CN111773437A

CN111773437A - Lubricated condom and preparation method thereof

Info

Publication number: CN111773437A
Application number: CN202010668464.5A
Authority: CN
Inventors: 熊进; 张华�; 李亚男; 李脉; 陈达凯; 李月俏; 陈禹兴; 杨创钦; 陈聪伟; 邓远倪; 连培荣; 龙志东; 黄金锐; 黄嘉麒; 郭秋霞; 梁玲娜; 柏松; 李福生; 吕秋军; 梁佳潮
Original assignee: Guangzhou Qilong Biotechnology Co ltd
Current assignee: Guangzhou Qilong Biotechnology Co ltd
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-10-16
Anticipated expiration: 2040-07-13
Also published as: CN111773437B

Abstract

The application belongs to the technical field of birth control supplies, and particularly relates to a lubricating condom and a preparation method thereof. The application provides a lubricated condom, comprising a condom body and a lubricant; the lubricant is adhered to the inner surface or/and the outer surface of the condom body; the lubricant comprises the following components: human lysozyme and a solvent. The application also provides a preparation method of the lubricating condom, which comprises the following steps: step 1, mixing human lysozyme and a solvent to prepare a lubricant; and 2, soaking the condom body in the lubricant to enable the lubricant to be attached to the inner surface or/and the outer surface of the condom body, and thus obtaining the lubricated condom. The application provides a lubricating condom and a preparation method thereof, which can effectively overcome the technical defect that the traditional rubber condom can only effectively block particles with the diameter similar to that of human sperm, but can not effectively defend bacteria or viruses with the aperture smaller than 120 nanometers.

Description

Lubricated condom and preparation method thereof

Technical Field

The application belongs to the technical field of birth control supplies, and particularly relates to a lubricating condom and a preparation method thereof.

Background

The condom is a reproductive health product, aims at contraception, blocking viruses and bacteria and preventing sexually transmitted diseases, extends vibration, has fragrance and the like, and can also assist sexual function. However, the results of the studies published in the international famous medical journal are: the medical journal of New England reports that the failure rate of condom against AIDS is 16.7%, and the medical journal of UK society science and medicine reports that the failure rate of condom against AIDS is as high as 31%. The protective effect of condoms on 9 sexually transmitted diseases such as hepatitis B, AIDS, gonorrhea, chlamydia, syphilis, chancroid, lymphogranuloma venerum, genital herpes, condyloma acuminatum and the like is researched by a scientific special group which consists of National Institute of Health (NIH), Food and Drug Administration (FDA), center for disease prevention and control (CDC) and the American international development agency (US-AID), and the condoms widely used at present are found to be incapable of safely and effectively preventing the transmission of any sexual disease.

The reason for this is that the viruses in semen, such as AIDS, hepatitis B, human papilloma, etc., are much smaller than those in sperm, and condoms can block sperm, but cannot block various viruses and bacteria. The traditional condoms each have more than one hundred million pores with the diameter of 120 nanometers (the latex film body has the quality problem that natural fissures between 5000 and 70000 nanometers exist), can only effectively block particles with the diameter similar to the size of human sperm (the diameter is about 5000 nanometers), and can not completely block particles with the diameter equal to or less than 120 nanometers. That is, 42 nm of hepatitis B virus, 50-55 nm of human papilloma virus, 120 nm of HIV and 180 nm of type II herpesvirus are all likely to penetrate through the traditional natural latex condom. The research and development of condoms with the function of preventing bacteria, viruses and microorganisms is urgent.

In conclusion, because the condom can only effectively block particles with the diameter similar to that of human sperm, and can also block pathogens such as chlamydia, mycoplasma and the like, the condom can not effectively prevent bacteria or viruses with the pore diameter smaller than 120 nanometers. Therefore, the traditional rubber condom can only obstruct most sperms, but has poor effect on preventing the transmission of viruses and germs. Therefore, the condom is not equal to a condom, and the development of the condom which can directly resist bacteria and viruses and block sperms has great social value.

Disclosure of Invention

In view of this, the application provides a lubricating condom and a preparation method thereof, which can effectively overcome the technical defect that the existing traditional rubber condom can only effectively block particles with the diameter similar to that of human sperm, but can not effectively defend bacteria or viruses with the pore diameter less than 120 nanometers.

A first aspect of the application provides a lubricated condom comprising a condom body and a lubricant;

the lubricant is adhered to the inner surface or/and the outer surface of the condom body;

the lubricant comprises the following components:

human lysozyme and a solvent.

The lysozyme can be human lysozyme extracted from a human body or recombinant human lysozyme, the human lysozyme and the recombinant human lysozyme extracted from the human body contain 130 amino acids, the molecular weight is about 14700Da, and the amino acid sequences are as follows:

KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV；

the recombinant human lysozyme can also be recombinant human lysozyme with more than four amino acids, the recombinant human lysozyme with more than four amino acids has 134 amino acids, the molecular weight is about 15,100Da, and the amino acid sequence is as follows:

EAEAKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV；

the recombinant human lysozyme can also be a recombinant human lysozyme with six more amino acids, wherein the recombinant human lysozyme with six more amino acids has 136 amino acids, and the amino acid sequence is as follows:

EAEAEAKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV。

wherein, the effect of the recombinant human lysozyme with more than four amino acids and the recombinant human lysozyme with more than six amino acids is equivalent to that of the recombinant human lysozyme, and the purity and the activity of the recombinant human lysozyme with more than four amino acids and the recombinant human lysozyme with more than six amino acids are higher than that of the recombinant human lysozyme.

Preferably, the pH value of the lubricant is 4.5-5.5. The lubricant is acidic, so that the lubricating condom is acidic, the pH environment of the lubricating condom is equivalent to that of a vagina, the vaginal mucosa and the like cannot be damaged, and the human lysozyme can have activity in an acidic environment.

Preferably, the lubricating condom further comprises a lubricating liquid, an emulsifier, a preservative, a disinfectant and a thickening agent.

Preferably, the lubricating fluid is selected from glycerol and/or dimethicone; the emulsifier is selected from tween-80; the preservative is selected from bis (hydroxymethyl) imidazolidinyl urea; the disinfectant is selected from polyhexamethylene biguanide; the thickener is selected from cationic acrylamide polymerization emulsions.

Specifically, the product selected by the thickener is polyquaternium Fs 222; the cationic acrylamide polymerization emulsion selected by the application does not influence the activity of the recombinant human lysozyme, so that the lubricant has certain viscosity, and the lubricant is kept in a uniform and stable suspension state or an opacified state.

Preferably, the lubricant comprises per 1000ml of solvent:

preferably, the lubricant comprises per 1000ml of solvent:

preferably, the condom body is a male condom body. The male condom body can be an existing common male condom.

Preferably, the condom body is a female condom body. The female condom body can be a common female condom.

In a second aspect, the present application provides a method of making a lubricated condom, comprising the steps of:

step 1, mixing human lysozyme and a solvent to prepare a lubricant;

and 2, soaking the condom body in the lubricant to enable the lubricant to be attached to the inner surface or/and the outer surface of the condom body, and thus obtaining the lubricated condom.

Preferably, the lubricant further comprises a lubricating liquid, an emulsifier, a preservative, a disinfectant and a thickener; the preparation method of the lubricant comprises the following steps:

step one, adding a lubricating liquid, an emulsifier, a preservative and a disinfectant into a solvent for mixing to obtain a mixture 1;

step two, adding human lysozyme into the mixture 1 and mixing to obtain a mixture 2;

step three, adding a thickening agent into the mixture 2, and mixing to obtain a lubricant;

the lubricant contains the following components in every 1000ml of solvent:

the purpose of the application is to overcome the technical defect that the existing traditional rubber condom can only effectively block particles with the diameter similar to the size of human sperm, but can not effectively defend bacteria or viruses with the pore diameter smaller than 120 nanometers. Accordingly, the present application provides a novel lubricated condom comprising a condom body and a lubricant; the lubricant is adhered to the inner surface or/and the outer surface of the condom body; the lubricant comprises the following components: human lysozyme and a solvent. Generally, human lysozyme is basically used as a medicine in a human body, the human lysozyme is creatively used in condoms, and the human lysozyme is coated on the inner surface or/and the outer surface of the condoms, and because the human lysozyme is a strong cation and has positive charge, the human lysozyme can directly act with virus proteins with negative charge to form double salts with DNA, RNA and apoprotein, so that viruses are inactivated; meanwhile, human lysozyme is an alkaline hydrolase capable of hydrolyzing mucopolysaccharide, and because mucopolysaccharide is one of the main components of bacterial cell walls, human lysozyme can catalyze and hydrolyze beta-1, 4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the cell walls, so that insoluble polysaccharide of the cell walls is decomposed into soluble glycopeptides, and bacterial contents escape to dissolve the cell walls. Therefore, when the condom is used, the human lysozyme rapidly reacts with bacteria and/or viruses in semen or vagina, hydrolyzes the bacteria, neutralizes the viruses and inactivates the viruses, so that the condom has antibacterial and antiviral effects, can effectively block the viruses and/or bacteria in the semen and/or vagina and can effectively prevent the transmission of venereal diseases caused by the viruses and/or bacteria passing through the nano through holes of the rubber condom. The lubricating condom is put in a community in a certain city in the middle of Guangdong province, and before the test, the morbidity of syphilis, gonorrhea, condyloma acuminatum and genital herpes in the community is as follows: 55.60/10 ten thousand, 27.16/10 ten thousand, 22.41/10 ten thousand and 10.56/10 ten thousand. After the lubricating condom is put into use in the community for one year, according to the statistics of health departments, the morbidity of syphilis, gonorrhea, condyloma acuminatum and genital herpes of the community is 50.21/10 ten thousand, 24.30/10 ten thousand, 20.21/10 ten thousand and 9.15/10 ten thousand, the incidence rate of venereal diseases in the area is reduced by about 10 percent, and the lubricating condom has great social value.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph showing the change of the total number of colonies in a condom after a permeation sterilization test of recombinant human lysozyme, egg white lysozyme, which is provided in example 1 of the present application, from the outer surface of the condom to the inner surface of the condom;

FIG. 2 shows the enzyme activity loss of recombinant human lysozyme, egg white lysozyme, provided in example 1 of the present application, after the permeation sterilization test from the outer surface of the condom to the inner surface of the condom;

FIG. 3 shows the total number of colonies in a masturbator after a permeation sterilization test of recombinant human lysozyme, egg white lysozyme, provided in example 2 of the present application, from the inner surface of a condom to the outer surface of the condom;

FIG. 4 shows the enzyme activity loss change of the recombinant human lysozyme, the egg white lysozyme, provided in example 2 of the present application, after the permeation sterilization test from the inner surface of the condom to the outer surface of the condom.

Detailed Description

The application provides a lubricating condom and a preparation method thereof, which are used for solving the technical defect that the existing traditional rubber condom can only effectively block particles with the diameter similar to that of human sperm, but can not effectively defend bacteria or viruses with the pore diameter less than 120 nanometers.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The human lysozyme used in the following examples was all recombinant human lysozyme.

Wherein, the recombinant human lysozyme: lot number 20170319, purity 98.50% activity unit 50000U/mg, supplied by Guangzhou Qilong Biotech, Inc.; the recombinant human lysozyme contains 130 amino acids, the molecular weight is about 14700Da, and the amino acid sequence is as follows:

the recombinant human lysozyme is obtained through fermentation engineering, and the method comprises the following steps: preparing 200 ml of culture medium, adding 6 ml of phosphoric acid, 3 g of magnesium sulfate, 4 g of potassium sulfate, 1g of potassium hydroxide and 1.5 g of calcium sulfate into the culture medium, adding distilled water to 200 ml, inoculating glycerol tube seeds after autoclaving, culturing for 36-48 hours at the culturing temperature of 20-35 ℃ with the rotation number of a shaking table of 250 revolutions per minute. Performing seeding tank culture, and finally performing production tank culture. Extracting and purifying the culture solution after the recombinant human lysozyme fermentation expression is completed, and detecting and storing the protein content, the purity and the activity of the extracted and purified protein concentrated solution. Mixing qualified recombinant human lysozyme with sterile pure water, and adjusting the pH value to 5 to obtain 10000U/ml, 30000U/ml and 50000U/ml recombinant human lysozyme solution;

examples 1-2 using Micrococcus muralis: batch number F819BD0354, a product of bio-engineering (shanghai) incorporated;

examples 1 to 2 the buffer solution used was a phosphate buffer solution (pH6.2) prepared by diluting 11.71g of sodium dihydrogen phosphate dihydrate and 7.85g of disodium hydrogen phosphate dodecahydrate to a constant volume of 1L, and sterilizing the mixture with steam under high pressure (121 ℃ C., 20 min);

the preparation method of the normal saline adopted in the embodiments 1 to 2 is that 0.9g of sodium chloride is added to 100ml of constant volume, 9ml of sodium chloride is respectively filled into test tubes, and high-pressure steam sterilization is carried out (121 ℃, 20 min);

the culture medium adopted in the embodiments 1 to 2 is a liquid LB culture medium, and the preparation method of the liquid LB culture medium comprises the steps of fully dissolving 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in a proper amount of deionized water, adjusting the pH value to 7.4 by using sodium hydroxide, fixing the volume to 1L, sterilizing by high pressure steam (121 ℃, 20min), storing at 4 ℃ for standby, and recovering to room temperature when used;

the LB agar slant used in examples 1 to 2 was prepared by dissolving tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g in deionized water, adjusting pH to 7.4 with sodium hydroxide, diluting to 1L, autoclaving (121 ℃ for 20min), packaging into agar slant using sterile test tube, storing at 4 ℃ for later use, and returning to room temperature;

examples 1-2 used LB agar plates prepared by a method comprising slant of LB agar: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 20g of agar, adding a proper amount of deionized water to fully dissolve, adjusting the pH value to 7.4 by using sodium hydroxide, fixing the volume to 1L, sterilizing by using high-pressure steam (121 ℃, 20min), subpackaging into agar plates by adopting sterile plates, storing at 4 ℃ for later use, and recovering to room temperature when in use;

the recovery method of the strains in the embodiments 1 to 2 comprises the following steps: taking 0.5ml of liquid LB culture medium in an aseptic operation mode, dripping the liquid LB culture medium into an ampoule bottle filled with freeze-dried powder of a wall-dissolving micrococcus standard strain, slightly shaking to dissolve and fully disperse freeze-dried bacteria, transplanting a bacteria suspension into an LB agar slant, culturing for 18-24 h in an incubator at 37 ℃, and storing for later use at 4 ℃;

the method for expanding the strain culture of the embodiment 1-2 comprises the following steps: in an aseptic operation mode, an appropriate amount of thalli is picked up to LB agar slant for streak purification, the thalli is cultured in an incubator at the temperature of (37 +/-1) DEG C for 24 hours, and the morphology of a single colony is observed. And selecting a wet, convex, smooth-edge and yellow colony, streaking and purifying the colony in an LB agar plate again, and culturing the colony in an incubator at the temperature of (37 +/-1) ℃ for 24 hours. Storing the plate at 4 deg.C for one month; and (3) selecting a single colony in an LB agar plate subjected to secondary streak purification, uniformly mixing the single colony in 50ml of liquid LB culture medium, shaking the bacteria in a constant temperature shaking table at 37 +/-1 ℃ for 18h at a speed of 110r/min, storing the bacteria at 4 ℃, and recovering the temperature to room temperature when the bacteria are used within one week. Uniformly mixing 500ul of bacterial liquid into 50ml of liquid LB culture medium, and shaking the bacterial liquid in a constant temperature shaking table at the temperature of 37 +/-1 ℃ for 18 hours at a speed of 110r/min to prepare bacterial suspension A;

the bacterial suspensions of examples 1-2 were prepared as follows: centrifuging (12000rpm, 5min) a bacterial suspension A prepared by the enlarged culture of the strain to collect thalli, and washing the thalli to a state without a culture medium by adopting a phosphate buffer solution; then, the ultraviolet spectrophotometer is turned on, the wavelength is adjusted to 450nm, the cell is preheated at the room temperature of (25 +/-1) ° C, a certain amount of the cell is suspended in phosphate buffer, and the concentration of the cell is adjusted by the phosphate buffer, so that the absorbance value at 450nm is 1.300.

Examples 1-2 the enzyme activity of recombinant human lysozyme was tested in different treatment groups by: accurately measuring 1ml (accurate to 0.01ml) of recombinant human lysozyme sample, fully dispersing the recombinant human lysozyme sample in a proper amount of phosphate buffer solution, fixing the volume to 100ml, keeping the variation of the absorbance value within 1min to be 0.025-0.125, and keeping the reading value at 1min to be not less than 1.000, and carrying out parallel test on the same sample. Then, 2.5ml of the prepared bacterial suspension is added into a reaction tube in a water bath kettle at room temperature or constant temperature of (25 +/-1) ° C, 0.5ml of a sample of recombinant human lysozyme is added, and reading A at 450nm is recorded when the reaction is carried out for 1min₁Record reading A at 450nm at 2min of reaction₂And converting the enzyme activity of the corresponding recombinant human lysozyme through a formula, and making two parallels for each recombinant human lysozyme sample.

The result calculation formula is:

wherein I is enzyme activity;

∣A₁-A₂| is the change in absorbance per minute at 450 nm;

ew is the volume (ml) of the original sample contained in 0.5ml of the sample for detection;

0.001 is an activity unit which decreases the absorbance value at 450nm by 0.001 per minute.

The method for counting the Micrococcus muralis plates of examples 1 to 2 was: using a pipette to pipette 1ml of sample into 9ml of physiological saline in an aseptic operation mode to prepare 1:10 sample diluent A; gradually diluting according to 10 times of gradient until the total number of the final plate colonies is 25-250 cfu; the counting step comprises the following steps:

preparing 100ml of LB agar culture medium by using a triangular flask, sterilizing the culture medium by using high-pressure steam (121 ℃, 20min), and taking the culture medium out of a water bath kettle at 55 ℃ for heat preservation for later use;

transferring 1ml of the prepared sample diluent A into three sterile empty culture dishes by using a liquid transfer gun in an aseptic operation mode, adding about 15ml of LB agar culture medium for standby heat preservation, quickly shaking up, and horizontally standing until the culture medium is solidified;

thirdly, after the flat plate is solidified, the flat plate is placed in a constant temperature incubator at 37 ℃ upside down, and the counting can be realized after the flat plate is cultured for 24 hours.

1.0 × 10 in examples 1 to 2⁷The cfu/ml wall-dissolving microballon bacterial suspension is prepared by accurately weighing 0.2g (to 0.01g) in aseptic manner, and counting by plate to 0.5 × 10¹¹The micrococcus muralis bacterial powder with cfu/g viable count is fully dissolved by adding a proper amount of normal saline, and the volume is fixed to 1L. The particle size of the Micrococcus muralis is close to that of sperm, so that the Micrococcus muralis is adopted to simulate the sperm, and the recombinant human lysozyme permeating the condom and the sterilization effect thereof are researched. In the test, 10000U/ml, 30000U/ml and 50000U/ml of recombinant human lysozyme with different enzyme activity gradients and 50000U/ml of egg white lysozyme are taken as experimental groups, and phosphate buffer is taken as a control group to explore the sterilization effect of the recombinant human lysozyme.

Examples 1-4 the female pacifier was a female pacifier, purchased from Ningbo long love adult products, Inc., condom, London, Leio, Inc.

Examples 3-4 the recombinant human lysozyme used in example 4 was supplied by Kyoho Biotech, Inc., purity 99.60%, activity 200000U/ml, lot 20190601. Positive drug acyclovir: chinese food & drug assay research institute, lot number: 170630 and 201703, the content: 99.8 percent; control egg white lysozyme: shanghai Yien chemical technology Co., Ltd., purity 98.50%, activity 40000U/mg, batch number 201806-88-3.

Example 3-example 4 a sample of HPV53 type was collected and provided by the first hospital affiliated to the university of zhongshan, and the sample mass was accurately weighed and homogenized with a glass homogenizer, and an appropriate amount of physiological saline was added to prepare an in vitro suspension of 5mg/ml for use. Taking 200. mu.L of the suspension, and extracting HPV viral DNA by using a virus RNA/DNA rapid purification kit (Code No.9766) of Takara corporation; the strain was identified as HPV53 subtype strain using the nucleic acid detection and genotyping kit (PCR-reverse dot blot) from Daan GenBank, Inc., university of Zhongshan.

Examples 3-4 BSC-1000-II-B2 biosafety cabinets (New Lanpu electronics, Inc., Shenzhen), ESJ780-4 electronic balances (New Lanpu electronics, Inc., Shenzhen), HH-1 constant temperature water bath (Changfeng instruments, Beijing), 7500 fluorescence quantitative PCR instruments (ThermoFisher).

The culture media in examples 3 to 4: RPMI-1640 complete medium, containing 10% fetal bovine serum, 2mM L-glutamine, 10mM HEPES, 50. mu.M 2-mercaptoethanol, 100,000IU penicillin preparation.

Example 1

The embodiment of the application provides a test for permeation sterilization of recombinant human lysozyme and egg white lysozyme with different enzyme activities from the outer surface of a condom to the inner surface of the condom, which comprises the following specific steps:

1. 2ml of 1.0 × 10 was added to the condom⁷cfu/ml wall-dissolving micrococcus bacterial liquid, and sleeving the condom in a vibrating rod;

2. sucking 5ml of 10000U/ml recombinant human lysozyme solution into a masturbation device;

3. inserting a vibration rod into the masturbation device for surging;

4. sampling at four sampling time points of 10min, 20min, 40min and 60min, taking 100 mul of the solution in the condom at each time point for counting the total number of the micrococcus lytica colonies, and taking 100 mul of the solution in the masturbator for counting the total number of the micrococcus lytica colonies;

5. repeating the steps to finish 30000U/ml, 50000U/m recombinant human lysozyme solution, 50000U/ml egg white lysozyme solution and a control group test. The results are shown in tables 1 to 3 and fig. 1 to 2, fig. 1 is a graph showing the change of the total number of colonies in the condom after the permeation sterilization test of the recombinant human lysozyme, egg white lysozyme provided in example 1 of the present application from the outer surface of the condom to the inner surface of the condom; FIG. 2 shows the recombinant human lysozyme provided in example 1 of the present application, in which the enzyme activity loss of the egg white lysozyme varies after the permeation sterilization test from the outer surface of the condom to the inner surface of the condom, and FIG. 1 shows a control (blank control is pH sterile pure water), a test 1 (i.e., 10000U/ml recombinant human lysozyme solution), a test 2 (i.e., 30000U/ml recombinant human lysozyme solution), a test 3 (i.e., 50000U/ml recombinant human lysozyme solution), and a test 4 (i.e., 50000U/ml egg white lysozyme solution), respectively; in FIG. 2, test 1 (i.e., 10000U/ml of recombinant human lysozyme solution), test 2 (i.e., 30000U/ml of recombinant human lysozyme solution), test 3 (i.e., 50000U/ml of recombinant human lysozyme solution), and test 4 (i.e., 50000U/ml of egg white lysozyme solution) are labeled, respectively.

TABLE 1 recombinant human lysozyme, egg white lysozyme varies in the total number of colonies in the condom after the osmotic sterilization test from the outer surface of the condom to the inner surface of the condom

Note: the total number of colonies for all samples was calculated on a 2ml volume basis.

TABLE 2 recombinant human lysozyme, egg white lysozyme from the outer surface of the condom to the inner surface of the condom after the penetration sterilization test of the bacterial colony inside the masturbator

TABLE 3 recombinant human lysozyme, egg white lysozyme from the condom surface to the inner surface of the condom after the penetration sterilization test of recombinant human lysozyme, egg white lysozyme enzyme activity loss

As can be seen from the above table 2, the Micrococcus muralis has a probability of 2.6% to permeate the condom, the quantity is small, and the sterilization rate of the recombinant human lysozyme and the egg white lysozyme to the Micrococcus muralis permeating the condom is as high as 100%;

as can be seen from the graph 1, the total number of the micrococcus muralis colonies in the condom is obviously reduced after the permeation sterilization test of the group added with the recombinant human lysozyme and the egg white lysozyme from the outer surface of the condom to the inner surface of the condom, but the sterilization effect of the recombinant human lysozyme is obviously better than that of the egg white lysozyme compared with the equivalent recombinant human lysozyme and the equivalent recombinant egg white lysozyme with the same activity, and the recombinant human lysozyme reduces and increases the total number of the micrococcus muralis colonies along with the improvement of the enzyme activity; as can be seen from FIG. 2, as the test is carried out, the activity of the recombinant human lysozyme and the activity of the egg white lysozyme are reduced, the activity of the recombinant human lysozyme is reduced slowly, the activity of the egg white lysozyme is reduced rapidly, and the reduction trend of the curve corresponding to FIG. 1 is consistent; it can be seen from the combination of fig. 1 and fig. 2 that both the recombinant human lysozyme and the egg white lysozyme can effectively permeate the condom, and the recombinant human lysozyme has a remarkable bactericidal effect which is obviously superior to that of the egg white lysozyme.

Example 2

The embodiment of the application provides a test for permeation sterilization of recombinant human lysozyme and egg white lysozyme with different enzyme activities from the inner surface of a condom to the outer surface of the condom, which comprises the following specific steps:

1. adding 2ml of 10000U/ml recombinant human lysozyme solution into a condom, and sleeving the condom into a vibrating rod;

2.5ml of 1.0 × 10 was aspirated⁷cfu/ml of a solution of Micrococcus muralis in a masturbator;

3. inserting a vibration rod into the masturbation device for surging;

4. sampling at four sampling time points of 10min, 20min, 40min and 60min, taking 100 mul of the solution in the condom at each time point for enzyme activity determination, and taking 100 mul of the solution in a masturbation device for counting the total number of the micrococcus muralis colonies;

5. repeating the steps to finish 30000U/ml, 50000U/m recombinant human lysozyme solution, 50000U/ml egg white lysozyme solution and a control group test. The results are shown in tables 4 to 5 and fig. 3 to 4. FIG. 3 shows the total number of colonies in a masturbator after a permeation sterilization test of recombinant human lysozyme, egg white lysozyme, provided in example 2 of the present application, from the inner surface of a condom to the outer surface of the condom; FIG. 4 shows the enzyme activity loss change of the recombinant human lysozyme, the egg white lysozyme, provided in example 2 of the present application, after the permeation sterilization test from the inner surface of the condom to the outer surface of the condom. In FIGS. 3-4, test 1 (i.e., 10000U/ml recombinant human lysozyme solution), test 2 (i.e., 30000U/ml recombinant human lysozyme solution), test 3 (i.e., 50000U/ml recombinant human lysozyme solution), and test 4 (i.e., 50000U/ml egg white lysozyme solution) are labeled, respectively.

TABLE 4 Total number of bacterial colonies of recombinant human lysozyme, egg white lysozyme after the osmotic sterilization test from the inner surface of the condom to the outer surface of the condom

Note: the total number of colonies for all samples was calculated on a volume of 5 ml.

TABLE 5 recombinant human lysozyme, egg white lysozyme from the inner surface of the condom to the outer surface of the condom after the penetration sterilization test of recombinant human lysozyme, egg white lysozyme enzyme activity loss

It can be known from the graphs 1-4 that the curves corresponding to the

graphs

1 and 3 and the

graphs

2 and 4 are almost consistent, that is, the recombinant human lysozyme and the egg white lysozyme have obvious effects no matter the recombinant human lysozyme and the egg white lysozyme penetrate from the inside to the outside of the condom to achieve the sterilization effect or penetrate from the outside to the inside of the condom to achieve the sterilization effect, and the sterilization effect of the recombinant human lysozyme in the experiment is obviously superior to that of the egg white lysozyme.

Example 3

The embodiment of the application provides a contrast test for the high-risk HPV virus resistance of recombinant human lysozyme, acyclovir and egg white lysozyme, which comprises the following specific steps:

1. preparing a lubricating condom: the formula is prepared from the following raw materials, glycerol, tween-80, polyhexamethylene biguanide and simethicone are added into 1000ml of sterile water and stirred uniformly, then recombinant human lysozyme with the purity of 90% -99% is added into the sterile water and mixed and stirred uniformly, finally, polyquaternium Fs222 is added into the sterile water and mixed and stirred uniformly, and the pH value is adjusted to 5, so that the lubricant is obtained, wherein each 1000ml of sterile water in the lubricant contains:

50 per mill of glycerol; 1% tween-80; 4% o of bis (hydroxymethyl) imidazolidinyl urea; 2 per mill of polyhexamethylene biguanide; 20 per mill of polyquaternium Fs 222; 50 per mill of dimethyl silicone oil; 3000 million units of recombinant human lysozyme.

2. The test is provided with a blank group, a recombinant human lysozyme, a positive drug acyclovir and a control drug egg white lysozyme group. 3ml of the in vitro suspension (containing the virus particles of the HPV53 subtype strain) at 5mg/ml is pipetted into 4 10ml EP tubes, shaken uniformly and cultured in an incubator at 37 ℃.

Setting the positive control substance diluted by times: application liquid of recombinant human lysozyme (30000U/ml); preparing acyclovir into 500 mu g/ml application liquid; egg white lysozyme (30000U/ml) was used as a liquid with phosphate buffered saline as a control.

The specific operation method is that 4 masturbation devices are respectively filled with 2ml of the prepared virus suspension; the condom is sleeved on a shaker, 1ml of liquid of three positive control products (recombinant human lysozyme, acyclovir and egg white lysozyme) is respectively and uniformly coated on the outer surface of the condom, the three vibrators coated with the positive control products and sleeved with the condom are inserted into a masturbation device for surging, and are respectively marked as an acyclovir group, a recombinant human lysozyme group and an egg white lysozyme group, and the blank group is obtained by replacing the positive control products with phosphate buffer solution for the same test. Samples of 10. mu.l were taken at 10min, 20min, 40min and 60min for four sampling times, respectively, and HPV viral DNA was extracted using a viral RNA/DNA rapid purification kit (Code No.9766) from Takara. The HPV53 type nucleic acid detection kit is used for making and detecting an HPV53 type fluorescent PCR standard curve. The results are shown in Table 5.

Table 5 fluorescent PCR assay for HPV53 type

As can be seen from Table 5, the comparison among the acyclovir group, the recombinant human lysozyme group and the egg white lysozyme group shows that the in vitro degradation effect of the acyclovir, the recombinant human lysozyme and the egg white lysozyme on the HPV nucleic acid is gradually increased along with the increase of the treatment time, the effect of the acyclovir and the recombinant human lysozyme group is similar, the effect of the acyclovir and the recombinant human lysozyme group is superior to that of the egg white lysozyme, and the recombinant human lysozyme serving as the human-derived protein has a good in vitro degradation effect on the HPV nucleic acid.

Example 4

The embodiment of the application provides a comparison test of the high-risk HPV virus resistance of recombinant human lysozyme with different enzyme activities, the steps are similar to those of embodiment 3, and the specific steps are as follows:

1. preparing a lubricating condom: the formula is prepared from the following raw materials, glycerol, tween-80, polyhexamethylene biguanide and simethicone are added into 1000mL of sterile water and uniformly stirred, then 3000U-30 ten thousand U/mL of recombinant human lysozyme with the purity of 90-99% is added into the sterile water and uniformly mixed, finally, polyquaternium Fs222 is added into the sterile water and uniformly mixed, the pH value is adjusted to 5, and the lubricant is obtained, wherein each 1000mL of sterile water in the lubricant contains:

50 per mill of glycerol; 1% tween-80; 4% o of bis (hydroxymethyl) imidazolidinyl urea; 2 per mill of polyhexamethylene biguanide; 20 per mill of polyquaternium Fs 222; 50 of dimethyl silicone oil; recombinant human lysozyme. The enzyme activity of the recombinant human lysozyme group is 1 ten thousand units per milliliter; the medium-enzyme activity recombinant human lysozyme group is 3 ten thousand units of recombinant human lysozyme per milliliter; the high-enzyme-activity recombinant human lysozyme group is 5 ten thousand units of recombinant human lysozyme per milliliter

2. The test is provided with a blank group, a low-enzyme activity recombinant human lysozyme group, a medium-enzyme activity recombinant human lysozyme group and a high-enzyme activity recombinant human lysozyme group. 3ml of the suspension in vitro (containing HPV53 subtype strain virus particles) is pipetted into 4 10ml of EP tubes, shaken well in 10ml of EP tubes and cultured in an incubator at 37 ℃.

The low-enzyme activity recombinant human lysozyme group is 1 ten thousand units of recombinant human lysozyme per milliliter; the medium-enzyme activity recombinant human lysozyme group is 3 ten thousand units of recombinant human lysozyme per milliliter; the high-enzyme-activity recombinant human lysozyme group is an application liquid of 5 ten thousand units of recombinant human lysozyme per milliliter; phosphate buffer was used as control.

The specific operation method is that three masturbation devices are respectively filled with 2ml of the prepared virus suspension; sheathing the condom on a vibrator; respectively sucking 1ml of three medicine liquids with different enzyme activities (a low-enzyme activity recombinant human lysozyme group, a medium-enzyme activity recombinant human lysozyme group and a high-enzyme activity recombinant human lysozyme group), respectively and uniformly smearing the medicine liquids on the outer surface of a condom, inserting the three medicine-smeared vibrators sleeved with the condom into a masturbation device for surging, wherein the blank pair group is similar to the above group, and the difference is only that the medicine liquids with different enzyme activities are replaced by phosphate buffer solutions. Samples of 10. mu.l were taken at 10min, 20min, 40min and 60min for four sampling times, respectively, and HPV viral DNA was extracted using a viral RNA/DNA rapid purification kit (Code No.9766) from Takara. The HPV53 type nucleic acid detection kit is used for making and detecting an HPV53 type fluorescent PCR standard curve.

TABLE 6 fluorescent PCR detection results of low, medium and high enzyme activities of recombinant human lysozyme on HPV53 type

As can be seen from Table 6, the effect of recombinant human lysozyme on HPV nucleic acid degradation in vitro gradually increased with the increase of the treatment time, the effects of the recombinant human lysozyme in the high-dose group and the medium-dose group were close, and the effect of the recombinant human lysozyme in the low-dose group was inferior. The recombinant human lysozyme lubricating fluid has the best effect in a high-dose group, and provides a new choice for the antibacterial and antiviral effects of the condom and the barrier effect.

Example 5

The embodiment of the application provides a vaginal mucosa irritation test that the lubricant for lubricating condoms is recombinant human lysozyme-containing lubricant, common lubricating liquid and egg white lysozyme lubricating liquid, which comprises the following specific steps:

according to the regulations of GB 15979-2002 disposable sanitary article and the 2008 edition of the disinfection technical Specification, SPF SD rats are selected for the vaginal mucosa stimulation test, and the lubricant containing the recombinant human lysozyme in the embodiment of the application contains the following components in each 1000ml of sterile water: 50 per mill of glycerol; 1% tween-80; 4% o of bis (hydroxymethyl) imidazolidinyl urea; 2 per mill of polyhexamethylene biguanide; 20 per mill of polyquaternium Fs 222; 50 per mill of dimethyl silicone oil; 2000 ten thousand units of recombinant human lysozyme; the common lubricating liquid is commercially available, and the formula of the common lubricating liquid comprises water, glycerol, sodium hydroxide, sodium polyacrylate, benzyl alcohol, p-hydroxybenzoic acid, polyquaternium Fs222, tween-80, germabp, polyhexamethylene biguanide and simethicone; the formula of the egg white lysozyme lubricating fluid is similar to that of the lubricating fluid containing the recombinant human lysozyme in the embodiment of the application, and the difference is only that the recombinant human lysozyme is replaced by the egg white lysozyme.

9 SPF grade SD rats were tested per lubricant. The results of the assay are shown in Table 7. The results show that the lubricant containing the recombinant human lysozyme in the example of the application has no irritation (no hyperemia and edema) to the vaginal mucosa of rats, and the other two lubricants (a common lubricant and an egg white lysozyme lubricant) have different irritation to the vaginal mucosa of rats.

The egg white lysozyme lubricating oil test group comprises three animals, and the visual observation shows that the vaginal mucosa of one animal can be obviously congested, edematous, ulcer and slight erosion, and the mucosal surface is covered with milky mucus; in the other two animals, slight congestion and edema of the vaginal mucosa were observed, and a small amount of milky mucus was present on the mucosal surface. When observed under an optical microscope (HE, multiplied by 100), the animal vaginal mucosa epithelial cells can be necrotized and shed, the mucosa is obviously congested and edematous, and a large amount of inflammatory cells can infiltrate;

no obvious pathological changes are observed in the animal vaginal mucosa of the common lubricating fluid under the visual observation and the optical microscope observation. The test egg white lysozyme lubricating oil condoms have an irritation index of 9.88 to the vaginal mucosa and irritation to the vaginal mucosa of New Zealand rabbits, scored according to the vaginal mucosa irritation response standard. The histopathological results are shown in table 7.

The result shows that the lubricant containing the recombinant human lysozyme has no stimulation and pathological change on vaginal mucosa of SPF SD rats, which indicates that the humanized recombinant human lysozyme has good affinity and is more suitable for being used as a lubricating fluid for human condoms. However, the lubricating fluid containing egg white lysozyme has slight stimulation to vaginal mucosa of SPF SD rats and hyperemia and edema, which are related to the species source of the protein.

TABLE 7 vaginal mucosa irritation test results of different lubricants on SD rats

The traditional condoms have more than one hundred million and more than 120 nanometers of pore diameter each (the latex membrane body has quality problems, and natural cracks between five thousand and seventy thousand nanometers exist), and experiments show that the rate of the cytolytic micrococcus with the size equivalent to that of sperms to penetrate through the condoms is high, and viruses and bacteria smaller than the sperms are more likely to penetrate through the condoms to infect people. Test data prove that the recombinant human lysozyme condom lubricating liquid can kill bacteria and viruses and neutralize the viruses in semen, so that the bacteria and/or the viruses in the semen or the vagina cannot pass through the nano through holes of the rubber condom, and the recombinant human lysozyme is attached to the condom lubricating liquid, so that the condom lubricating liquid has obvious protective effects on preventing virus and bacterial infection of both men and women.

The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A lubricated condom, comprising a condom body and a lubricant;

the lubricant comprises the following components:

human lysozyme and a solvent.

2. The lubricated condom of claim 1, wherein the lubricant has a pH of 4.5 to 5.5.

3. The lubricated condom of claim 1, further comprising a lubricating fluid, an emulsifier, a preservative, a disinfectant, and a thickener.

4. The lubricated condom of claim 3, wherein the lubricating fluid is selected from glycerol or/and dimethicone; the emulsifier is selected from tween-80; the preservative is selected from bis (hydroxymethyl) imidazolidinyl urea; the disinfectant is selected from polyhexamethylene biguanide; the thickener is selected from cationic acrylamide polymerization emulsions.

5. The lubricated condom of claim 4, wherein the lubricant comprises, per 1000ml of solvent:

6. the lubricated condom of claim 5, wherein the lubricant comprises, per 1000ml of solvent:

7. the lubricated condom of claim 1, wherein the condom body is a male condom body.

8. The lubricated condom of claim 1, wherein the condom body is a female condom body.

9. A method of making a lubricated condom, comprising the steps of:

step 1, mixing human lysozyme and a solvent to prepare a lubricant;

10. The method of manufacturing according to claim 9, wherein the lubricant further comprises a lubricating liquid, an emulsifier, a preservative, a disinfectant and a thickener; the preparation method of the lubricant comprises the following steps:

the lubricant contains the following components in every 1000ml of solvent: