CN115637272A - Composition for candida vaginitis and preparation method and application thereof - Google Patents

Abstract

The invention provides a composition for candida vaginitis, a preparation method and application thereof, wherein a composition for candida vaginitis is further obtained by preparing polypeptide ECBN-RGD and preparing beta-N-acetylglucosaminidase DspB, the application of DspB can rapidly degrade candida albicans biofilm, candida albicans are exposed and are in a free state, and are convenient to recognize, kill and clear, ECBN-RGD can efficiently recognize candida albicans and kill candida albicans in a targeted manner, and after the lotion is prepared, the acidic environment is utilized to inhibit the growth of candida albicans, protect epithelial cells and repair mucosal injury.

Description

Composition for candida vaginitis and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a composition for candida vaginitis, and a preparation method and application thereof.

Background

Candida albicans, also known as Candida albicans, belongs to the genus Cryptococcus. Candida albicans is a symbiotic bacterium of human body and has 3 forms of yeast phase, pseudomycelial phase and mycelial phase. Normally colonized candida albicans is in yeast phase, is only present on the surface layer of epithelial cells, does not invade cells, and is transformed from symbiont to pathogen to infect and penetrate host cells to grow when the immunity of the body is low or the local microecological balance is destroyed. Candida albicans in a free state is easily recognized and killed by a host immune system or a drug, and immune cells and the drug sensitivity of the Candida albicans after a biofilm is formed are seriously hindered. The biofilm is a large amount of extracellular matrix generated when microorganisms are attached to a biological material or the surface of human tissue to grow, wraps a bacterial cell population with a specific structure, and is a unique form of non-free bacterial cells formed for adapting to the living environment in the microbial growth process. Biofilm formation can help candida albicans avoid host immune cell invasion and drug attack, and 80% of human microbial infections are related to biofilm formation according to literature reports. The formation process of candida albicans biofilm on vaginal mucosa is divided into 3 stages: 1) Early adhesion and morphogenesis, budding of Candida albicans in the epithelial layer of the vaginal wall, with essentially no extracellular matrix; 2) The medium hyphae grow and begin to form extracellular matrix, and the yeast layer is under the elongated hyphae and only forms a small amount of extracellular matrix; 3) The maturation stage produces a large amount of extracellular matrix which forms a smooth, intact biofilm on the surface.

The biofilm formation after candida albicans infection greatly increases the difficulty of anti-infection treatment, and the biofilm acts as a barrier to protect bacterial cells from being recognized and eliminated by a host immune system and prevent antibiotic drugs from contacting the bacterial cells. The formation of biofilm structures increases the resistance of pathogenic bacteria to the host immune system and antibiotic drugs, and is an important reason for the failure of clinical anti-infective therapy and the recurrence of infection.

It is assumed that a product capable of degrading candida albicans biofilm is developed, pathogenic bacteria are exposed after the biofilm is degraded, and then the pathogenic bacteria are effectively killed. At present, clinically, antibiotics or antimycotics including fluconazole, clarithromycin, itramycin, amphotericin B and the like are generally adopted for treating candida albicans infection. Candida albicans has developed resistance to the above drugs, and the antibacterial effect is sometimes not ideal.

Develops a product for degrading candida albicans biofilm, effectively killing candida albicans and inhibiting candida albicans adhesion infection, and improves the treatment efficiency of the mycotic vaginitis.

Disclosure of Invention

Aiming at the limitations and drug resistance of the existing medicines or other types of products to candida albicans infection after biofilm formation, the invention provides a composition for candida vaginitis, a preparation method and application thereof, which can degrade biofilm exposed bacteria cells, apply newly synthesized high antibacterial active substances, effectively kill candida albicans when the candida albicans contacts the high antibacterial active substances after the candida albicans biofilm is degraded, and simultaneously can inhibit the adhesion of candida albicans on tissue mucosa to further prevent infection. The invention can effectively break down candida albicans biofilm, efficiently kill candida albicans, and has high safety and no toxic or side effect.

