WO2021208913A1 - 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用 - Google Patents

一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用 Download PDF

Info

Publication number
WO2021208913A1
WO2021208913A1 PCT/CN2021/086927 CN2021086927W WO2021208913A1 WO 2021208913 A1 WO2021208913 A1 WO 2021208913A1 CN 2021086927 W CN2021086927 W CN 2021086927W WO 2021208913 A1 WO2021208913 A1 WO 2021208913A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant
protein
lactobacillus
vector
cov
Prior art date
Application number
PCT/CN2021/086927
Other languages
English (en)
French (fr)
Inventor
董伟
谢小红
文利新
王吉
胡意
李鑫
张琳玉
马朝阳
Original Assignee
文利新
董伟
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 文利新, 董伟 filed Critical 文利新
Publication of WO2021208913A1 publication Critical patent/WO2021208913A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention belongs to the technical field of antiviral drugs, and specifically relates to a recombinant plasmid, a recombinant Lactobacillus expression system and applications thereof for preventing and treating neocoronavirus infection.
  • New crown pneumonia (COVID-19) is caused by a new type of coronavirus (named by the World Health Organization as 2019-nCOV; the International Committee for Classification of Viruses named SARS-CoV-2).
  • Coronavirus (CoV) is a positive-stranded single-stranded RNA virus with a diameter of 80 to 120 nm, divided into four types: ⁇ , ⁇ , ⁇ , and ⁇ .
  • SARS-CoV the 2003 SARS (SARS-CoV), the 2012 Middle East Respiratory Syndrome (MERS-CoV) and the 2019 new crown pneumonia virus are all ⁇ -coronaviruses, which have caused severe acute respiratory infections worldwide.
  • SARS-CoV-2 The new coronary pneumonia virus (SARS-CoV-2) has been confirmed by Chinese scientists that its genome includes 29891 nucleotides, including untranslated regions (UTR) genes at both ends and a complete open reading frame gene (open reading frame). frame, ORF), can encode at least four structural proteins: spike glycoprotein (spike, S), small envelope glycoprotein (envelope, E), membrane glycoprotein (membrane, M), nucleocapsid protein (nucleocapsid, N), its genome sequence has high homology with MERS-CoV.
  • UTR untranslated regions
  • M membrane glycoprotein
  • N nucleocapsid protein
  • SARS-CoV-2 is mainly spread by respiratory droplets, and very few are spread through aerosols and feces. How to quickly curb the global spread and spread of SARS-CoV-2?
  • the various biosecurity measures currently adopted such as home isolation, reduction of personnel movement, and various disinfection measures, are difficult and have many loopholes for the whole society. The price paid is huge. Except for China, the world has not stopped the rapid spread of the virus so far.
  • the second is to speed up the development of vaccines, but the vaccine development cycle is long, the production technology is more complicated, and the cost is high.
  • the injection requires professional and technical personnel. It is difficult to achieve the effect of herd immunity in a short time.
  • the purpose of the present invention is to provide a recombinant plasmid, recombinant Lactobacillus expression system for the prevention and treatment of new coronavirus infection and its application in the preparation of drugs for the prevention of new coronavirus infection, by blocking and interfering with the new coronavirus and mucosa Specific receptor binding can prevent and reduce new coronary pneumonia and quickly stop its global spread.
  • the present invention provides a recombinant vector for preventing and treating new coronavirus infection, including the coding sequence of the RBD region protein in the codon-optimized new coronavirus S1 protein; the nucleotide sequence of the coding sequence is as shown in the sequence table SEQ ID NO:1 Shown.
  • the basic vector of the recombinant vector includes a lactobacillus secretory expression vector.
  • the lactobacillus secretory expression vector includes pVE5523.
  • the multiple cloning sites where the coding sequence is inserted into pVE5523 are SalI and EcoRV.
  • the invention provides a recombinant lactobacillus expression system containing the recombinant vector.
  • the present invention provides a recombinant S1 protein secreted and expressed by the recombinant lactobacillus expression system.
  • the present invention provides a method for preparing the recombinant S1 protein, which includes the following steps:
  • the recombinant lactobacillus expression system is inoculated into a special medium for lactobacillus in a sterile environment, and fermented at 28-35° C. for 68-75 hours, and the obtained fermentation broth contains the recombinant S1 protein.
  • the present invention provides the application of the recombinant vector, the recombinant Lactobacillus expression system, the recombinant S1 protein or the fermentation broth prepared by the preparation method in the preparation of drugs for preventing and treating neocoronavirus infection.
  • the present invention provides a mucosal blocking agent for preventing and treating neocoronavirus infection, which includes the recombinant S1 protein or the fermentation broth obtained by the preparation method.
  • the mucosal blocking agent includes oral, nasal spray or eye drops.
  • the recombinant vector and recombinant Lactobacillus expression system for preventing and treating the new coronavirus infection provided by the present invention is to insert the coding sequence of the RBD region protein in the new coronavirus S1 protein into the vector through codon optimization, and then use Lactobacillus to express safely and efficiently.
  • S1 protein which can be used as a mucosal infection blocking spray, which binds to the human respiratory tract, conjunctiva, digestive tract and other mucosal surface specific receptors (ACE2), thus the site where the new coronavirus binds to the mucosal surface of the body It is preferentially occupied by the S1 protein to effectively block and interfere with the binding of the new coronavirus to the human mucosal specific receptor (ACE2), thereby preventing the occurrence of new coronary pneumonia, reducing the number of people infected with new coronary pneumonia, and quickly stopping its global spread.
  • ACE2 mucosal infection blocking spray
  • the invention provides a mucosal blocking agent for preventing and treating neocoronavirus infection. Since SARS-CoV-2 is mainly spread by respiratory droplets, very few are spread through aerosols, feces, etc., cutting off the S1 protein on the surface of the new coronavirus and the respiratory tract, conjunctiva, digestive tract and other mucosal surfaces ACE2 The binding of the receptor can effectively reduce the infection of the new coronavirus by blocking the path of viral infection.
