CN114404564A - Application of lipopeptide compound in resisting measles virus - Google Patents
Application of lipopeptide compound in resisting measles virus Download PDFInfo
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- CN114404564A CN114404564A CN202210005472.0A CN202210005472A CN114404564A CN 114404564 A CN114404564 A CN 114404564A CN 202210005472 A CN202210005472 A CN 202210005472A CN 114404564 A CN114404564 A CN 114404564A
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Abstract
The invention belongs to the field of life science, and particularly relates to application of a high-efficiency lipopeptide compound in resisting measles virus. The lipopeptide compound is a crude lipopeptide extract of lipopeptide epilin or/and sodium salt; the crude lipopeptide extract of the lipopeptide biological epilin or/and the sodium salt is a mixture containing two or three of C13, C14 and C15 fat side chains; and the carboxymethyl position thereof may be H+Or Na+Or mixtures thereof. The lipopeptide compound of the invention is mainly a bacillus subtilis substituteThe lipopeptide or the sodium salt thereof generated by the metabolism has strong activity of resisting the measles virus, and can be widely used for the aspects of resisting the measles virus, such as skin external medicaments, protective agents, cleaning agents, detergents, skin care products, personal care products, sprays, in-vitro nano administration, clothes washing, medical product washing, preventing adsorption of the measles virus on furniture or solid surfaces, industrial washing and the like, inhibiting or killing the virus and the like.
Description
Technical Field
The invention belongs to the field of life science, and particularly relates to application of a high-efficiency lipopeptide compound in resisting measles virus.
Background
Lipopeptide biosurfactants are bioactive substances produced by gram-positive bacillus and are one of the surfactant types with the strongest surface activity reported so far. The compound is a cyclic peptide formed by connecting 1 long-chain hydrophobic alkyl amino acid with other 7-10 amino acids in an amido bond or lactone form through carboxyl and amino of the amino acid, and contains an anionic biosurfactant with free carboxyl. Bonmatin and the like determine the three-dimensional structure of Surfactin by using high-resolution nuclear magnetic resonance hydrogen spectroscopy and a molecular dynamics technology. The Surfactin is saddle-shaped in aqueous solution, and the amino acid residues L-Leu are positioned at the same side2And D-L eu6Facing each other, 2 acidic amino acids L-Glu1And L-Asp5A small polar domain is formed, and the hydrophilic ability is certain. On the other side, amino acid residuesD-Leu3、L-Val4And L-Leu7And fatty acid chains constitute the major hydrophobic domain. The amphiphilic chemical structure enables Surfactin to show good surface activity and stability. In recent years, lipopeptides have been widely paid attention and studied, and are widely used in the fields of washing, textile, printing and dyeing, paper making, cosmetics, oil extraction, agriculture, and the like because of their excellent surface activity, low skin irritation, and biodegradability.
The World Health Organization (WHO) has listed measles as the next infectious disease to be eliminated following the global elimination of smallpox and the imminent elimination of polio. The core of the measles virus is single negative strand RNA, and the measles virus is wrapped by nucleocapsid which is spirally symmetrical and HAs two spikes on the surface, namely HA and Hemolysin (HL), and the components of the two spikes are glycoproteins. The main current measure for the prevention of measles is the live attenuated measles vaccine, which, although proven to be a highly immunogenic, safe and effective vaccine, presents temporary skin eruptions in about 2% of recipients (WHO's opinion on measles vaccine [ J ]. biological products for foreign medical preventive diagnostics, 2005,28(1): 8-12).
To date, no report has been found on the effect of using lipopeptide compounds for the inhibition or killing of measles virus. The invention fills the international blank of the substances in the prevention and treatment of measles, can be effectively used for reducing the infectivity of measles virus, helps the nation and even the world to overcome the difficulty and protects the life safety of people.
Disclosure of Invention
The invention aims to provide application of a high-efficiency lipopeptide compound in resisting measles virus.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the use of a lipopeptide compound against measles virus, said lipopeptide compound being a crude lipopeptide extract of the epilipin or/and sodium salt thereof;
the crude lipopeptide extract of the lipopeptide biological epilin or/and the sodium salt is a mixture containing two or three of C13, C14 and C15 fat side chains; and the carboxymethyl position thereof may be H+Or Na+Or mixtures thereof.
Obtaining a crude lipopeptide extract of the lipopeptide epilin or/and sodium salt: obtaining a genome which takes bacillus subtilis BITAFS-BS2020 as a starting strain to extract BS02 from an expression product of the genetic engineering bacteria, then obtaining a recombinant bacteria BITAFS-BS2020 by the genetic engineering technology together with pHT01-p43 plasmid, fermenting and culturing the obtained recombinant bacteria, and purifying after culture to obtain the lipopeptide crude extract of lipopeptide epiletin or/and sodium salt.
The recombinant strain BITAFS-BS2020 takes Bacillus subtilis BITAFS-BS2020 as an original strain, extracts a genome (strain conserved region) of BS02, adopts a primer (SEQ ID NO.1-2) and takes a BS02 genome as a template to perform PCR of srfa gene to obtain BIT-Sr (SEQ ID NO. 3);
the promoter PgroE of plasmid pHT01 was then replaced with p43(SEQ ID NO. 4); carrying out double enzyme digestion on BIT-Sr obtained by PCR amplification and pHT01-p43 respectively by using BamH1 and Xho1, recovering enzyme digestion products, and connecting the enzyme digestion products by using a T4 ligase system to form a recombinant plasmid pHT 01-AFS;
the obtained recombinant plasmid pHT01-AFS is transferred into a B.subtilis 168 strain (ATCC 23857), and after the transfer, double-antibody screening of ampicillin sodium and chloramphenicol is carried out, and finally, a recombinant bacterium BITAFS-BS2020 is obtained.
The construction method of the pHT01-P43 comprises the steps of carrying out BamH I and Kpn I double enzyme digestion on P43 and pHT01, recycling enzyme digestion products, and connecting the recycled products to obtain pHT 01-P43.
The recombinant strain BITAFS-BS2020 is subjected to fermentation culture, namely the obtained recombinant strain is inoculated into a liquid fermentation culture medium according to the inoculation amount of 0.1 percent, and the temperature is 32-37 ℃ and the pH value is 6.7-7.5 under the aerobic condition; performing fermentation culture for 21-32 h;
the liquid fermentation culture medium comprises (by weight percentage) (g/L) 5-20 parts of soybean flour and/or soybean protein, 0.5-2 parts of yeast extract, 10-40 parts of cane sugar and/or maltose, 0.1-8 parts of K2HPO 41, 0.1-1 part of magnesium sulfate heptahydrate, 0.005-0.04 part of anhydrous calcium chloride, 0.005-0.04 part of ferrous sulfate, 0.01-0.1 part of manganese chloride and the balance of water.
The obtained fermentation liquor is further refined by using an acid precipitation method, and then purified by using polar or non-polar ion exchange resin after refining to obtain a lipopeptide crude extract of lipopeptide epi-active elements or/and sodium salts, wherein the purity of the lipopeptide epi-active elements or/and sodium salts containing C13, C14 and C15 fat side chains in the crude extract is more than 95%.
The refining is realized by an acid precipitation method, the fermentation liquor obtained by shake flask culture is centrifuged at 8000rpm for 10min to obtain supernatant, the precipitate is discarded, the pH is adjusted to 2 by 5mol/l hydrochloric acid, the supernatant is placed in a refrigerator at 4 ℃ overnight, the acid precipitation solution is centrifuged at 8000rpm for 20min on the next day, the precipitate is collected, and the purity of the lipopeptide crude extract reaches about 70% by HPLC.
And (3) further purifying the refined product, namely separating the refined product by using one of nonpolar ion exchange resins D101, X-7 and X-5 in a pre-column manner, eluting the refined product by using 50-100% ethanol as a mobile phase at an elution flow rate of 2ml/min, collecting eluted components, and detecting that the lipopeptide epi-activin or/and sodium salt of the fat side chains containing C13, C14 and C15 can reach the purity of more than 95%.
The lipopeptide compound can be used for preparing an antiviral medicament or preparation for resisting measles virus MeV-AH18-1/P6 strain.
The test for the inactivation/inhibition of measles virus of the lipopeptide compound was evaluated by the viral disease prevention control of the chinese center for disease prevention control.
Wherein the crude extract of lipopeptide containing lipopeptide epiactivin or/and sodium salt described in the structure of formula 1 has a concentration of 150-300 μ M in water phase, and can kill measles virus 100% after 15min or more in the environment of 4-37 ℃.
The antiviral agent is in the form of liquid, lotion, cream, paste, solid or spray for external protection against measles virus infection; alternatively, the antiviral agent for personal care products is water or milk or spray for cleaning teeth, face, skin, bathing.
The invention has the beneficial effects that:
1. the invention provides application of a specific lipopeptide-containing compound in inhibiting measles virus, and provides a novel medicinal composition and a novel method for preventing and inhibiting the measles virus.
2. The invention is expected to simplify the differential treatment measures for individual characteristics of measles patients in clinic, and can effectively kill and protect measles virus by adopting the expression forms of environmental disinfectant, mask internal coating, ointment, washing and protecting products and the like as a protective article for measles virus.
The compound 1 can be used for preparing a series of anti-measles protective products, wherein the anti-measles protective products comprise effective components of skin external drugs for inhibiting measles virus, skin protective products, personal care products, cosmetics, sprays and in-vitro nano drug delivery systems, and have wide application prospects in the field of anti-measles virus.
Drawings
FIG. 1 BIT-BS02 genome electropherogram
FIG. 2 PCR obtaining target fragment BIT-Sr electropherogram
FIG. 3 electropherogram of enzyme digestion product recovery
FIG. 4 pHT01-srfA plasmid construction map
FIG. 5 recombinant transfer-coated blood agar plates to form lysoloops
FIG. 6 shows the result of sequencing and identification of recombinant colonies on blood plates (EMBOSS _002 is BIT-Sr)
FIG. 7P 43 promoter amplification product
FIG. 8 shows the results of the lipopeptide MS identification, the lipopeptide MS identification (a), and the lipopeptide compound C13/C14/C15 MS identification (b);
FIG. 9 measurement of lipopeptide content by HPLC method.
Detailed Description
The embodiments of the present invention are described below by specific examples, which are only for illustrating the present invention and are not intended to limit the scope of the present invention. Other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein, and various changes and modifications may be made thereto, and equivalents may be made thereto, which fall within the scope of the appended claims.
Example 1
Obtaining crude lipopeptide extract containing lipopeptide epi-active elements and/or sodium salts described in the structure of formula 1:
1) construction of the genetic recombinant Bacillus subtilis BITAFS-BS2020 for high lipopeptide yield:
the Bacillus subtilis (named as BITAFS-BS2020) is a genetically engineered Bacillus subtilis BIF-S, and the construction method thereof is as follows: extracting a genome of BS02 (with a strain preservation number of CGMCC No.2947) (extracting the genome by using a rapid extraction kit of genome DNA of a raw bacterium, wherein a specific method is shown in a kit use instruction manual) (shown in figure 1, a band shown by the genome DNA is an extracted BS02 genome), designing a primer (SEQ ID No.1-2) according to a sequence of a lipopeptide synthesis key gene srfa in NCBI, and carrying out PCR of the srfa gene by using a BS02 genome as a template to obtain BIT-Sr (SEQ ID No.3) (shown in figure 2, a band shown by a PCR result is BIT-Sr). The promoter PgroE of plasmid pHT01 (purchased from Kohler biosciences, Ltd., product number kl-zl-0940) was replaced with p43(SEQ ID NO. 4). BIT-Sr and pHT01-p43 were each double-digested with BamH1 and Xho1, and the products were recovered with a restriction recovery kit of TAKARA (see FIG. 3, sequentially from left to right, pHT01 after digestion, pHT01 without digestion, the restriction products of BIT-Sr PCR products, and BIT-Sr PCR products without digestion), and the restriction products were ligated with a T4 ligase system (see FIG. 4, pHT01-AFS plasmid map after ligation) to form a recombinant plasmid pHT01-AFS, which was transferred to B.subtilis 168 strain (ATCC 23857), spread on Columbia blood agar medium (purchased from Kahlin Co., Ltd.), blood plates containing ampicillin sodium (final concentration 25. mu.g/ml), chloramphenicol (final concentration 25. mu.g/ml) resistance, and cultured with 37 ℃ transformant (see FIG. 5, and (3) selecting a strain with a lysoloop larger than 1mm according to the growth condition of the transformant on a blood plate), selecting a colony with a large clearing loop for sequencing identification (as shown in figure 6, EMBOSS-001 is a base sequence of BS02-srfa, EMBOSS-002 is a base sequence of BIT-Sr), and comparing the base sequences to obtain a recombinant strain BITAFS-BS 2020.
The pHT01-P43 construction method comprises the steps of obtaining a P43 sequence (SEQ ID NO.4) according to NCBI, carrying out chemical synthesis of the sequence, designing a primer (SEQ ID NO.5-6) by taking P43 as a template, amplifying P43 (shown in figure 7, 1 is a P43 PCR product, and M is a DNA marker) in a PCR mode, and identifying that the size of P43 is correct. Carrying out BamH I and Kpn I double enzyme digestion on P43 and pHT01, recovering the enzyme digestion product, and connecting the recovered product to obtain pHT 01-P43.
SrfA-F CGGGATCCATGGAAATAACTTTTTACCCTTTAACG(SEQ ID NO.1)
SrfA-R CCGCTCGAGTTGACCGCTCGCATAAGACAG(SEQ ID NO.2)
ATGGAAATAACTTTTTACCCTTTAACGGATGCACAAAAACGAATTTGGTACACAGAAAAATTTTATCCTCACACGAGCATTTCAAATCTTGCGGGGATTGGTAAGCTGGTTTCAGCTGATGCGATTGATTATGTGCTTGTTGAGCAGGCGATTCAAGAGTTTATTCGCAGAAATGACGCCATGCGCCTTCGGTTGCGGCTAGATGAAAACGGGGAGCCTGTTCAATATATTAGCGAGTATCGGCCTGTTGATATAAAACATACTGACACTACTGAAGATCCGAATGCGATAGAGTTTATTTCACAATGGAGCCGGGAGGAAACGAAGAAACCTTTGCCGCTATACGATTGTGATTTGTTCCGTTTTTCCTTGTTCACCATAAAGGAAAATGAAGTGTGGTTTTACGCAAATGTTCATCACGTGATTTCTGATGGTATCTCCATGAATATTCTCGGGAATGCGATCATGCACATTTATTTAGAATTAGCCAGCGGCTCAGAGACAAAAGAAGGAATCTCGCATTCATTTATCGATCATGTTTTATCTGAACAGGAATATGCTCAATCGAAGCGGTTTGAAAAGGACAAGGCGTTTTGGAACAAACAATTTGAATCGGTGCCTGAACTTGTTTCCTTGAAACGGAATGCATCCGCAGGGGGAAGTTTAGATGCTGAGAGGTTCTCTAAAGATGTGCCTGAAGCGCTTCATCAGCAGATTCTGTCGTTTTGTGAGGCGAATAAAGTCAGTGTTCTTTCGGTATTTCAATCGCTGCTCGCCGCCTATTTGTACAGGGTCAGCGGCCAGAATGATGTTGTGACGGGAACATTTATGGGCAACCGGACAAATGCGAAAGAGAAGCAGATGCTTGGCATGTTTGTTTCTACGGTTCCGCTTCGGACAAACATTGACGGCGGGCAGGCGTTTTCAGAATTTGTCAAAGACCGGATGAAGGATCTGATGAAGACACTTCGCCACCAAAAGTATCCGTATAATCTCCTAATCAACGATTTGCGTGAAACAAAGAGCTCTCTGACCAAGCTGTTCACGGTTTCTCTTGAATATCAAGTGATGCAGTGGCAGAAAGAAGAGGATCTTGCCTTTTTGACTGAGCCGATTTTCAGCGGCAGCGGATTAAATGATGTCTCAATTCATGTAAAGGATCGATGGGATACTGGGAAACTCACCATAGATTTTGATTACCGCACTGATTTATTTTCACGTGAAGAAATCAACATGATTTGTGAGCGCATGATTACCATGCTGGAGAACGCGTTAACGCATCCAGAACATACAATTGATGAATTAACACTGATTTCTGATGCGGAGAAAGAGAAGCTGCTTGCGAGGGCCGGCGGTAAATCTGTGAGCTACCGTAAGGACATGACGATACCAGAGCTGTTCCAAGAAAAGGCTGAACTGCTTTCTGATCATCCAGCGGTTGTATTTGAAGATCGCACATTGTCCTATCGAACGTTACATGAGCAATCTGCACGCATCGCCAATGTGCTGAAACAGAAAGGGGTTGGCCCGGACAGTCCTGTCGCGGTTTTGATTGAACGCTCTGAACGGATGATTACAGCTATCATGGGAATTTTAAAAGCCGGCGGAGCCTATGTGCCGATTGATCCGGGTTTTCCTGCTGAGCGCATTCAATATATTTTGGAGGACTGCGGGGCGGATTTCATCCTGACTGAATCGAAGGTTGCGGCGCCTGAAGCCGATGCTGAGCTGATTGACTTAGATCAGGCGATTGAGGAAGGTGCAGAAGAAAGCCTGAATGCAGATGTGAACGCTCGGAACCTTGCCTACATTATTTACACATCGGGAACAACCGGACGCCCGAAAGGCGTTATGATCGAGCATCGCCAGGTTCATCATTTGGTTGAATCTCTGCAGCAGACGATTTATCAAAGCGGCAGCCAAACCCTGCGGATGGCATTGCTTGCGCCGTTCCACTTTGATGCGTCAGTGAAGCAGATCTTCGCGTCGCTTCTTTTGGGCCAAACCCTTTATATCGTACCGAAGAAAACAGTGACGAACGGGGCCGCCCTTACTGCATATTATCGGAAGAACAGCATTGAGGCGACGGACGGAACACCGGCTCATTTGCAAATGCTGGCAGCAGCAGGCGATTTTGAAGGCCTAAAACTGAAGCACATGCTGATCGGAGGAGAAGGCCTGTCATCTGTTGTTGCGGACAAGCTGCTGAAGCTGTTTAAAGAAGCCGGCACAGCGCCGCGTTTGACTAATGTGTACGGGCCGACTGAAACGTGCGTTGACGCGTCTGTTCATCCGGTTATCCCTGAGAATGCAGTTCAATCAGCGTATGTGCCGATCGGGAAAGCGCTGGGGAATAACCGCTTATATATTTTGGATCAAAAAGGCCGGCTGCAGCCTGAAGGCGTGGCGGGTGAGCTTTATATCGCGGGAGACGGTGTGGGCCGAGGCTATTTACATTTGCCTGAATTAACGGAAGAGAAGTTTTTACAAGATCCATTCGTGCCGGGCGATCGCATGTACCGGACCGGGGACGTGGTGCGCTGGCTTCCAGATGGAACAATCGAATATTTAGGCAGAGAGGATGACCAGGTCAAAGTCCGCGGATACCGGATTGAGCTTGGGGAAATTGAAGCCGTGATTCAGCAGGCGCCAGACGTTGCAAAAGCCGTTGTTTTGGCACGCCCTGACGAACAGGGAAATCTTGAGGTTTGCGCATATGTTGTGCAGAAGCCTGGAAGCGAATTTGCGCCAGCCGGTTTGAGGGAGCATGCGGCCAGACAGCTTCCTGACTATATGGTGCCGGCTTACTTTACAGAAGTGACAGAAATTCCGCTTACACCAAGCGGCAAAGTCGACCGCCGCAAGCTGTTTGCACTAGAGGTGAAGGCTGTCAGCGGCACTGCCTATACAGCGCCGCGAAATGAGACTGAAAAAGCAATCGCAGCCATTTGGCAGGACGTGCTGAACGTTGAGAAGGCGGGGATCTTTGACAATTTCTTTGAAACTGGCGGACATTCATTAAAAGCCATGACCCTTTTAACAAAGATTCATAAGGAAACAGGCATTGAGATTCCGCTTCAATTTTTGTTTGAGCATCCGACGATTACGGCTCTTGCAGAGGAAGCTGATCACAGAGAAAGCAAAGCTTTTGCGGTGATTGAACCTGCTGAAAAACAGGAGCATTACCCGCTTTCATTGGCACAGCAGCGAACATATATCGTCAGCCAGTTCGAGGATGCGGGAGTCGGCTATAACATGCCAGCAGCAGCAATTCTGGAAGGGCCTTTAGATATTCAAAAGCTGGAGCGCGCATTTCAGGGATTAATCCGACGCCACGAGTCATTGAGAACATCATTTGTTCTTGAAAACAGCACGCCGAGACAGAAAATTCACGATAGCGTTGATTTCAACATCGAAATGATTGAAAGAGGCGGCCGCTCAGATGAGGCAATTATGGCTTCATTCGTTCGGACATTTGATTTGGCGAAAGCTCCGCTGTTCAGAATCGGTTTGCTGGGGCTTGAAGAGAACCGTCATATGCTGCTGTTTGACATGCACCATTTGATTTCTGACGGTGTATCCATTGGCATTATGCTGGAGGAGTTAGCACGCATTTATAAAGGCGAACAGCTTCCTGATCTTCGTCTCCAGTATAAGGACTACGCTGTATGGCAAAGCAGACAGGCTGCTGAAGGGTACAAGAAGGACCAGGCTTATTGGAAGGAAGTCTTTGCAGGCGAGCTCCCGGTGCTTCAGCTTCTGTCCGATTACCCAAGACCACCTGTTCAAAGCTTTGAAGGGGATCGGGTGTCAATCAAGCTGGATGCGGGGGTAAAGGATCGCCTCAATCGTTTGGCTGAACAAAACGGCGCCACTTTATATATGGTGATGCTTTCCGCTTACTATACGCTTTTGTCAAAGTATACGGGGCAGGATGACATCATTGTCGGGACACCGTCAGCGGGCAGAAATCACTCCGATACAGAGGGCATTATCGGGATGTTCGTCAATACGCTTGCGATTCGCAGTGAGGTGAAGCAGAATGAGACGTTTACCCAATTGATCTCGCGTGTCCGCAAACGGGTGCTGGATGCCTTTTCTCATCAGGACTATCCGTTTGAGTGGCTTGTTGAAGATTTGAACATCCCGCGTGATGTTAGCAGGCATCCGCTGTTTGACACGATGTTCAGCCTTCAAAACGCGACAGAGGGCATTCCGGCTGTCGGCGATCTTTCCTTGTCTGTTCAAGAGACCAATTTCAAGATTGCCAAATTTGATTTGACGGTGCAGGCGAGAGAAACCGATGAAGGCATTGAGATTGATGTGGATTACAGCACAAAGCTGTTTAAACAAAGCACGGCAGACAGGCTGCTTACGCATTTTGCGCGTTTGCTTGAAGATGCTGCGGCTGATCCAGAGAAGCCGATTTCTGAGTATAAGCTTCTTTCTGAAGAGGAGGCTGCTTCGCAAATTCAGCAGTTTAACCCGGGCAGAACACCTTATCCGAAAGATAAAACAATTGTTCAGCTGTTTGAGGAGCAAGCGGCGAATACGCCAGACCACACTGCGCTTCAATATGAAGGCGAATCACTCACTTATCGTGAACTGAATGAACGGGCCAATCGTTTAGCCCGCGGCATTCTTTCTCTTGGAGCTGGCGAAGGCAGAACTGCGGCTGTCTTATGCGAGCGGTCAA(SEQ ID NO.3)
TGTCGACGTGCATGCAGGCCGGGGCATATGGGAAACAGCGCGGACGGAGCGGAATTTCCAATTTCATGCCGCAGCCGCCTGCGCTGTTCTCATTTGCGGCTTCCTTGTAGAGCTCAGCATTATTGAGTGGATGATTATATTCCTTTTGATAGGTGGTATGTTTTCGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAATAACTGACAAACATCACCCTCTTGCTAAAGCGGCCAAGGACGCTGCCGCCGGGGCTGTTTGCGTTTTTACCGTGATTTCGTGTATCATTGGTTTACTTATTTTTTTGCCAAAGCTGTAATGGCTGAAAATTCTTACATTTATTTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTAAAATATAAAGTGATAGCGGTACCATTATA(SEQ ID NO.4)
P43-F CCTCTAGATGATAGGTGGTATGTTTTCC(SEQ ID NO.5)
P43-R CTCTACATTCCTCTCTTACCTATAATGGTACCCT(SEQ ID NO.6)
2) Acid precipitation treatment of lipopeptide-containing fermentation liquor to obtain lipopeptide crude extract
Performing shake flask culture on BITAFS-BS2020 by adopting an LB culture medium under the shake flask culture condition: culturing at 37 deg.C, 180rpm, 16h constant temperature. LB medium composition: 5g/l of yeast powder, 10g/l of peptone, 10g/l of sodium chloride and the balance of water.