In order to achieve the above objects, the present invention provides a genetic engineering method for preparing beta-N-acetylglucosaminidase DspB, which comprises the steps of,

subjecting Haemophilus actinomycete to IS903 transposon mutagenesis, inserting transposon into dspB gene sequence DspB gene, and cloning and amplifying dspB gene by trans PCR; the upstream primer is 5-GCGCGCCATatgAATTGTTGCGTAAAAGGCAATTCC-3 an NdeI restriction site and an ATG initiation codon were added at the underlined position, and the downstream primer was 5-GCGGTACCCTCATCCCCATTCGTCTTATGAATC-3, a KpnI restriction site is added in place of the stop codon of the dsp gene at the underlined position; accessing the plasmid pET296 to construct a target plasmid pRC1;

and (3) transfecting pRC1 into escherichia coli BL21 (DE 3), culturing overnight, incubating in a culture medium, resuspending, ultrasonically treating, eluting, collecting protein, and digesting with thrombin.

In one aspect of the invention, the invention provides a beta-N-acetylglucosaminidase DspB which can degrade Candida albicans biofilm to expose Candida albicans in a free state.

In one aspect, the invention provides a pharmaceutical composition for candidal vaginitis, which comprises beta-N-acetylglucosaminidase DspB and ECBN-RGD.

Preferably, the ECBN-RGD is prepared by activating carboxyl of RGD, adding it into ECBN, and stirring for reaction.

Preferably, the beta-N-acetylglucosaminidase DspB is 0.1-100 parts by mass, and the ECBN-RGD is 0.5-500 parts by mass.

Preferably, the mass ratio of the beta-N-acetylglucosaminidase DspB to the ECBN-RGD is 1: (0.5-10).

Preferably, the mass ratio of the beta-N-acetylglucosaminidase DspB to the ECBN-RGD is 1: (1-5).

Preferably, the composition also comprises lactic acid, sodium lactate, sodium hyaluronate, glycine, L-serine, epidermal growth factor EGF, phenoxyethanol, ethylhexyl glycerol and water.

Preferably, the mass portions of the sodium hyaluronate are 2000-20000g/L, the glycine 10000-30000g/L, L-serine is 1000-10000g/L, and the epidermal growth factor EGF is 10000-50000mg/L.

Preferably, the pH after dissolution is between 3.5 and 5.5.

In one aspect of the invention, the invention provides a composition for treating candida vaginitis, which comprises 0.1-100 parts of DspB0.5-500 parts of ECBN-RGD.

As one aspect of the invention, the invention provides a preparation method of a lotion prepared from a composition for treating candida vaginitis, which comprises the steps of adding sodium hyaluronate into water to completely dissolve the sodium hyaluronate, then adding lactic acid, sodium lactate, glycine and L-serine, stirring the solution at a high temperature of 80 ℃ for 20 minutes, then cooling to 45 ℃, adding phenoxyethanol and ethylhexyl glycerol, stirring uniformly, continuously cooling to within 40 ℃, adding DspB, ECBN-RGD and EGF, and stirring uniformly.

Preferably, the relative molecular mass of the sodium hyaluronate is 20000 to 20000Da.

The invention has the following beneficial effects:

the invention provides a biological lotion which can degrade candida albicans biofilm, effectively kill candida albicans and eliminate infection. The method has the following beneficial effects:

1. the application of DspB can rapidly degrade the Candida albicans biofilm, expose the Candida albicans and enable the Candida albicans to be in a free state, and is convenient for identification, killing and removal.

