的诱导分泌表达
1)将菌株接入M17液体培养基中，30℃静置培养过夜，第二天转接至新的M17肉汤培养基中，待OD600数值达到0.4～0.6时，加入诱导剂乳酸链球菌素Nisin继续培养5h；
2)培养结束后10000rpm离心20min，取上清，用0.22μm的过滤膜过滤上清，加入N-月桂酰肌氨酸钠至终浓度0.1％，室温15min；
3)加入三氯醋酸至终浓度7.5％，混匀，冰上2h；
4)10000rpm离心10min，弃上清，加入2ml四氢呋喃，10000rpm离心10min；
5)10000rpm离心10min，弃上清，加入2ml四氢呋喃，10000rpm离心10min；
6)弃上清，晾干，加入1ml 8M尿素溶解。
2、电泳
1)配制10％SDS-PAGE凝胶；
2)收集的蛋白样品中加入浓度5×SDS-PAGE蛋白上样缓冲液，100℃加热5～10min；
3)冷却到室温后，把蛋白样品直接上样到SDS-PAGE胶加样孔内；100V电泳90～120min；
4)按照Pierce银染试剂盒(Thermo，#24600)要求，对上述PAGE胶进行银染。
3、转印与免疫印迹显影
1)无水甲醇浸泡PVDF转印膜1～2min，使其充分活化；
2)按照“电转印负极-固定海绵-定性滤纸-PAGE胶-PVDF转印膜-定性滤纸-固定海绵-电转印正极”的顺序制备转印模块，100V湿法转印45min，将转印后的PVDF转印膜小心取出，TBST缓冲液室温洗涤一次；
3)5％脱脂奶粉-TBST缓冲液室温封闭上述PVDF转印膜3h，然后将上述PVDF转印膜4℃过夜孵育人源LL-37单克隆抗体(Santa Cruz#sc-166770)；
4)第二天使用TBST缓冲液对上述PVDF转印膜室温洗涤三次，每次10min，然后孵育辣根过氧化物酶标记的小鼠源二抗，室温震荡1h；
5)使用TBST缓冲液对上述PVDF转印膜室温洗涤三次，每次10min，去除二抗后按照Pierce高灵敏度底物显色试剂盒(Thermo，#34096)的要求，对上述PVDF转印膜进行显影。
结果如图2-a所示，PNZ8149-7×LL-37表达菌株的上清中特异性表达7×LL-37，且随着诱导剂浓度的增加，分子量为34kda的目的条带(图2-a左箭头)逐渐增多。免疫印迹结果如图2-b所示，相对于空载体对照菌株，采用200ng/ml Nisin诱导剂诱导的PNZ8149-7×LL-37表达菌株在分子量34kda附近呈现明显的条带。
实施例4 PNZ8149-7×LL-37表达菌株的药物代谢动力学
1、选取250～300g的雄性SD大鼠，单次灌胃PNZ8149-7×LL-37表达菌株(每只大鼠给药量为3.5E10个活菌，即CFU＝3.5E10)。
2、分别于灌胃前(0h)和灌胃后1、2、4、6h经尾静脉取血。
3、分离血清，利用LL-37Elisa试剂盒(Hycult biotechnology Cat#HK321)检测血清中LL-37浓度。
结果如图3所示：灌胃后2h，血液中LL-37浓度显著(p<0.05)高于灌胃前血液中浓度。
实施例5 PNZ8149-7×LL-37表达菌株的抗模拟型冠状病毒(SARS)效果
1、S蛋白是冠状病毒的主要免疫原也是介导病毒感染的主要蛋白；通过将VSV的G蛋白替换为SARS的S蛋白构建rVSV-SARS重组病毒，将S 蛋白展示到VSV病毒的表面；该重组病毒可以模拟SARS病毒的感染过程；
2、为了验证LL-37对SARS病毒的抑制效果，将包装好rVSV-SARS模拟病毒稀释成2000ffu/ml浓度的病毒液，用病毒液将LL-37稀释成0ng/ml，10ng/ml，100ng/ml，1000ng/ml，10000ng/ml，100000ng/ml浓度的处理液，室温孵育30min。