Acid precipitation: centrifuging the fermentation liquor obtained by shake flask culture at 10 ℃ and 8000rpm for 10min to obtain a supernatant, discarding the precipitate, dropwise adding 5mol/l sodium hydroxide into the supernatant until the pH of the solution is 11, centrifuging at 10 ℃ and 5000rpm for 30min to collect the supernatant, adjusting the pH to 2 by using 5mol/l hydrochloric acid, standing overnight in a refrigerator at 4 ℃, centrifuging the acid precipitation solution at 10 ℃ and 8000rpm for 20min the next day, and collecting the precipitate to obtain the lipopeptide crude extract.
3) Mass spectrometry for identifying metabolite of recombinant strain BIT AFS-BS2020
And (3) purifying the obtained acid precipitation sample by using a semi-preparative liquid phase (elett, UV1201), and identifying the mass spectrum of the purified lipopeptide sample by using a specific method which comprises the following steps: the lipopeptide purified sample was mixed with a methanol solution to prepare a 30mg/ml aqueous solution, and the solution was purified using an Elite Sinochrom ODS AP(10X 250mm,5 μm) or equivalent chromatography columns. The mobile phase contains CH with a volume concentration of 0.1 percent3MeOH of COOH H2O (5:1, V: V) mixed solution. The column temperature was room temperature, the flow rate was 4.8ml/min, the injection volume was 0.5ml, and the run time was 40 min. Finally, a purified sample of the lipopeptide is collected.
And performing MALDI-TOF-MS identification. MALDI-TOF-MS records by a Bruker Reflex-TOF instrument, and uses a 337nm nitrogen source light source for desorption and ionization, and the matrix is alpha-cyano-4-hydroxycinnamic acid (alpha-cyanoo-4-hydroxycinnamic acid). Mass analyser analysis was performed using MALDI-TOF Post source decay (PDS) mass spectrometry.
The structure of the lipopeptide compound obtained by fermentation of the recombinant strain BIT AFS-BS2020 was identified to be consistent with the surfactin structure described in the literature "Down treatment of surfactin produced by Bacillus subtilis ATCC 21332". (see FIG. 8, a shows the mass spectrometric identification results of lipopeptide compounds obtained by fermentation of BIT AFS-BS2020, and b shows the MS identification results of lipopeptide compounds C13, C14 and C15)
HPLC is adopted to measure the content of lipopeptide which is a metabolite of the recombinant strain BIT AFS-BS2020
The acid precipitate obtained in example 2 was dissolved in chromatographic grade methanol at a ratio of 1:1(w/v), filtered through a 0.22 μm filter, and the filtrate was filtered through the filter to prepare a sample. The experimental conditions for HPLC were:
1) mobile phase: a is an aqueous solution and B is an acetonitrile solution containing TFA at a volume concentration of 0.1%
2) And (3) equal elution gradient: a: b is 15%: 85% (v/v)
3) Temperature: room temperature (15 ℃); sample introduction amount: 10 mu l of the mixture; flow rate: 1 ml/min; detection wavelength: 210nm
Lipopeptide obtained by shake flask culture of the recombinant strain BIT AFS-BS2020 is subjected to acid precipitation and then is quantified by HPLC (see figure 9), and the yield of the lipopeptide can be stabilized at 4.19 g/L.
Then the purification process is shown in patent No. 202010270189.1, publication No. CN111518163A, the name of the invention is the contents of examples 1-6 in the patent documents of the application of lipopeptide compounds in resisting novel coronavirus).
Example 2
0.05g of crude extract of the lipopeptide compound obtained by the preparation in the above example was lyophilized by a conventional method to obtain a dry powder of the preparation, which was dissolved in 100mL of sterile PBS to prepare a lipopeptide-containing preparation having an original concentration of 500. mu.M.
Example 3
0.03g of crude extract of the lipopeptide compound obtained by the preparation in the above example was lyophilized by a conventional method to obtain a dry powder of the preparation, which was dissolved in 100mL of sterile PBS to prepare a lipopeptide-containing preparation having an original concentration of 300. mu.M.
Example 4
To verify the cytotoxicity of lipopeptide-containing preparations, the cytotoxicity of lipopeptide preparations (dry powders) was determined in Vero/SLAM cell plates using the CPE method of cell morphology change, and the median toxic concentration (TD50) and the maximum non-toxic concentration (TD0) of the product on cells were calculated.
Experimental Material
Lipopeptide-containing preparation example 2
(II) cells: african green monkey kidney cell (Vero/SLAM)
(III) measles Virus (MeV-AH18-1/P6 Strain)
Step 1) Vero/SLAM cells were cultured in MEM (10% fetal bovine serum, 1% P.S.) at 37 ℃ in 5% CO2,passage was carried out for 5 days. Cells were plated into 96-well culture plates at a density of 1 × 105mL, incubated at 37 ℃ for 24 hours with 5% CO2, and changed to 2% maintenance solution until the cells were confluent with 75% monolayer.
Step 2) preparation of lipopeptide of a sample to be detected: 0.05g of crude extract of lipopeptide compound obtained by the preparation of the above example was dissolved in 100mL of sterile PBS by freeze-drying the crude extract by a conventional method to obtain a dry powder of the preparation.
Step 3) diluting each test agent by PBS multiple ratio: sample lipopeptide dry powder, the concentration to be measured is 500 mu M, 400 mu M, 300 mu M, 200 mu M, 100 mu M, 50 mu M and 25 mu M respectively; the half poisoning concentration (TD50) and the maximum non-toxic concentration (TD0) of the product were calculated by a Reed-Muench method, by inoculating 8 wells of cells at each concentration, 10. mu.l per well, and simultaneously setting a cell control, incubating the wells at 37 ℃ and 5% CO2 for 3-5 days, observing the change of cell shape CPE under an inverted microscope every day, wherein the change of cell shape CPE is less than 25% to "+", the change of cell shape CPE is between 26% and 50% to "+", the change of cell shape CPE is between 51% and 75% to "+ +", and the change of cell shape CPE is between 76% and 100% to "+ + +". Experiments were performed in triplicate and data from each experiment was recorded.
I.e. distance ratio (more than 50% mortality-50%)/(more than 50% mortality-less than 50% mortality)
TD50 log + distance ratio of greater than 50% virus dilution log of dilution factor
The experimental results are as follows: on Vero/SLAM cells, detecting the cytotoxicity of the lipopeptide preparation on Vero/SLAM cells by adopting a virus CPE method, and calculating the maximum non-toxic concentration TD0 and the half toxic concentration TD50, wherein the specific results are as follows:
TABLE 1
And (4) conclusion: according to the data analysis in Table 1, the lipopeptide preparation has no toxic effect on Vero/SLAM cells when the concentration is 300 μ M or less.
Example 5
Experiments to verify that lipopeptide compounds inactivate measles virus.
1) The preparation obtained in example 3 was diluted to the desired concentration (150. mu.M) with sterile PBS and kept in a water bath at 20 ℃. + -. 1 ℃ until use.
2) Preparation of virus suspension: measles virus (MeV-AH18-1/P6 strain) was removed from the cells and thawed at room temperature before use at-80 ℃.
3) And (3) disinfection test: and (3) adding 1 part of the virus suspension into 1 part of the disinfectant to be detected, mixing in equal proportion, immediately mixing uniformly and timing. Acting at 4 deg.C, 25 deg.C and 37 deg.C for a predetermined time (15min, 30min, 1h, 2h), immediately taking out 0.1mL, adding 0.9mL cell maintenance solution, and mixing to stop acting.
4) Control group test: the positive control group is a virus control group, and whether the virus growth is good or not and whether the virus titer meets the test requirements or not are observed; in the negative control group, the cell maintenance solution was used as a negative control, and the presence or absence of contamination and the growth of cells were observed.
5) The virus titer was measured for each of the above groups by the end point dilution method. The test was repeated 3 times (P1, P2, P3).
6) End point dilution method: serial dilution of the sample to be titrated with cell maintenance solution by 10 times, sucking 100. mu.L of the sample solution, and inoculating on a monolayer cell culture plate with 8 wells for each titer. After being placed at 36 +/-1 ℃ for 1-2 h, the culture plate is taken out and the cell maintenance liquid is replaced. Continuously placing the mixture into a CO2 incubator (36 ℃ plus or minus 1 ℃,5 percent plus or minus 1 percent CO2) to culture for 3 to 4 days, observing the growth state of the cells under a microscope, and recording the pathological change condition of the cells.
7) And (3) culturing the effect product virus: the product of the action of the disinfectant and the virus is immediately inoculated into 24-hole plate cells, each hole is 100 mul, the 24-hole plate cells are put into a CO2 incubator (36 ℃ plus or minus 1 ℃ and 5 percent plus or minus 1 percent CO2) to be cultured for 3 to 5 days, the growth state of the cells is observed under a microscope, and the pathological change condition of the cells is recorded.
8) Determination of results
The test result meets all the following conditions and can be judged to be qualified:
a) the negative control group has normal cell growth;
b) the positive control group virus titer logarithmic value is more than or equal to 4.0;
c) the cells of the disinfection test group have no pathological changes and are consistent with the growth condition of the cells of the negative control group;
d) both virus culture and blind third-generation cytopathic effects were negative.
TABLE 3 suspension measles Virus inactivation assay lipopeptide formulations
As shown in Table 3, it is known that the lipopeptide products with a concentration of 150. mu.M or more can completely kill measles virus when exposed to the environment of 4 ℃, 25 ℃ and 37 ℃ for 15min or more.
Sequence listing
<110> Dalianzhen ao pharmaceutical industry Co., Ltd
DALIAN BITEOMICS Inc.
Chinese Center for Disease Control and Prevention
<120> use of lipopeptide compounds against measles virus
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgggatccat ggaaataact ttttaccctt taacg 35
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccgctcgagt tgaccgctcg cataagacag 30
<210> 3
<211> 4696
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggaaataa ctttttaccc tttaacggat gcacaaaaac gaatttggta cacagaaaaa 60
ttttatcctc acacgagcat ttcaaatctt gcggggattg gtaagctggt ttcagctgat 120
gcgattgatt atgtgcttgt tgagcaggcg attcaagagt ttattcgcag aaatgacgcc 180
atgcgccttc ggttgcggct agatgaaaac ggggagcctg ttcaatatat tagcgagtat 240
cggcctgttg atataaaaca tactgacact actgaagatc cgaatgcgat agagtttatt 300
tcacaatgga gccgggagga aacgaagaaa cctttgccgc tatacgattg tgatttgttc 360
cgtttttcct tgttcaccat aaaggaaaat gaagtgtggt tttacgcaaa tgttcatcac 420
gtgatttctg atggtatctc catgaatatt ctcgggaatg cgatcatgca catttattta 480
gaattagcca gcggctcaga gacaaaagaa ggaatctcgc attcatttat cgatcatgtt 540
ttatctgaac aggaatatgc tcaatcgaag cggtttgaaa aggacaaggc gttttggaac 600
aaacaatttg aatcggtgcc tgaacttgtt tccttgaaac ggaatgcatc cgcaggggga 660
agtttagatg ctgagaggtt ctctaaagat gtgcctgaag cgcttcatca gcagattctg 720
tcgttttgtg aggcgaataa agtcagtgtt ctttcggtat ttcaatcgct gctcgccgcc 780
tatttgtaca gggtcagcgg ccagaatgat gttgtgacgg gaacatttat gggcaaccgg 840
acaaatgcga aagagaagca gatgcttggc atgtttgttt ctacggttcc gcttcggaca 900
aacattgacg gcgggcaggc gttttcagaa tttgtcaaag accggatgaa ggatctgatg 960
aagacacttc gccaccaaaa gtatccgtat aatctcctaa tcaacgattt gcgtgaaaca 1020
aagagctctc tgaccaagct gttcacggtt tctcttgaat atcaagtgat gcagtggcag 1080
aaagaagagg atcttgcctt tttgactgag ccgattttca gcggcagcgg attaaatgat 1140
gtctcaattc atgtaaagga tcgatgggat actgggaaac tcaccataga ttttgattac 1200
cgcactgatt tattttcacg tgaagaaatc aacatgattt gtgagcgcat gattaccatg 1260
ctggagaacg cgttaacgca tccagaacat acaattgatg aattaacact gatttctgat 1320
gcggagaaag agaagctgct tgcgagggcc ggcggtaaat ctgtgagcta ccgtaaggac 1380
atgacgatac cagagctgtt ccaagaaaag gctgaactgc tttctgatca tccagcggtt 1440
gtatttgaag atcgcacatt gtcctatcga acgttacatg agcaatctgc acgcatcgcc 1500
aatgtgctga aacagaaagg ggttggcccg gacagtcctg tcgcggtttt gattgaacgc 1560
tctgaacgga tgattacagc tatcatggga attttaaaag ccggcggagc ctatgtgccg 1620
attgatccgg gttttcctgc tgagcgcatt caatatattt tggaggactg cggggcggat 1680
ttcatcctga ctgaatcgaa ggttgcggcg cctgaagccg atgctgagct gattgactta 1740
gatcaggcga ttgaggaagg tgcagaagaa agcctgaatg cagatgtgaa cgctcggaac 1800
cttgcctaca ttatttacac atcgggaaca accggacgcc cgaaaggcgt tatgatcgag 1860
catcgccagg ttcatcattt ggttgaatct ctgcagcaga cgatttatca aagcggcagc 1920
caaaccctgc ggatggcatt gcttgcgccg ttccactttg atgcgtcagt gaagcagatc 1980
ttcgcgtcgc ttcttttggg ccaaaccctt tatatcgtac cgaagaaaac agtgacgaac 2040
ggggccgccc ttactgcata ttatcggaag aacagcattg aggcgacgga cggaacaccg 2100
gctcatttgc aaatgctggc agcagcaggc gattttgaag gcctaaaact gaagcacatg 2160
ctgatcggag gagaaggcct gtcatctgtt gttgcggaca agctgctgaa gctgtttaaa 2220
gaagccggca cagcgccgcg tttgactaat gtgtacgggc cgactgaaac gtgcgttgac 2280
gcgtctgttc atccggttat ccctgagaat gcagttcaat cagcgtatgt gccgatcggg 2340
aaagcgctgg ggaataaccg cttatatatt ttggatcaaa aaggccggct gcagcctgaa 2400
ggcgtggcgg gtgagcttta tatcgcggga gacggtgtgg gccgaggcta tttacatttg 2460
cctgaattaa cggaagagaa gtttttacaa gatccattcg tgccgggcga tcgcatgtac 2520
cggaccgggg acgtggtgcg ctggcttcca gatggaacaa tcgaatattt aggcagagag 2580
gatgaccagg tcaaagtccg cggataccgg attgagcttg gggaaattga agccgtgatt 2640
cagcaggcgc cagacgttgc aaaagccgtt gttttggcac gccctgacga acagggaaat 2700
cttgaggttt gcgcatatgt tgtgcagaag cctggaagcg aatttgcgcc agccggtttg 2760
agggagcatg cggccagaca gcttcctgac tatatggtgc cggcttactt tacagaagtg 2820
acagaaattc cgcttacacc aagcggcaaa gtcgaccgcc gcaagctgtt tgcactagag 2880
gtgaaggctg tcagcggcac tgcctataca gcgccgcgaa atgagactga aaaagcaatc 2940
gcagccattt ggcaggacgt gctgaacgtt gagaaggcgg ggatctttga caatttcttt 3000
gaaactggcg gacattcatt aaaagccatg acccttttaa caaagattca taaggaaaca 3060
ggcattgaga ttccgcttca atttttgttt gagcatccga cgattacggc tcttgcagag 3120
gaagctgatc acagagaaag caaagctttt gcggtgattg aacctgctga aaaacaggag 3180
cattacccgc tttcattggc acagcagcga acatatatcg tcagccagtt cgaggatgcg 3240
ggagtcggct ataacatgcc agcagcagca attctggaag ggcctttaga tattcaaaag 3300
ctggagcgcg catttcaggg attaatccga cgccacgagt cattgagaac atcatttgtt 3360
cttgaaaaca gcacgccgag acagaaaatt cacgatagcg ttgatttcaa catcgaaatg 3420
attgaaagag gcggccgctc agatgaggca attatggctt cattcgttcg gacatttgat 3480
ttggcgaaag ctccgctgtt cagaatcggt ttgctggggc ttgaagagaa ccgtcatatg 3540
ctgctgtttg acatgcacca tttgatttct gacggtgtat ccattggcat tatgctggag 3600
gagttagcac gcatttataa aggcgaacag cttcctgatc ttcgtctcca gtataaggac 3660
tacgctgtat ggcaaagcag acaggctgct gaagggtaca agaaggacca ggcttattgg 3720
aaggaagtct ttgcaggcga gctcccggtg cttcagcttc tgtccgatta cccaagacca 3780
cctgttcaaa gctttgaagg ggatcgggtg tcaatcaagc tggatgcggg ggtaaaggat 3840
cgcctcaatc gtttggctga acaaaacggc gccactttat atatggtgat gctttccgct 3900
tactatacgc ttttgtcaaa gtatacgggg caggatgaca tcattgtcgg gacaccgtca 3960
gcgggcagaa atcactccga tacagagggc attatcggga tgttcgtcaa tacgcttgcg 4020
attcgcagtg aggtgaagca gaatgagacg tttacccaat tgatctcgcg tgtccgcaaa 4080
cgggtgctgg atgccttttc tcatcaggac tatccgtttg agtggcttgt tgaagatttg 4140
aacatcccgc gtgatgttag caggcatccg ctgtttgaca cgatgttcag ccttcaaaac 4200
gcgacagagg gcattccggc tgtcggcgat ctttccttgt ctgttcaaga gaccaatttc 4260
aagattgcca aatttgattt gacggtgcag gcgagagaaa ccgatgaagg cattgagatt 4320
gatgtggatt acagcacaaa gctgtttaaa caaagcacgg cagacaggct gcttacgcat 4380
tttgcgcgtt tgcttgaaga tgctgcggct gatccagaga agccgatttc tgagtataag 4440
cttctttctg aagaggaggc tgcttcgcaa attcagcagt ttaacccggg cagaacacct 4500
tatccgaaag ataaaacaat tgttcagctg tttgaggagc aagcggcgaa tacgccagac 4560