2. The newly synthesized ECBN-RGD can efficiently identify the Candida albicans and kill the Candida albicans in a targeted manner. Candida albicans adheres to vaginal mucosal tissue first, the adhesion process is mediated by mannan-protein complex on bacterial cell wall, namely glycoprotein, glycoprotein bound with the surface of mammalian cell membrane is CR3, and the ligand structure which can be recognized by CR3 as complement receptor on the surface of Candida albicans is arginine-glycine-aspartic acid tripeptide structure, namely RGD. RGD is widely present on cell membrane proteins such as fibronectin, collagen fibers and fibrinogen, which is also responsible for the wide adhesion range and strong adhesion of Candida albicans. The RGD structure in ECBN-RGD competitively binds to CR3 of candida albicans wall, and reduces the binding of the candida albicans and mucosal epithelial cells to generate adhesion. After the RGD specifically recognizes Candida albicans, ECB molecules can non-competitively inhibit beta-1 and 3-glucan synthetase of fungal cells, prevent the synthesis of important components beta-1 and 3-glucan of the fungal cell walls, influence the integrity of the cell walls, cause the imbalance of osmotic pressure in the fungal cells and cause the rupture and death of thalli. Because human cells do not have cell walls and do not need beta-1 and 3-glucan synthetase, the ECB molecules have no toxic or side effect on human bodies. From the above, ECBN-RGD can efficiently kill Candida albicans and block Candida albicans adhesion infection.

3. Further, the acidic pH environment of the lotion inhibits the growth and proliferation of Candida albicans, and avoids the repeated attack of infection.

4. Furthermore, the strong water molecule binding capacity of the sodium hyaluronate and the wettability of the serine are utilized to enhance the wettability of the mucous membrane tissue, and the hydrophobic environment is avoided to promote the formation of candida albicans biofilm.

5. Further, cell damage caused by candida albicans is repaired by glycine and EGF, and cell protection and epithelial layer repair effects are exerted.

The materials prepared by the method are safe and have no cytotoxicity.

Drawings

FIG. 1 is a chemical structure diagram of RGD peptide;

FIG. 2 is a chemical structure diagram of ECBN;

FIG. 3 is ECBN grafting RGD to form ECBN-RGD;

FIG. 4 is a chemical structural diagram of ECBN-RGD prepared by the present invention;

FIG. 5 is a microscopic view of the cell status of the negative control group;

FIG. 6 is a microscope photograph of the cell status of the positive control group;

FIG. 7 is a microscopic image of the cell status of the experimental group (DspB group);

FIG. 8 is a high performance gel chromatogram of a standard;

FIG. 9 is a high performance gel chromatogram of a sample prepared according to the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present application will be further described with reference to specific examples.

The ECBN for synthesizing the ECBN-RGD in the technical scheme is obtained by cutting off a fatty acid side chain which is easy to generate hemolytic toxicity from Echinocandin (ECB), the ECBN is not stable but toxic, the RGD peptide is clinically used for tumor characterization and drug loading, and the safety is verified. The ECBN-RGD formed after the ECBN is grafted with the RGD has enhanced stability and is non-toxic. Lactic acid, sodium lactate, sodium hyaluronate, glycine, tryptophan and epidermal growth factor are common components of human medicines or skin care products, and phenoxyethanol and ethylhexylglycerin are relatively mild and safe medical or cosmetic preservatives. The safety of the components for human bodies is verified, and DspB is not widely used in clinic and human bodies, so that DspB biosafety, namely cytotoxicity is evaluated.

Example 1: preparation of DspB

DspB is prepared by a genetic engineering method, and comprises 3 processes of plasmid construction, dspB expression in escherichia coli and DspB protein purification.

The plasmid construction comprises the following steps: 1) The DspB gene is from Haemophilus actinomycetemcomitans. First, the clinical wild strain CU1000 of Haemophilus actinomycetemcomitans was treated with BD medium supplemented with 6g/L yeast extract and 8g/L glucose at 37 ℃ with 10% CO ₂ Culturing under the condition; 2) CU1000 strain IS IS903 transposon mutagenesis, transposon insertion into the dspB gene sequence; 3) The dspB gene is cloned and amplified by trans-PCR, and NdeI and KpnI restriction enzyme cutting sites are respectively introduced into the upstream primer and the downstream primer, namely the upstream primer is 5-GCGCGCGCCATatgAATTGTTGCGTAAAAGGCAATTCC-3, an NdeI restriction site and an ATG initiation codon are added at the underlined position, and the downstream primer is 5-GCGGTACCCTCATCCCCATTCGTCTTATGAATC-3, a KpnI restriction site is added in place of the stop codon of the dsp gene at the underlined position; 4) The PCR product was ligated to NdeI and KpnI sites of plasmid pET296 using NdeI and KpnI sites, and the objective plasmid pRC1 was constructed.