3、将不同浓度的处理液处理培养于96孔板的vero细胞，每孔100μl，每个浓度设置3个复孔，将细胞放置于37℃细胞培养箱中。
4、病毒感染2h后更换成含20mM NH 4Cl的新鲜培养基，将细胞放置于28℃细胞培养箱中，感染24h后荧光显微镜下统计荧光信号阳性的细胞数。
5、24h之后，在显微镜下观察有绿色荧光的细胞数量。最后根据对比试验，鉴定LL-37的抗病毒能力。
结果如图4所示，LL-37在10μg/ml浓度处理的时候，最大抑制率能达到85％。
实施例6 PNZ8149-7×LL-37表达菌株的抗模拟型新型冠状病毒(SARS-CoV2)效果
1、将VSV的G蛋白替换为SARS-CoV2的S蛋白构建rVSV-SARS-CoV2重组病毒，将S蛋白展示到VSV病毒的表面；此重组病毒可以模拟SARS-CoV2的入侵过程，可以广泛用于病毒入侵机制研究，以及抗体和小分子抗病毒药物的筛选；
2、为了验证LL-37对SARS-CoV2病毒的抑制效果，将包装好rVSV-SARS-CoV2模拟病毒稀释成2000ffu/ml浓度的病毒液，用病毒液将LL-37稀释成0ng/ml，10ng/ml，100ng/ml，1000ng/ml浓度的处理液，室温孵育30min；
3、将不同浓度的处理液处理培养于96孔板的vero细胞，每孔100μl，每个浓度设置3个复孔，将细胞放置于37℃细胞培养箱中；
4、病毒感染2h后更换成含20mM NH 4Cl的新鲜培养基，将细胞放置于28℃细胞培养箱中，感染24h后荧光显微镜下统计荧光信号阳性的细胞数；
5、24h之后，在显微镜下观察有绿色荧光的细胞数量。最后根据对比试验，鉴定LL-37的抗病毒能力；
结果如图5所示：LL-37在100ng/ml浓度处理的时候，最大抑制率能达到41.5％。
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。

Claims (10)

  1. 一种表达LL-37多肽的重组表达载体，其特征在于，所述重组表达载体的构建用骨架质粒为PNZ8149；所述重组表达载体中插入有融合基因；所述融合基因的核苷酸序列如SEQ ID NO：1所示。
  2. 根据权利要求1所述的重组表达载体，其特征在于，所述融合基因插入在PNZ8149的SphI和XbaI限制性酶切位点之间。
  3. 一种包含权利要求1或2所述重组表达载体的重组乳酸乳球菌。
  4. 根据权利要求1所述的重组乳酸乳球菌，其特征在于，所述重组乳酸乳球菌的原始菌株包括乳酸乳球菌NZ3900。
  5. 权利要求3或4所述重组乳酸乳球菌的构建方法，包括以下步骤：
    利用权利要求1或2所述重组表达载体转化乳酸乳球菌感受态细胞，得到重组乳酸乳球菌。
  6. 权利要求1或2所述重组表达载体或者权利要求3或4所述重组乳酸乳球菌在制备抗细菌和/或抗病毒的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述病毒包括冠状病毒。
  8. 根据权利要求7所述的应用，其特征在于，所述冠状病毒包括SARS冠状病毒和/或SARS-CoV2冠状病毒。
  9. 一种抗细菌和/或抗病毒的药物，其特征在于，所述药物包括权利要求3或4所述重组乳酸乳球菌。
  10. 根据权利要求9所述的药物，其特征在于，所述药物的剂型包括片剂、颗粒剂、丸剂、胶囊剂或口服液。
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