  • the mucosal blocking agent has the following characteristics:
  • the recombinant new coronavirus S1 protein contains only viral functional proteins, no virus infection genes exist, and it will not cause virus mutation. It is a topical preparation with high safety;
  • Recombinant S1 protein is secreted into the cell wall of bacteria and is a soluble expression product.
  • Lactobacillus is a common probiotic in the human body and is recognized as the safest food-grade microorganism.
  • the S1 protein expressed by lactic acid bacteria does not need to be extracted and purified, so it is omitted.
  • the complicated purification process and renaturation process after the expression of the conventional prokaryotic expression system greatly reduces the production cost of the mucosal blocking agent, which is conducive to rapid industrial production;
  • the mucosal blocking agent can be used repeatedly, is not only non-toxic and harmless, but also can induce mucosal immunity and further reduce the risk of virus infection. It can be used by professional medical staff before taking up their jobs, or in closed transportation and office places. Or use in communities and families to prevent infection.
  • Figure 1 is a vector map of the recombinant vector pVE5523-SARS-CoV-2-S1;
  • Figure 2 shows the sequencing result of the recombinant vector and the sequence comparison result of the inserted nucleotide fragment
  • Figure 3 shows the fluorescence quantitative PCR amplification curve of the recombinant vector pVE5523-SARS-CoV-2-S1;
  • Figure 4-1 shows the Western Blot identification of recombinant S1 protein; where M: Marker 26616; 1: Recombinant Lactobacillus supernatant 20 ⁇ l; 2: Recombinant Lactobacillus precipitation 20 ⁇ l;
  • Figure 4-2 shows the results of recombinant S1 protein Coomassie brilliant blue staining analysis; among them, M: Marker 26616;
  • the present invention provides a recombinant vector for preventing and treating new coronavirus infection, including the coding sequence of the RBD region protein in the codon-optimized new coronavirus S1 protein; the nucleotide sequence of the coding sequence is as shown in the sequence table SEQ ID NO:1 Shown.
  • the basic vector of the recombinant vector preferably includes a lactobacillus secretory expression vector.
  • a lactobacillus secretory expression vector well known in the art can be used.
  • the lactobacillus secretory expression vector is pVE5523.
  • the multiple cloning sites where the coding sequence is inserted into pVE5523 are preferably SalI and EcoRV.
  • the coding sequence is obtained by optimizing the RBD region protein in the S1 protein of the new coronavirus according to the codon preference of Lactobacillus. It is repeated twice at the same time, which is beneficial to increase the protein expression.
  • the nucleotide sequence is shown in the sequence table.
  • the SEQ ID NO:1 shown in () is convenient for subsequent insertion into a lactobacillus secretory expression vector for high-efficiency expression.
  • the RBD region protein of the new coronavirus S1 protein is used as the new coronavirus-specific protein bound to the human mucosal surface ACE2 (Angiotensin Converting Enzyme 2) receptor, which can effectively compete for the binding site of the new coronavirus and is safe to use , To prevent the infection of the new coronavirus.
  • ACE2 Angiotensin Converting Enzyme 2 receptor
  • the method for constructing the recombinant vector preferably includes the following steps:
  • the restriction endonuclease digestion method is not particularly limited, as long as the restriction endonuclease digestion method is well-known in the art.
  • the basic vector is pVE5523
  • the types of the restriction endonucleases are preferably SalI and EcoRV, and the map of the recombinant vector is shown in Figure 1.
  • connection method preferably uses T4 ligase connection.
  • the screening method preferably introduces the recombinant vector into Escherichia coli, extracts the plasmid after culturing, and performs sequencing and comparison of the plasmid.
  • the recombinant vector consistent with the inserted coding sequence is the recombinant vector pVE5523-SARS-CoV- 2-S1 (sequence is SEQ ID NO: 2), used for subsequent operations.
  • the invention provides a recombinant lactobacillus expression system containing the recombinant vector.
  • the type of Lactobacillus in the recombinant Lactobacillus expression system is not particularly limited, as long as the Lactobacillus well known in the art can be used.
  • the type of Lactobacillus is tested using ATCC393 Lactobacillus casei.
  • the method for preparing the recombinant Lactobacillus expression system preferably transforms the recombinant vector into a Lactobacillus competent state and screens to obtain the recombinant Lactobacillus expression system.
  • the transformation method is not particularly limited, and a transformation method well-known in the art can be used.
  • the conversion adopts an electric conversion method.
  • the screening method preferably spreads E. coli on an MRS solid culture plate containing erythromycin and cultures it in a static state; picks positive clones and inoculates them into an MRS liquid medium containing erythromycin for static culture; extracts recombinant plasmids Plasmids were identified by fluorescence quantitative PCR.
  • the primers used for fluorescent quantitative PCR are SEQ ID NO: 3 and SEQ ID NO: 4.
  • the nucleotide sequence of the amplified product of the fluorescence quantitative PCR reaction is SEQ ID NO: 5.
  • the positive recombinant plasmid has a typical amplification curve.
  • the ATCC393 competent control has no amplification curve, indicating that the pVE5523-SARS-CoV-2-S1 plasmid was successfully transformed into ATCC393 competent cells.
  • the present invention provides a recombinant S1 protein secreted and expressed by the recombinant lactobacillus expression system.
  • the present invention provides a method for preparing the recombinant S1 protein, which includes the following steps:
  • the recombinant Lactobacillus expression system was inoculated into Lactobacillus liquid culture medium under aseptic environment, fermented at 28 ⁇ 35°C for 68 ⁇ 75h, the obtained fermentation broth was centrifuged at 5000 ⁇ 6000rpm for 15 ⁇ 30min, and the supernatant was taken Solution to obtain recombinant S1 protein.
  • the Lactobacillus liquid culture medium is MRS liquid culture.
  • the culture temperature of the recombinant lactobacillus expression system is 34-36°C, and the fermentation time is preferably 72h.
  • the temperature of the centrifugation is preferably 4°C.
  • the quality of the recombinant S1 protein accounts for 10% of the total protein quality of Lactobacillus.