DspB expression in E.coli comprises the following steps: 1) 2 liter Erlenmeyer flasks containing 500ml LB medium were supplemented with 30ug/ml natamycin, inoculated with 5ml pRC1 transfected and overnight cultured E.coli BL21 (DE 3); 2) The Erlenmeyer flasks were incubated at 37 ℃ with shaking (200 rpm) until the optical density of the culture at 600nm reached 0.6 (ca.3h); 3) 0.2mM isopropyl-. Beta. -D-thiogalactoside (IPTG) was added, and the flask was further incubated with shaking for 5 hours, and 6000g was centrifuged for 15 minutes to collect cells, and the precipitated cells were stored at-80 ℃.

DspB protein purification comprises the following steps: 1) The cell pellet was thawed at room temperature and resuspended in 2mg/ml lysozyme buffer (20 mM Tris-HCl [ pH8.0], 500mM NaCl, 1mM phenylmethylsulfonyl fluoride, 0.1% ethylphenylpolyethylene glycol); 2) Sonicating the cell suspension on ice for 30 seconds using an ultrasonic cell disruptor equipped with a microprobe, with the processing parameters 30% capacity with a 30% capacity cycle (30% capacity at 30% duty cycle); 3) The cell debris was centrifuged at 15000g at 4 ℃ for 20 min and the supernatant was collected and loaded onto 3ml (bed volume) activated Ni affinity column; 4) Washing the column with 50ml of a buffer containing 5mM imidazole (20mM Tris [ pH7.5],500mM NaCl), and then washing the column with 25ml of a buffer containing 50mM imidazole; 5) Eluting the protein with 25ml of an eluent (2 mM Tris [ pH8.0] -500mM NaCl-100mM imidazole); 6) Collecting and analyzing the condition of eluted protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining; 7) Dialyzing the protein-containing fragment in water overnight using a 10000kDa dialysis membrane; 8) The dialyzed protein was digested with 5U/mg thrombin (Novagen) for 1 hour at room temperature and the thrombin was removed using the thrombin cleavage Capture kit (Novagen) according to the instructions provided in the kit; 9) Undigested protein was removed by loading onto a Ni affinity column as described in 3) and washing the column with 10ml of wash buffer containing 5mM imidazole; 10 ) the fragments containing the target protein are collected, then freeze-dried into solid powder, and stored under refrigeration for later use.

DspB security verification

The experimental method comprises the following steps: 1. digesting the L929 cells with good state and spreading rate reaching 80%, counting the cells to prepare 20000/ml cell suspension, inoculating the cell suspension on a 96-well plate according to 200 ul/well, reserving 1 column, adding a culture solution without cells as a blank group, and culturing in a CO2 incubator for 24 hours to allow the cells to adhere to the wall; 2. the experimental group takes the conventional cell culture solution (MEM +10% fetal bovine serum +100U/ml penicillin +100ug/ml streptomycin + 1%L-glutamine) and adds the prepared DspB powder to dissolve, and the final concentration of the DspB in the culture solution is 100ug/ml;3. extracting the culture solution from a 96-well plate, adding 200ul of cell culture solution containing DspB into each well of an experimental group, culturing for 72h, adding conventional cell culture solution into a blank control group, adding 10ul of DMSO into a positive control group to kill cells, and adding conventional cell culture solution into a negative control group; 4. observing the growth condition and the morphological change condition of the cells under a microscope; 5. addition of MTT (reduction of viable cells with MTT): 20ul of MTT at a concentration of 5mg/ml was added to each well and incubated for 4h in a CO2 incubator. Pumping out the liquid on a 96-well plate, adding 150ul DMSO into each well, and oscillating for 10min;6, detection: detecting OD value (representing the number of living cells) at 492nm by using a microplate reader; 7. analyzing the data, calculating the relative proliferation rate of the cells: relative cell proliferation% = [ (experimental absorbance-blank absorbance)/(negative control absorbance-blank absorbance) ] + 100.