cacactgcgc ttcaatatga aggcgaatca ctcacttatc gtgaactgaa tgaacgggcc 4620
aatcgtttag cccgcggcat tctttctctt ggagctggcg aaggcagaac tgcggctgtc 4680
ttatgcgagc ggtcaa 4696
<210> 4
<211> 426
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgtcgacgtg catgcaggcc ggggcatatg ggaaacagcg cggacggagc ggaatttcca 60
atttcatgcc gcagccgcct gcgctgttct catttgcggc ttccttgtag agctcagcat 120
tattgagtgg atgattatat tccttttgat aggtggtatg ttttcgcttg aacttttaaa 180
tacagccatt gaacatacgg ttgatttaat aactgacaaa catcaccctc ttgctaaagc 240
ggccaaggac gctgccgccg gggctgtttg cgtttttacc gtgatttcgt gtatcattgg 300
tttacttatt tttttgccaa agctgtaatg gctgaaaatt cttacattta ttttacattt 360
ttagaaatgg gcgtgaaaaa aagcgcgcga ttatgtaaaa tataaagtga tagcggtacc 420
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cctctagatg ataggtggta tgttttcc 28
<210> 6
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctctacattc ctctcttacc tataatggta ccct 34
Claims (8)
1. Use of a lipopeptide compound against measles virus, wherein: the lipopeptide compound is a crude lipopeptide extract of lipopeptide epilin or/and sodium salt;
the crude lipopeptide extract of the lipopeptide biological epilin or/and the sodium salt is a mixture containing two or three of C13, C14 and C15 fat side chains; and the carboxymethyl position thereof may be H+Or Na+Or mixtures thereof.
2. Use of a lipopeptide compound according to claim 1 against measles virus, characterized in that: obtaining a crude lipopeptide extract of the lipopeptide epilin or/and sodium salt: obtaining a genome which takes bacillus subtilis BITAFS-BS2020 as a starting strain to extract BS02 from an expression product of the genetic engineering bacteria, then obtaining a recombinant bacteria BITAFS-BS2020 by the genetic engineering technology together with pHT01-p43 plasmid, fermenting and culturing the obtained recombinant bacteria, and purifying after culture to obtain the lipopeptide crude extract of lipopeptide epiletin or/and sodium salt.
3. Use of a lipopeptide compound according to claim 2 against measles virus, characterized in that: the recombinant strain BITAFS-BS2020 takes Bacillus subtilis BITAFS-BS2020 as an original strain, extracts the genome of BS02, adopts a primer (SEQ ID NO.1-2) and takes a BS02 genome as a template to perform PCR of srfa gene, and obtains BIT-Sr (SEQ ID NO. 3);
the promoter PgroE of plasmid pHT01 was then replaced with p43(SEQ ID NO. 4); carrying out double enzyme digestion on BIT-Sr obtained by PCR amplification and pHT01-p43 respectively by using BamH1 and Xho1, recovering enzyme digestion products, and connecting the enzyme digestion products by using a T4 ligase system to form a recombinant plasmid pHT 01-AFS;
the obtained recombinant plasmid pHT01-AFS is transferred into a B.subtilis 168 strain (ATCC 23857), and after the transfer, double-antibody screening of ampicillin sodium and chloramphenicol is carried out, and finally, a recombinant bacterium BITAFS-BS2020 is obtained.
4. Use of a lipopeptide compound according to claim 2 against measles virus, characterized in that: the construction method of the pHT01-P43 comprises the steps of carrying out BamH I and Kpn I double enzyme digestion on P43 and pHT01, recycling enzyme digestion products, and connecting the recycled products to obtain pHT 01-P43.
5. Use of a lipopeptide compound according to claim 2 against measles virus, characterized in that: the recombinant strain BITAFS-BS2020 is subjected to fermentation culture, namely the obtained recombinant strain is inoculated into a liquid fermentation culture medium according to the inoculation amount of 0.1 percent, and the temperature is 32-37 ℃ and the pH value is 6.7-7.5 under the aerobic condition; performing fermentation culture for 21-32 h;
the liquid fermentation culture medium comprises (by weight percentage) (g/L) 5-20 parts of soybean flour and/or soybean protein, 0.5-2 parts of yeast extract, 10-40 parts of cane sugar and/or maltose, 0.1-8 parts of K2HPO 41, 0.1-1 part of magnesium sulfate heptahydrate, 0.005-0.04 part of anhydrous calcium chloride, 0.005-0.04 part of ferrous sulfate, 0.01-0.1 part of manganese chloride and the balance of water.
6. Use of a lipopeptide compound according to claim 2 against measles virus, characterized in that: the obtained fermentation liquor is further refined by using an acid precipitation method, and then purified by using polar or non-polar ion exchange resin after refining to obtain a lipopeptide crude extract of lipopeptide epi-active elements or/and sodium salts, wherein the purity of the lipopeptide epi-active elements or/and sodium salts containing C13, C14 and C15 fat side chains in the crude extract is more than 95%.
7. Use of a lipopeptide compound according to claim 1 against measles virus, characterized in that: the lipopeptide compound can be used for preparing an antiviral medicament or preparation for resisting measles virus MeV-AH18-1/P6 strain.
8. Use of a lipopeptide compound according to claim 7 against measles virus, wherein: the antiviral agent is in the form of liquid, lotion, cream, paste, solid or spray for external protection against measles virus infection; alternatively, the antiviral agent for personal care products is water or milk or spray for cleaning teeth, face, skin, bathing.
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