  • the present invention provides the application of the recombinant vector, the recombinant Lactobacillus expression system, the recombinant S1 protein or the fermentation broth prepared by the preparation method in the preparation of drugs for preventing and treating neocoronavirus infection.
  • the present invention provides a mucosal blocking agent for preventing and treating neocoronavirus infection, including the recombinant S1 protein or the recombinant S1 protein prepared by the preparation method.
  • the mucosal blocking agent preferably includes oral and nasal sprays and eye drops.
  • the S1 protein concentration range is preferably 10ng/ml-50ng/ml, more preferably 20-40ng/ml, most preferably 30ng/ml.
  • the human dosage of the mucosal blocking agent is preferably 0.2ml/person/time, twice a day, wherein the nasal spray dosage is preferably 0.1ml/person/time, and the eye drop dosage is preferably 0.1ml/person/time.
  • the present invention has no special restrictions on the preparation methods of the oral and nasal sprays and/or eye drops, as long as the preparation methods are well known in the art.
  • the codon-optimized S1 gene of the new coronavirus SARS-CoV-2 (SARS-CoV-2-S1)
  • the S1 protein of the new coronavirus SARS-CoV-2 is a key surface protein for the virus to bind to the ACE2 receptor on the host surface and mediate the virus into the host cell. Screen the gene sequence of the SARS-CoV-2 S1 protein that specifically binds to ACE2, and optimize the sequence with Lactobacillus codons.
  • the modified sequence was artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd., SARS-CoV-
  • the nucleotide sequence of 2-S is shown in SEQ ID NO:1.
  • Restriction endonucleases SalI and EcoRV were purchased from NEB; Taq enzyme, dNTP, DNA Marker DL2000, DL15000, Agarose Gel DNA Purification Kit, Mini BEST Plasmid Purification Kit were purchased from Dalian Bao Biological Company.
  • the cloning vector pVE5523 was provided by Nanjing GenScript Biotechnology Co., Ltd.
  • the small fragments of the cloning vector pVE5523 cut with SalI/EcoRV double enzymes were ligated with the SARS-CoV-2-S1 gene fragments cut with the same double enzymes, and the ligation products were transformed into E. coli. After culture, the plasmid was extracted with the kit , And send the extracted plasmid to Nanjing GenScript Biotechnology Co., Ltd. for sequencing verification.
  • Recombinant plasmid sequencing results After gene sequencing, the recombinant plasmid is compared with the inserted S1 gene fragment (single repeat sequence) (see Figure 2). The sequencing result is consistent with expectations, indicating the synthesized SARS-CoV-2-S1 gene fragment It was successfully inserted into the Lactobacillus secretory vector pVE5523, the recombinant plasmid was successfully constructed, and the positive plasmid was named pVE5523-SARS-CoV-2-S1.
  • Erythromycin was purchased from Baierdi Biotechnology Co., Ltd.
  • the pVE5523-SARS-CoV-2-S1 recombinant vector was electrotransformed into Lactobacillus ATCC393 competent, and the electro-transformed Lactobacillus ATCC393 was spread on the MRS solid culture plate containing 5 ⁇ g/ml erythromycin at 30°C. Cultivate in an incubator for 72 hours, pick the colonies on the plate and inoculate them into MRS liquid medium containing 5 ⁇ g/ml erythromycin, and incubate at 30°C for 72 hours; extract the plasmids from the bacteria and use ATCC393 competence as a control. Fluorescence quantitative PCR was used for identification, and the identification primers and amplification sequences were as follows:
  • Upstream primer ccaaccatacagagtagtagta (SEQ ID NO: 3)
  • Downstream primer gttagactcagtaagaacacct (SEQ ID NO: 4);
  • Fluorescence quantitative PCR reaction program pre-denaturation 95°C5min; cyclic reaction 95°C10s, 60°C30s, 40 cycles; melting curve 95°C15s, 60°C60s, 95°C15s; test according to the above parameters on the fluorescence quantitative PCR instrument .
  • the amplified sequence is as follows:
  • Fluorescence quantitative PCR was used to detect the amplified recombinant plasmid, and the results are shown in Figure 3. It can be seen from the amplification curve that the positive recombinant plasmid has a typical amplification curve, and the CT value varies within the range of 19-22, while the ATCC393 competent control does not have an amplification curve. The results showed that pVE5523-SARS-CoV-2-S1 plasmid was successfully transformed into ATCC393 competent cells.
  • the recombinant Lactobacillus expression system was inoculated with Lactobacillus MRS liquid medium at a ratio of 1%, and the fermentation broth was harvested after 72 hours at 35°C.
  • Recombinant S1 protein content detection Lactobacillus plate surface spread method live bacteria technology, and then calculate that the S1 protein content accounts for 10% of the total protein content of Lactobacillus.
  • the recombinant S1 protein prepared in Example 4 was diluted to a solution of 10ng/ml.
  • the diluted S1 protein solution was administered to SPF New Zealand rabbits and SD rats in the form of sprays.
  • the negative control group A was sprayed with the same volume of normal saline and the control group B was sprayed with the same volume of MRS culture medium.
  • Each group of New Zealand rabbits and Four SD rats were observed for 2 weeks. No abnormalities such as abnormal body temperature, allergies, and respiratory tract infections occurred in 24 animals. This indicates that the recombinant S1 protein prepared by the present invention is safe and has no side effects.
  • the recombinant lactobacillus expression system was fermented using the method of Example 4, and the harvested fermentation broth was centrifuged at 4° C. and 8000 rpm for 15 minutes to harvest the bacteria.
  • the recombinant S1 protein was quantitatively analyzed: the polyacrylamide gel was decolorized by Coomassie brilliant blue staining, and the decolorized gel was photographed with a protein gel imager.
  • Recombinant S1 protein concentration ( ⁇ g/ml) target band concentration ⁇ 500 ⁇ l ⁇ 20ml
  • the calculated concentration of the recombinant S1 protein is 3.53 ⁇ g/ml, indicating that the recombinant S1 protein can be efficiently expressed in the Lactobacillus expression system.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Otolaryngology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Nutrition Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Ophthalmology & Optometry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用，属于抗病毒药物技术领域。通过密码子优化新冠病毒S1蛋白中的RBD区域蛋白的编码序列***载体中，再利用乳酸杆菌安全高效地表达，得到重组表达S1蛋白，使其能作为粘膜感染阻断喷剂，与人体呼吸道、眼结膜、消化道及其它粘膜表面特异性受体（ACE2）结合，从而使得新冠病毒与机体黏膜表面结合的位点被S1蛋白优先占位封闭，进而高效阻断和干扰新冠病毒与人粘膜特异性受体结合，预防新冠肺炎发生，减少新冠肺炎感染人群数量，快速阻遏其全球漫延。