The experimental results are as follows: the cell morphology and growth conditions are shown in the following figures 5-7, the cells of the negative control group are spindle-shaped, normal in morphology, good in growth and 100% in relative proliferation rate; the positive control group has a large amount of broken and dead cells, a small number of cells and a relative proliferation rate of 17.8 percent; the cells in the experimental group are spindle-shaped, normal in shape, good in growth and 97.9% in relative proliferation rate. The relative proliferation rates of the cells are detailed in table 1 below.

TABLE 1 results of relative cell proliferation rates

Group of	Average value of absorbance	Relative cell proliferation rate
			Blank group	0.100	/
Negative control group	1.034	100％
			Positive control group	0.266	17.8％
Experimental group	1.014	97.9％

From the above experimental results, cells added into 100ug/ml DspB cell culture fluid are spindle-shaped, normal in morphology, 97.9% in relative cell proliferation rate, and good in cell growth, so that DspB has no cytotoxicity and good cell compatibility at the concentration.

Example 2: preparation of ECBN-RGD

The specific method for preparing ECBN-RGD comprises the following steps: 1) Weighing 10mg RGD peptide (RGD structure is shown in figure 1), and dissolving with 1ml PBS buffer (pH 4.0); 2) Adding 50mg EDC.HCL and 25mg NHS into the RGD solution, magnetically stirring at 4 ℃ for 12 hours, and activating the carboxyl of RGD; 3) 24mg of ECBN (in the form of echinocandin B mother nucleus hydrochloride, the structure of which is shown in FIG. 2) is weighed and dissolved by 1ml of PBS buffer (pH 4.0); 4) Slowly dropping the activated RGD solution into the ECBN solution under stirring, and continuously stirring for 24 hours at 4 ℃ to generate ECBN-RGD, wherein a reaction schematic diagram is shown in figure 3; 5) Transferring the reaction solution into a dialysis bag (1 KD), dialyzing with deionized water for 72 hours, changing water every 12 hours, freeze-drying after dialysis to obtain ECBN-RGD (the structure of ECBN-RGD is shown in figure 4) solid, and refrigerating for later use.

Verification of ECBN-RGD Synthesis

The synthesis condition of ECBN-RGD is verified by detecting the molecular weight by using high-efficiency gel chromatography. Sample preparation: dissolving 1mg of prepared ECBN-RGD or standard peptide with 1ml of PBS buffer solution (pH4.0), centrifuging at 10000rpm for 10min, taking supernate and passing through a 0.2um microporous filter membrane, and completing sample preparation. And (3) chromatographic column: TSKgel2000 SWXL; mobile phase: acetonitrile/water/trifluoroacetic acid =45/55/0.1 (V/V); flow rate: 0.5ml/min; column temperature: 30 ℃; sample introduction amount: 10ul; and (3) standard substance: 1) Thymopentin peptide (Mr679.8), flag peptide (Mr 1013), malanide peptide (Mr 1633.9) and Systemin peptide (Mr 2010.3).

Firstly, determining the peak time of a standard sample, using lgMr of the logarithm of the molecular weight of each standard sample, and drawing a graph with retention time to obtain a linear relation, wherein the regression equation is y = -0.079X +4.522; r ² =0.959. Root of herbaceous plantAccording to the standard curve, the molecular weight of 3 peaks of the sample is respectively 350, 820 and 1200 through calculation of a regression equation.

From the results of the molecular weight measurement, most of the samples were synthesized ECBN-RGD molecules, and a small amount of RGD peptide and ECBN molecules were incompletely reacted during the synthesis reaction, but most of the samples were synthesized ECBN-RGD molecules.

In vitro verification shows that DspB depolymerizes Candida albicans biofilm and ECBN-RDG kills and dissolves Candida albicans.

The experimental method comprises the following steps: taking 96-well plate, adding 200ul of 10-contained single well ² To 10 ⁴ CFU of Candida albicans unicellular suspension, at 37 deg.C, 10% CO ² Incubating for 24h to form a biological membrane;

1) The plate holes of the 96-hole plate containing the bacterial liquid are divided into 4 groups of a control group, a DspB group, an ECBN-RGD group and a DspB synergistic ECBN-RGD group, and the specific steps are as follows: 10ul of phosphate buffer was added to each well of the control group; adding 10ul DspB solution (DspB 1mg/ml, prepared with phosphate buffer solution, and DspB final concentration of 50ug/ml in 96-well plate) into each well of DspB group; adding 10ul of ECBN-RGD solution (ECBN-RGD 0.1mg/ml, prepared by phosphate buffer solution, and the final concentration of ECBN-RGD in a 96-well plate is 5 ug/ml) into each hole of the ECBN-RGD group; dspB in cooperation with ECBN-RGD group 10ul DspB and ECBN-RGD solution (DspB 1mg/ml, ECBN-RGD 0.1mg/ml, prepared with phosphate buffer solution, final DspB concentration of 50ug/ml and final ECBN-RGD concentration of 5ug/ml in 96-well plate) were added per well. Incubating for another 6 hours;

2) The plate holes of the control group and each test group are washed under running water, bacterial cells remained on the surfaces after washing are stained by crystal violet, then the floating color is removed by washing with the running water, and finally, the adherent bacterial cells are decolorized by 95% ethanol.

3) Measuring the absorbance of four groups of ethanol solutions at 590nm, wherein the absorbance value is in direct proportion to the number of adherent bacteria cells, and analyzing the bacteria cell residue.

The experimental results are as follows:

the average absorbance at 590nm of the control group was 1.698, and the average absorbance at 590nm of the test group containing DspB, ECBN-RGD and DspB in combination with ECBN-RGD were 0.765, 1.412 and 0.232, respectively.

TABLE 2.590 nm Absorbance value of ethanol solution

The reduction rate R1 of DspB group well plate bottom adhesion bacteria cells is:

R1＝(1.698-0.765)/1.698＝54.9％

the reduction rate R2 of ECBN-RGD group pore plate bottom adhesion bacteria cells is as follows:

R2＝(1.698-1.412)/1.698＝16.8％

the reduction rate R3 of DspB in cooperation with ECBN-RGD group pore plate bottom adhesion bacteria cells is as follows:

R3＝(1.698-0.232)/1.698＝86.3％

from the above calculation, it can be seen that DspB and ECBN-RGD can be added to kill part of Candida albicans, wherein the DspB group has a higher cell reduction rate than the ECBN-RGD group, and the probable reason is that DspB has a depolymerization effect on the biofilm and a liquid shear force can remove a certain part of bacterial cells, while ECBN-RGD has a weaker effect in the presence of the biofilm. When DspB cooperates with ECBN-RGD, the ECBN-RGD can be combined on bacterial cells in time to quickly kill the Candida albicans after the DspB dissolves the Candida albicans biofilm, and most of the bacterial cells lose activity. Compared with the effect of respectively using DspB and ECBN-RGD, the action of cooperation of DspB and ECBN-RGD greatly improves the removal capability of Candida albicans.

Example 3

The embodiment of the invention provides a biological lotion for candida albicans infection, which comprises the following components in concentration: beta-N-acetylglucosaminidase (DspB, prepared in example 1) 0.1-100mg/L, ECBN-RGD (prepared in example 2) 0.5-500mg/L, lactic acid 2g/L, sodium lactate 5g/L, sodium hyaluronate 2-20g/L, glycine 10-30g/L, L-serine 1-10g/L, epidermal Growth Factor (EGF) 10-50mg/L, phenoxyethanol 2.7g/L, ethylhexylglycerol 0.3g/L, and purified water to make up the balance.

DspB is a glycoside hydrolase family 20 protein that hydrolyzes poly β - (1.6) -N-acetylglucosamine in bacterial and fungal biofilm matrices. Candida albicans biofilm polysaccharide is composed of alpha-mannan, beta-1.6-glucan and beta-1.3-glucan. The addition of DspB can degrade the network structure of existing biofilm of Candida albicans, and prevent new biofilm formation. After DspB depolymerizes or disperses the Candida albicans biomembrane, the Candida albicans is in a free state and is easy to recognize or kill and clear.

ECBN-RGD is a polypeptide molecule synthesized by hexacyclic lipopeptide ECBN and tripeptide RGD, wherein the RGD can specifically recognize candida albicans cell wall glycoprotein and is combined with the candida albicans cell wall glycoprotein, the ECBN can inhibit beta-1 and 3-glucan synthetase, so that synthesis of important cell wall components beta-1 and 3-glucan is blocked, bacterial cells are dissolved and killed, and the fungal cells are killed in a targeted manner. ECBN is poor in stability and water solubility, and water solubility is also improved by greatly enhanced molecular stability after binding to RGD. Meanwhile, after RGD is combined with a bacterial cell wall receptor, the adhesion of the candida albicans on a mucous membrane tissue is competitively inhibited, and the infection and the invasion of the candida albicans on the mucous membrane tissue are prevented.

The lactic acid and the sodium lactate in the proportion can ensure that the pH value of the product is in a range of 3.5-5.5, and are not beneficial to the growth of candida albicans. The sodium hyaluronate (with molecular weight of 20000-20000 Da) has strong water molecule binding capacity, and can moisten mucosal tissues by combining the wettability of L-serine. The moist environment can promote the repair of mucosal epithelial cells and simultaneously avoid the reformation of candida albicans biofilm in a hydrophobic environment. EGF promotes epithelial cell proliferation and repairs damaged epithelial tissue. Glycine is the amino acid with the simplest structure, participates in various metabolic and pathophysiological processes in vivo, and has the functions of cell protection and immunoregulation.

Furthermore, the product in the form of the lotion can remove the free candida albicans through liquid shearing force on one hand, and can prevent the free candida albicans from being infected in a larger area due to the diffusion of free pathogenic bacteria by efficiently killing and timely removing the free candida albicans after the biofilm is damaged on the other hand.

The preparation method of the biological lotion comprises the following steps:

taking a certain amount of purified water at room temperature, adding sodium hyaluronate to completely dissolve, then adding lactic acid, sodium lactate, glycine and L-serine, stirring the solution at the high temperature of 80 ℃ for 20 minutes, then cooling to 45 ℃, adding phenoxyethanol and ethylhexylglycerin, stirring uniformly, continuously cooling to within 40 ℃, adding DspB, ECBN-RGD and EGF, and stirring uniformly to obtain the biological lotion.

Lactic acid and sodium lactate are contained so that the pH of the buffer solution ranges from 3.5 to 5.5.

The sodium hyaluronate has relative molecular mass of 20000-20000 Da.

The biological lotion prepared in example 3 was used in combination with a vaginal douche to verify the therapeutic effect on candidal vaginitis.

60 cases of candida vaginitis (VVC) patients are outpatient in China and Western medicine combined hospital outpatient service in Tung Xiang Rejin in 2021-2021 in 5 months. The clinical manifestations of 60 patients are vulvar pruritus or causalgia, large leucorrhea, bean curd residue like leucorrhea, congestion and red swelling of vulva or vaginal wall mucous membrane, and positive candida albicans detected by vaginal secretion under microscope. These patients were randomized into 2 groups and given conventional drug treatment (control group, 30 cases) and biological lotion treatment (experimental group, 30 cases), respectively.

The patients in the control group were given fluconazole orally at 150 mg/dose/day for 7 consecutive days. The biological lotion is used by the patients in the experimental group, 50ml of the biological lotion is added into a vaginal irrigator bottle, 450ml of purified water is added for dilution, the temperature is raised to 40 ℃ for use, the irrigator bottle body is squeezed, and the vulva and the vagina are fully irrigated by an irrigator head for 1 time/day for 7 days continuously.

And 7-14 days after the treatment, judging the curative effect according to clinical symptoms and microscopic examination results. The cure standard is clinical symptom disappearance, vaginal mucosa damage disappearance and negative candida albicans microscopic examination; effective standards are that clinical symptoms are reduced, vaginal mucosa damage is reduced, and candida albicans is negative in microscopic examination; the ineffective standard is that the clinical symptoms and the vaginal mucosa damage condition are not improved or even aggravated, and the candida albicans is positive through microscopic examination.

The experimental result shows that the control group has 23.3 percent of cured 7 cases, 50 percent of effective 15 cases and 26.7 percent of ineffective 8 cases; the experimental group has 23 cases of cure 76.7%, 6 cases of effective 20% and 1 case of ineffective 3.3%, and the results are shown in the following table 3:

TABLE 3 comparison of treatment results

From the table, the biological lotion prepared by the invention greatly improves the cure rate compared with the conventional medicine, greatly reduces the ineffective rate of treatment, and shows that the invention improves the limitation of the existing medicine to the treatment of candida vaginitis.

The invention is applied to local tissues in the form of lotion, dspB component is contacted with Candida albicans infected tissues to rapidly degrade Candida albicans biofilm, RGD in ECBN-RGD can be specifically combined with mannan-protein complex on the cell wall of Candida albicans, then ECB structure can non-competitively inhibit beta-1, 3-glucan synthetase of Candida albicans cell wall, so that the synthesis of key components beta-1, 3-glucan of the Candida albicans cell wall is blocked, the integrity of the cell wall is destroyed, and the Candida albicans cell is dissolved and killed. The acidic environment of the lotion inhibits the growth of Candida albicans. Candida albicans causes damage to mucosal epithelium, and sodium hyaluronate, glycine, L-serine and EGF protect epithelial cells and repair mucosal damage.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A gene engineering method for preparing beta-N-acetylglucosaminidase DspB is characterized in that: comprises the following steps of (a) carrying out,

subjecting Haemophilus actinomycete to IS903 transposon mutagenesis, inserting transposon into dspB gene sequence DspB gene, and cloning and amplifying dspB gene by trans PCR; upstream primerIs 5-GCGCGCCATatgAATTGTTGCGTAAAAGGCAATTCC-3 an NdeI restriction site and an ATG initiation codon were added at the underlined position, and the downstream primer was 5-GCGG TACCCTCATCCCCATTCGTCTTATGAATC-3, a KpnI restriction site is added at the underlined position in place of the stop codon of the dsp gene; accessing the plasmid pET296 to construct a target plasmid pRC1;

transfecting Escherichia coli BL21 (DE 3) by using pRC1, culturing overnight, incubating in a culture medium, carrying out resuspension, ultrasonic treatment and elution, collecting protein, and then digesting by using thrombin.

2. The beta-N-acetylglucosaminidase DspB obtained by the method of claim 1, which is characterized in that: degrading Candida albicans biofilm to expose Candida albicans, and making Candida albicans in free state.

3. A pharmaceutical composition for candida vaginitis, which is characterized in that: including beta-N-acetylglucosaminidase DspB and ECBN-RGD.

4. The pharmaceutical composition against candida vaginitis recited in claim 3 wherein: the preparation method of the ECBN-RGD comprises the steps of activating carboxyl of RGD, adding the activated carboxyl into ECBN, and stirring for reaction.

5. The pharmaceutical composition against candida vaginitis recited in claim 3 wherein: according to the mass parts, the beta-N-acetylglucosaminidase DspB is 0.1-100 parts, and the ECBN-RGD is 0.5-500 parts.

6. The pharmaceutical composition against candida vaginitis recited in claim 3 wherein: the mass ratio of the beta-N-acetylglucosaminidase DspB to the ECBN-RGD is 1: (0.5-10).

7. The pharmaceutical composition against candida vaginitis recited in claim 3 wherein: the mass ratio of the beta-N-acetylglucosaminidase DspB to the ECBN-RGD is 1: (1-5).

8. The pharmaceutical composition against candida vaginitis recited in claim 3 wherein: also comprises lactic acid, sodium lactate, sodium hyaluronate, glycine, L-serine, epidermal growth factor EGF, phenoxyethanol, ethylhexyl glycerol and water.

9. The pharmaceutical composition against candida vaginitis recited in claim 8 wherein: 2000-20000g/L sodium hyaluronate, 10000-30000g/L, L-serine 1000-10000g/L glycine and 10000-50000mg/L epidermal growth factor EGF.

10. The pharmaceutical composition against candida vaginitis recited in claim 8 wherein: the pH after dissolution is between 3.5 and 5.5.

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