Description

一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用
本申请要求于2020年04月14日提交中国专利局、申请号为202010288793.7、发明名称为“一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用”的中国专利申请的优先权，同时本申请要求于2021年03月12日提交中国专利局、申请号为2021102677115、发明名称为“一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于抗病毒药物技术领域，具体涉及一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用。
背景技术
新冠肺炎(COVID-19)是由一种新型冠状病毒(世界卫生组织将其命名为2019-nCOV；国际病毒分类委员会命名为SARS-CoV-2)感染所致。冠状病毒(CoV)为直径80～120nm的正链单股RNA病毒，分为α、β、δ和γ等四个类型。其中，2003年非典(SARS-CoV)和2012年中东呼吸综合征(MERS-CoV)和2019年新冠肺炎病毒都属于β-冠状病毒，均引发了全球的严重的急性呼吸道感染疫病。新冠肺炎病毒(SARS-CoV-2)经中国科学家的研究证实，其基因组包括29891个核苷酸，包含两端非翻译区(untranslated regions，UTR)基因和一个完整的开放阅读框基因(open reading frame，ORF)，至少可以编码四种结构蛋白：刺突糖蛋白(spike，S)、小包膜糖蛋白(envelope，E)、膜糖蛋白(membrane，M)、核衣壳蛋白(nucleocapsid，N)，其基因组序列与MERS-CoV具有较高的同源性。
SARS-CoV-2的传播方式主要是以呼吸道飞沫传播为主，极少数是通过气溶胶、粪便等途径传播。如何快速遏制SARS-CoV-2在全球的传播和漫延，一是目前采取的各种生物安全措施，如居家隔离、减少人员流动以及各种消毒措施等，但其难度大，漏洞多，全社会付出的代价巨大，全 世界除了中国，到目前为止还没有阻止病毒的快速漫延；二是加快疫苗研制，但疫苗研制周期长，生产工艺技术较复杂，成本较高，注射需要专业技术人员，很难在短时间内达到群体免疫效果。
发明内容
有鉴于此，本发明的目的在于提供一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其在制备预防新冠病毒感染的药物中的应用，通过阻断和干扰新冠病毒与粘膜特异性受体结合，从而预防和减少新冠肺炎，快速阻遏其全球漫延。
本发明提供了一种用于防治新冠病毒感染的重组载体，包括密码子优化的新冠病毒S1蛋白中RBD区域蛋白的编码序列；所述编码序列的核苷酸序列如序列表SEQ ID NO:1所示。
优选的，所述重组载体的基础载体包括乳酸杆菌分泌型表达载体。
优选的，所述乳酸杆菌分泌型表达载体包括pVE5523。
优选的，所述编码序列***pVE5523中的多克隆位点为SalI和EcoRV。
本发明提供了一种包含所述重组载体的重组乳酸杆菌表达***。
本发明提供了由所述重组乳酸杆菌表达***中分泌表达的重组S1蛋白。
本发明提供了所述重组S1蛋白的制备方法，包括以下步骤：
将所述重组乳酸杆菌表达***在无菌环境下接种至乳酸杆菌专用培养基，在28～35℃条件下发酵68～75h，得到的发酵液中含重组S1蛋白。
本发明提供了所述重组载体、所述重组乳酸杆菌表达***、所述重组S1蛋白或所述制备方法制备的发酵液在制备防治新冠病毒感染的药物中的应用。
本发明提供了一种防治新冠病毒感染的粘膜阻断剂，包括所述重组S1蛋白或所述制备方法得到的发酵液。
优选的，所述粘膜阻断剂包括口、鼻喷剂或滴眼液。
本发明提供的用于防治新冠病毒感染的重组载体和重组乳酸杆菌表 达***，是通过密码子优化新冠病毒S1蛋白中的RBD区域蛋白的编码序列***载体中，再利用乳酸杆菌安全高效地表达，得到重组表达S1蛋白，使其能作为粘膜感染阻断喷剂，与人体呼吸道、眼结膜、消化道及其它粘膜表面特异性受体(ACE2)结合，从而新冠病毒与机体黏膜表面结合的位点被S1蛋白优先占位封闭，进而高效阻断和干扰新冠病毒与人粘膜特异性受体(ACE2)结合，从而预防新冠肺炎发生，减少新冠肺炎感染人群数量，快速阻遏其全球漫延。
本发明提供的一种防治新冠病毒感染的粘膜阻断剂。由于SARS-CoV-2的传播方式主要是以呼吸道飞沫传播为主，极少数是通过气溶胶、粪便等传播，切断新冠病毒表面的S1蛋白与呼吸道、眼结膜、消化道及其它粘膜表面ACE2受体的结合，阻断病毒感染路径就可有效降低新冠病毒感染，所述粘膜阻断剂具有以下特点：
相比于疫苗，重组新冠病毒S1蛋白，只包含病毒功能性蛋白，无病毒感染基因存在，不会导致病毒变异，且是外用制剂，安全性高；
重组S1蛋白分泌到细菌的细胞壁上，属于可溶性的表达产物，同时乳酸杆菌是人体内常见的益生菌，被公认为最安全的食品级微生物，用乳酸菌表达的S1蛋白无需提取纯化，省去了常规原核表达***表达后的复杂的纯化过程和复性过程，大大降低了粘膜阻断剂的生产成本，利于快速工业化生产；
所述粘膜阻断剂可多次重复使用，不仅无毒、无害，而且可诱导粘膜免疫，进一步减少病毒感染风险，可用于专业医务人员上岗前使用，或乘坐密闭交通工具和办公场所使用，或社区、家庭预防感染使用。
附图说明
图1为重组载体pVE5523-SARS-CoV-2-S1载体图谱；
图2为重组载体的测序结果与***的核苷酸片段进行序列比对结果；
图3为重组载体pVE5523-SARS-CoV-2-S1的荧光定量PCR扩增曲线；
图4-1为重组S1蛋白Western Blot鉴定；其中M：Marker 26616；1：重组乳酸杆菌上清20μl；2：重组乳酸杆菌沉淀20μl；
图4-2为重组S1蛋白考马斯亮蓝染色分析结果；其中，M:Marker 26616；
1：重组乳酸杆菌上清20μl；2：重组乳酸杆菌沉淀20μl；3：BCA标准品8.8ng/μl；4：BCA标准品17.59ng/μl；5：BCA标准品35.19ng/μl；6：BCA标准品70.375ng/μl；7：BCA标准品140.75ng/μl；8：BCA标准品281.5ng/μl；9：BCA标准品563ng/μl。
具体实施方式
本发明提供了一种用于防治新冠病毒感染的重组载体，包括密码子优化的新冠病毒S1蛋白中RBD区域蛋白的编码序列；所述编码序列的核苷酸序列如序列表SEQ ID NO:1所示。
在本发明中，所述重组载体的基础载体优选包括乳酸杆菌分泌型表达载体。本发明对所述乳酸杆菌分泌型表达载体的种类没有特殊限制，采用本领域所熟知的乳酸杆菌分泌型表达载体即可。为了举例说明重组载体的种类，在本发明实施例中，所述乳酸杆菌分泌型表达载体为pVE5523。所述编码序列***pVE5523中的多克隆位点优选为SalI和EcoRV。
在本发明中，所述编码序列是新冠病毒S1蛋白中RBD区域蛋白按照乳酸杆菌密码子偏好进行优化得到的，同时进行两次重复，有利于提高蛋白表达量，其核苷酸序列如序列表中SEQ ID NO:1所示(gtcgacatgtacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagatattgcttaaacaagatgcggtcgacgtcgacatgtacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacaccatgatatc)，便于后续***到乳 酸杆菌分泌型表达载体中高效表达。以新冠病毒S1蛋白中RBD区域蛋白作为人体粘膜表面ACE2(Angiotensin Converting Enzyme 2，血管紧张素转换酶2)受体结合的新冠病毒特异性蛋白，能够有效竞争新冠病毒的结合位点，同时使用安全，防止新冠病毒的感染。
在本发明中，所述重组载体的构建方法，优选包括以下步骤：
A.将编码序列和基础载体采用相同的限制性内切酶酶切，分别得到带有粘性末端的编码序列和基础载体；
B.将所述带有粘性末端的编码序列和基础载体连接，筛选，得到重组载体。
在本发明中，所述限制性内切酶酶切的方法没有特殊限制，采用本领域所熟知的酶切方法即可。当所述基础载体为pVE5523时，所述限制性内切酶的种类优选为SalI和EcoRV，重组载体的图谱见图1。
在本发明中，所述连接的方法优选采用T4连接酶连接。
在本发明中，所述筛选方法优选将重组载体导入大肠杆菌中，经培养后提取质粒，将质粒进行测序、比对，与***的编码序列一致的重组载体为重组载体pVE5523-SARS-CoV-2-S1(序列为SEQ ID NO:2)，用于后续操作。
本发明提供了一种包含所述重组载体的重组乳酸杆菌表达***。
在本发明中，所述重组乳酸杆菌表达***中乳酸杆菌的种类没有特殊限制，采用本领域所熟知的乳酸杆菌即可。为了举例说明重组乳酸杆菌表达***的组成，本发明实施例中，所述乳酸杆菌的种类采用ATCC393干酪乳杆菌进行实验。
在本发明中，所述重组乳酸杆菌表达***的制备方法，优选将所述重组载体转化乳酸杆菌感受态中，筛选，得到重组乳酸杆菌表达***。所述转化的方法没有特殊限制，采用本领域所熟知的转化方法即可。在本发明实施例中，所述转化采用电转化方法。
所述筛选方法优选将大肠杆菌涂布在含红霉素的MRS固体培养板上，静置培养；挑取阳性克隆，接种到含红霉素的MRS液体培养基中静置培养；提取重组质粒质粒，采用荧光定量PCR进行鉴定。用于荧光定 量PCR的引物为SEQ ID NO:3和SEQ ID NO:4。荧光定量PCR反应的扩增产物的核苷酸序列为SEQ ID NO:5。阳性重组质粒具有典型的扩增曲线，ATCC393感受态对照没有扩增曲线，表明pVE5523-SARS-CoV-2-S1质粒成功转化到了ATCC393感受态细胞中。
本发明提供了由所述重组乳酸杆菌表达***中分泌表达的重组S1蛋白。
本发明提供了所述重组S1蛋白的制备方法，包括以下步骤：
将所述重组乳酸杆菌表达***在无菌环境下接种至乳酸杆菌液体培养基，在28～35℃条件下发酵68～75h，将得到的发酵液在5000～6000rpm离心15～30min，取上清液得到重组S1蛋白。
本发明对所述乳酸杆菌液体培养基的种类没有特殊限制，采用本领域所熟知的培养乳酸杆菌的培养基即可。在本发明实施例中，所述乳酸杆菌液体培养基为MRS液体培养。所述重组乳酸杆菌表达***的培养温度为34～36℃，所述发酵的时间优选为72h。所述离心的温度优选为4℃。
在本发明中，经检测，所述重组S1蛋白的质量占乳酸杆菌总蛋白质量的10％。
本发明提供了所述重组载体、所述重组乳酸杆菌表达***、所述重组S1蛋白或所述制备方法制备的发酵液在制备防治新冠病毒感染的药物中的应用。
本发明提供了一种防治新冠病毒感染的粘膜阻断剂，包括所述重组S1蛋白或所述制备方法制备得到的重组S1蛋白。所述粘膜阻断剂优选包括口、鼻喷剂和滴眼液。在粘膜阻断剂中，所述S1蛋白浓度范围优选为10ng/ml～50ng/ml，更优选为20～40ng/ml，最优选为30ng/ml。所述粘膜阻断剂的人用剂量优选为0.2ml/人/次，每天2次，其中喷鼻剂量优选为0.1ml/人/次，滴眼剂量优选为0.1ml/人/次。本发明对所述口、鼻喷剂和/或滴眼液的制备方法没有特殊限制，采用本领域所熟知的制备方法即可。
下面结合实施例对本发明提供的一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
密码子优化的新型冠状病毒SARS-CoV-2的S1基因(SARS-CoV-2-S1)
新型冠状病毒SARS-CoV-2的S1蛋白是病毒与宿主表面ACE2受体结合、介导病毒进入宿主细胞的关键表面蛋白。筛选SARS-CoV-2的S1蛋白中与ACE2特异性结合区域的基因序列，并对序列进行乳酸杆菌密码子优化，改造后序列由南京金斯瑞生物科技有限公司进行人工合成，SARS-CoV-2-S的核苷酸序列如SEQ ID NO:1所示。
实施例2
重组表达载体pVE5523-SARS-CoV-2-S1的构建方法
1材料与方法
1.1材料及来源
限制性内切酶SalI、EcoRV均购自NEB公司；Taq酶、dNTP、DNA Marker DL2000、DL15000、Agarose Gel DNA Purification Kit、Mini BEST Plasmid Purification Kit购自大连宝生物公司。克隆载体pVE5523由南京金斯瑞生物科技有限公司提供。
1.2试验方法
克隆载体pVE5523以SalI/EcoRV双酶切后的小片段，与用同样双酶切的SARS-CoV-2-S1基因片段连接，经连接产物转化入大肠杆菌中，经培养，采用试剂盒提取质粒，将提取的质粒送至南京金斯瑞生物科技有限公司进行测序验证。
2试验结果
重组质粒测序结果：重组质粒经基因测序后与***的S1基因片段(单个重复序列)进行序列比对(见图2)，测序结果与预期相符，说明合成的SARS-CoV-2-S1基因片段成功***到乳酸杆菌分泌型载体pVE5523中，重组质粒构建成功，阳性质粒命名为pVE5523-SARS-CoV-2-S1。
实施例3
重组乳酸杆菌表达***的制备及检测方法
1材料与方法
1.1材料及来源
红霉素(Emr)购自拜尔迪生物技术有限公司。
1.2试验方法
将pVE5523-SARS-CoV-2-S1重组载体经电转化进入乳酸杆菌ATCC393感受态中，电转化后的乳酸杆菌ATCC393涂布于含5μg/ml红霉素的MRS固体培养板上，在30℃培养箱中培养72h，将平板上的菌落挑斑后接种到含5μg/ml红霉素的MRS液体培养基中，30℃静置培养72h；提取细菌中的质粒，以ATCC393感受态为对照，采用荧光定量PCR进行鉴定，鉴定引物及扩增序列如下：
上游引物：ccaaccatacagagtagtagta(SEQ ID NO:3)
下游引物：gttagactcagtaagaacacct(SEQ ID NO:4)；
荧光定量PCR的反应体系共计25μl：
Figure PCTCN2021086927-appb-000001
荧光定量PCR的反应程序：预变性95℃5min；循环反应95℃10s，60℃30s，40循环；融解曲线95℃15s，60℃60s，95℃15s；按以上参数在荧光定量PCR仪上测试。
扩增序列如下：
Figure PCTCN2021086927-appb-000002
2试验结果
采用荧光定量PCR对扩增的重组质粒进行检测，结果如图3所示。由扩增曲线可以看出，阳性重组质粒具有典型的扩增曲线，CT值在19～22范围内变化，而ATCC393感受态对照没有扩增曲线。结果表明pVE5523-SARS-CoV-2-S1质粒成功转化到了ATCC393感受态细胞中。
实施例4
重组乳酸杆菌表达***的培养方法及重组S1蛋白的分离
将重组乳酸杆菌表达***以1％比例接种乳酸杆菌MRS液体培养基，35℃，72h后收获发酵液。
重组S1蛋白的分离及检测：将上述收获的发酵液在4℃，8000rpm条件下离心15min，收获上清液。将上清液分别进行4℃透析和冷冻干燥以50倍浓缩蛋白，浓缩后加入1×SDS凝胶加样缓冲液(含DTT)，充分混匀，煮沸10min，1000g离心10min，取上清10μl上样进行SDS-PAGE电泳分析。
重组S1蛋白含量检测：乳酸杆菌平板表面散布法活菌技术，然后计算，S1蛋白含量占乳酸杆菌总蛋白含量的10％。
重组S1蛋白定性分析：利用S1蛋白特异性抗体(北京义翘神州SARS-Cov-2 Spike Neutralizing antibody,Cat:40592-R001)对聚丙烯酰胺凝胶进行Western blot分析。
结果如图4-1所示，重组S1蛋白不能产生明显的条带，说明重组S1蛋白在上清液中不表达或表达含量较低。
进一步对重组S1蛋白定量分析：将聚丙烯酰胺凝胶进行考马斯亮蓝染色脱色，脱色后的凝胶用蛋白凝胶成像仪进行拍摄。
结果如图4-2所示，通过结合Western blot图中目的蛋白条带，在脱色后的聚丙烯酰胺凝胶中上清样本未发现相应条带。进一步证实重组S1蛋白在上清液中不表达或表达含量较低。
实施例5
将实施例4制备的重组S1蛋白稀释至10ng/ml的溶液。将稀释S1蛋白溶液以喷剂的形式对SPF新西兰兔和SD大鼠给药，同时设置阴性对照组A喷雾同体积的生理盐水、对照组B喷雾同体积的MRS培养液，每组新西兰兔子和SD大鼠各4只，观察2周，24只动物均没有出现体温异常、过敏以及呼吸道感染等异常不良现象。这表明本发明制备的重组S1蛋白安全、无副作用。
实施例6
采用实施例4的方法进行重组乳酸杆菌表达***发酵，收获的发酵液在4℃，8000rpm条件下离心15min，收获菌体。向菌体中加入500μl细胞裂解液重悬菌体并使用超声破碎仪进行裂解；在菌体裂解液中加入1ⅹSDS凝胶加样缓冲液(含DTT)，同时将标准品蛋白(已知浓度)进行倍比稀释，加入1ⅹSDS凝胶加样缓冲液(含DTT)；将上述所以蛋白样品充分混匀，煮沸10min，1000g离心10min，取上清20μl上样进行SDS-PAGE电 泳，分别做考马斯亮蓝染色和Western blot分析。
重组S1蛋白定性分析：利用S1蛋白特异性抗体(北京义翘神州SARS-Cov-2 Spike Neutralizing antibody,Cat:40592-R001)对聚丙烯酰胺凝胶进行Western blot分析。
结果如图4-1所示，重组S1蛋白在重组乳酸杆菌中成功表达，蛋白主要存在于重组乳酸杆菌菌体蛋白中。
本实施例对重组S1蛋白定量分析：将聚丙烯酰胺凝胶进行考马斯亮蓝染色脱色，脱色后的凝胶用蛋白凝胶成像仪进行拍摄。
结果如图4-2所示，通过结合Western blot图中目的蛋白条带，在脱色后的聚丙烯酰胺凝胶中对应位置找到目的条带。
利用ImageJ软件对聚丙烯酰胺凝胶中的蛋白条带进行灰度分析，以标准品蛋白浓度为参照，对重组S1蛋白进行定量分析，结果如下表1所示，按照公式A计算重组S1蛋白在重组乳酸杆菌的表达量。
表1蛋白灰度分析
样品 标准品1 标准品2 标准品3 标准品4 标准品5 标准品6 目的条带
灰度值 1.464 3.916 7.028 15.16 25.346 50.192 15.19
浓度(ng/μl) 17.6 35.2 70.4 140.75 281.5 563 141.03
重组S1蛋白浓度(μg/ml)＝目的条带浓度×500μl÷20ml  式A
经计算所述重组S1蛋白的浓度为3.53μg/ml，说明所述重组S1蛋白实现在乳酸杆菌表达***中高效表达。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。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，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种用于防治新冠病毒感染的重组载体，其特征在于，包括密码子优化的新冠病毒S1蛋白中RBD区域蛋白的编码序列；所述编码序列的核苷酸序列如序列表SEQ ID NO：1所示。
  2. 根据权利要求1所述重组载体，其特征在于，所述重组载体的基础载体包括乳酸杆菌分泌型表达载体。
  3. 根据权利要求2所述重组载体，其特征在于，所述乳酸杆菌分泌型表达载体包括pVE5523。
  4. 根据权利要求3所述重组载体，其特征在于，所述编码序列***pVE5523中的多克隆位点为SalI和EcoRV。
  5. 一种包含权利要求1～4任意一项所述重组载体的重组乳酸杆菌表达***。
  6. 权利要求5所述重组乳酸杆菌表达***中分泌表达的重组S1蛋白。
  7. 权利要求6所述重组S1蛋白的制备方法，其特征在于，包括以下步骤：
    将权利要求5所述重组乳酸杆菌表达***在无菌环境下接种至乳酸杆菌液体培养基中，在28～35℃条件下发酵68～75h，得到的发酵液中含重组S1蛋白。
  8. 权利要求1～4任意一项所述重组载体、权利要求5所述重组乳酸杆菌表达***、权利要求6所述重组S1蛋白或权利要求7所述制备方法制备的发酵液在制备防治新冠病毒感染的药物中的应用。
  9. 一种防治新冠病毒感染的粘膜阻断剂，其特征在于，包括权利要求6所述重组S1蛋白或权利要求7所述制备方法得到的发酵液。
  10. 根据权利要求9所述粘膜阻断剂，其特征在于，所述粘膜阻断剂包括口、鼻喷剂或滴眼液。
PCT/CN2021/086927 2020-04-14 2021-04-13 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用 WO2021208913A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010288793 2020-04-14
CN202010288793.7 2020-04-14
CN202110267711.5 2021-03-12
CN202110267711.5A CN112877351A (zh) 2020-04-14 2021-03-12 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用

Publications (1)

Publication Number Publication Date
WO2021208913A1 true WO2021208913A1 (zh) 2021-10-21

Family

ID=76042453

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/086927 WO2021208913A1 (zh) 2020-04-14 2021-04-13 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用

Country Status (2)

Country Link
CN (1) CN112877351A (zh)
WO (1) WO2021208913A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113073106B (zh) * 2021-06-04 2021-09-07 北京华芢生物技术有限公司 新型冠状病毒b.1.525尼日利亚突变株rbd的基因及其应用
CN113186204B (zh) * 2021-06-11 2021-10-15 北京华芢生物技术有限公司 新型冠状病毒巴西株p．1突变株rbd的基因及其应用
CN115851727A (zh) * 2022-11-14 2023-03-28 江苏中方基因生物医学科技有限公司 一种靶向干扰新冠病毒s蛋白的小核酸rna干扰及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798844A (zh) * 2003-06-04 2006-07-05 生物领先公司 Sars病毒抗原的细胞表面表达载体和用该载体转化的微生物
KR102076917B1 (ko) * 2018-10-25 2020-02-13 연세대학교 산학협력단 자가조립 나노 입자를 제조하기 위한 유전자 재조합 발현 벡터 시스템 및 이를 이용하는 방법
CN110951756A (zh) * 2020-02-23 2020-04-03 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100567477C (zh) * 2007-01-09 2009-12-09 东北农业大学 表达猪传染性胃肠炎病毒s蛋白的重组乳酸乳球菌及制法
CN104988107B (zh) * 2015-05-19 2018-06-08 中国农业科学院兰州兽医研究所 一种高效表达***病毒抗原基因的重组乳酸杆菌及其制备方法和应用
CN111944837B (zh) * 2020-03-30 2023-09-08 河南师范大学 一种表达covid-19抗原的表达载体及基因工程乳酸菌口服疫苗的构建方法
CN111518740A (zh) * 2020-05-18 2020-08-11 军事科学院军事医学研究院军事兽医研究所 一种表达SARS-CoV-2 S基因的重组植物乳酸菌及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1798844A (zh) * 2003-06-04 2006-07-05 生物领先公司 Sars病毒抗原的细胞表面表达载体和用该载体转化的微生物
KR102076917B1 (ko) * 2018-10-25 2020-02-13 연세대학교 산학협력단 자가조립 나노 입자를 제조하기 위한 유전자 재조합 발현 벡터 시스템 및 이를 이용하는 방법
CN110951756A (zh) * 2020-02-23 2020-04-03 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LUO BO , DUAN SUNQUN , ZHENG XIAOLI , MAO YINGYU: "Construction of Expression Vector of Human Papillomavirus 16 L1 Gene in Lactobacillus", JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES), vol. 48, no. 1, 20 January 2013 (2013-01-20), pages 50 - 52, XP055858230 *
TANG LI-JIE , OU DI , WANG RONG-JUN , XU YI-GANG , ZHONG TAO , LI YI-JING: "Expression of Porcine Transmissible Gastroenteritis Coronavirus Spike Glycoprotein in Lactobacillus", BIOTECHNOLOGY, vol. 17, no. 6, 31 December 2007 (2007-12-31), pages 4 - 8, XP055858226, ISSN: 1004-311X, DOI: 10.16519/j.cnki.1004-311x.2007.06.029 *

Also Published As

Publication number Publication date
CN112877351A (zh) 2021-06-01

Similar Documents

Publication Publication Date Title
WO2021208913A1 (zh) 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达***及其应用
EP3012322B1 (en) Porcine pseudorabies virus gene deletion strain, vaccine composition, and preparation method therefor and application thereof
KR20220132588A (ko) 탈최적화된 SARS-CoV-2 및 이의 방법 및 용도
EP4056687A1 (en) F-genotype mumps virus attenuated strain and construction method therefor and application thereof
CN107098974B (zh) 一种融合蛋白及其应用
CN107058634A (zh) 鸭腺病毒2型和鸭腺病毒a型双重pcr检测引物及试剂盒
CN113943714B (zh) 一种猫杯状病毒毒株及其应用
WO2020238458A1 (zh) 用于表达e2蛋白的细胞株及其应用，e2蛋白及其应用
WO2021217959A1 (zh) 一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用
CN114958783B (zh) 一种三基因缺失的猫疱疹病毒i型重组病毒、猫传染性鼻气管炎活疫苗以及制备方法
CN110760511B (zh) 一种用于治疗杜氏肌营养不良症的gRNA、表达载体、CRISPR-Cas9***
CN116200347A (zh) 一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用
CN104059889A (zh) 伪狂犬病病毒变异株双基因缺失毒株及其构建方法和应用
CN113230394B (zh) 一种用于牛病毒性腹泻的rna疫苗及其构建方法
CN106939320A (zh) 一种伪狂犬病毒js‑2012株感染性克隆质粒、构建方法与应用
CN110016457B (zh) 一株重组细粒棘球蚴Eg95基因的粗糙型布鲁氏菌及其疫苗生产方法
Zhang et al. Recombinant hemagglutinin protein and DNA-RNA-combined nucleic acid vaccines harbored by yeast elicit protective immunity against H9N2 avian influenza infection
CN109867713B (zh) 一种犬瘟热基因工程亚单位疫苗
CN109022373B (zh) 鸭瘟病毒ul56基因3’端缺失和lorf5基因缺失突变株及其构建方法与应用
JP2021535759A (ja) 改変ｐｅｄｖスパイクタンパク質
WO2022122036A1 (zh) 一种SARS-CoV-2病毒的免疫原、药物组合物及其应用
CN112402598A (zh) 针对鸭疫里默氏杆菌感染引起的通用型亚单位疫苗
CN110013547B (zh) 一株重组小反刍兽疫病毒h基因的粗糙型布鲁氏菌及其疫苗生产方法
CN109939225B (zh) 一株重组鹦鹉热衣原体外膜蛋白momp基因的粗糙型布鲁氏菌及其疫苗生产方法
CN103820414B (zh) 变形链球菌重组亚单位基因工程疫苗候选抗原Glu的纯化方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21788212

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21788212

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC