CN110227156A - A kind of elimination gynaecological imflammation pathogenic bacteria and the lgY targeting preparation of HPV viruse and preparation method thereof - Google Patents

Abstract

This application discloses a kind of elimination gynaecological imflammation pathogenic bacteria and the lgY targeting preparation of HPV viruse and preparation method thereof, component including following parts by weight: 4 ~ 6 parts of lgY, 2 ~ 4 parts of targeting peptides, 30 ~ 60 parts of thermo-sensitive gels, preparation method includes the following steps: that lgY and targeting peptides are added in (1) in the container equipped with water, and it is spare that ultrasound forms it into mixed solution；(2) thermo-sensitive gel material is added in the mixed solution prepared to step (1), so that thermo-sensitive gel material dissolves, the solution of formation is lgY targeting preparation for stirring.Layer of gel is formed in vagina and uterine neck interior surface when lgY targeting preparation provided by the invention uses, extend the pharmacy effect time, inflammation pathogenic bacteria and HPV viruse of the lgY in addition to eliminating vagina and cervical mucosa surface in gel, more penetrate mucous membrane under the carrying of targeting peptides and enter mucous membrane and targeted with lower portion and remove depth virus infection, thus thoroughly, efficiently eliminate gynaecological imflammation pathogenic bacteria and HPV viruse.

Description

A kind of lgY targeting preparation and its preparation for eliminating gynaecological imflammation pathogenic bacteria and HPV viruse Method

Technical field

This application involves gynecological disease field of medicaments, in particular to a kind of elimination gynaecological imflammation pathogenic bacteria and HPV disease The lgY targeting preparation and preparation method thereof of poison.

Background technique

Cervical carcinoma is the gynecologic malignant tumor that China is only second to that breast cancer occupies the 2nd, but cervical carcinoma is also current generation simultaneously The specific cancer of unique cause of disease in boundary generally believes that human papilloma virus (HPV) infection is cause cervical carcinogenesis important at present Factor, but HPV infection is not the single factor for being uterine neck carcinogenesis, other than HPV infection, palace caused by reproductive system pathogen Neck chronic inflammation is also the independent hazard factor for inducing cervical carcinoma.Therefore, female gynecological inflammation pathogenic bacteria are eliminated and HPV viruse is The effectively most effective behave of prevention cervical carcinoma.

Currently, being related to eliminating there are mainly two types of the modes of female gynecological inflammation pathogenic bacteria or HPV viruse: inoculation HPV viruse Vaccine and antibiotic or drug therapy.

Inoculation HPV viruse vaccine is only capable of the HPV viruse for Partial Species, cannot generate and be immunized to all HPV viruses, more Female gynecological inflammation pathogenic bacteria cannot be eliminated, such as NEISSERIA GONORRHOEAE, chlamydia trachomatis, some aerobic bacterias, anaerobic bacteria, giant cell disease Poison, mycoplasma hominis and Ureaplasma urealyticum etc. cause a disease so inoculation HPV viruse vaccine cannot eliminate female gynecological inflammation comprehensively Bacterium or HPV viruse.

Antibiotic or drug therapy initial stage can achieve the effect that eliminate female gynecological inflammation pathogenic bacteria or HPV viruse, but with Antibiotic failure get worse, using the excessive direct result of antibiotic be exactly make pathogen generate drug resistance, cause it is tighter The gynaecological imflammation of weight.There are also some womens to tend to the drug therapy gynaecological imflammation using traditional Chinese medicine ingredients, but the medicine of traditional Chinese medicine ingredients Object is largely antipyretic and antidote functions, is commonly used as the adjuvant treatment of chronic gynaecological imflammation, cannot function as the head for HPV viruse Choosing treatment.Otherwise the pathogen for causing gynaecological imflammation is allowed to take advantage of the occasion to spread development with " slow soldier is to formidable opponent ", to delay the state of an illness. And the vagina of healthy women is acidic environment, there is self-cleaning function beneficial bacterium, the gel of long-time abuse of antibiotics and traditional Chinese medicine ingredients The method for treating gynaecological imflammation is improper, can kill vagina beneficial bacterium, destroys the acid-base balance of vagina.A large amount of anti-medicine is cultivated instead Bacterium often makes the concurrent multiple infection of single inflammatory patients, and repeated infection gynaecological imflammation often causes high-risk HPV infection, such as Fruit is not adopted an effective measure, canceration rate 1/5 after 5 years.

Summary of the invention

The main purpose of the application is to integrate existing shortage elimination gynaecological imflammation pathogenic bacteria or HPV viruse is effective The problem of means, provides a kind of new direct attack and preparation method thereof for eliminating gynaecological imflammation pathogenic bacteria or HPV viruse comprehensively.

To achieve the goals above, provide it is a kind of elimination gynaecological imflammation pathogenic bacteria and HPV viruse lgY targeting preparation and Preparation method, concrete scheme are as follows:

A kind of lgY targeting preparation for eliminating gynaecological imflammation pathogenic bacteria and HPV viruse, the component including following parts by weight: 4 ~ 6 parts LgY, 2 ~ 4 parts of targeting peptides, 30 ~ 60 parts of thermo-sensitive gels.

IgY is one kind of antibody, is the immunoglobulin extracted using biological high-tech.Combine potency and in and virus Or inhibit in terms of the ability of bacterium, IgY is similar to human antibodies such as IgG, IgE etc..IgY can change virus surface structure and It is combined with virus；It may be prevented from breeding in cell entry human cell in this way.IgY also can make it lose work in conjunction with pathogenic bacteria Property and lose pathogenecity.IgY can also be such that the phagocytosis of macrophage is substantially improved, and significantly increase body autoimmunity. Because IgY will not change bacterium or virus inhereditary material and biological characteristics, will not induce virus or mutant bacteria and Generate drug resistance；It fails increasingly serious today in antibiotic, this feature of IgY is of great practical significance.

LgY targeting preparation provided by the invention is solution shape medicament, usually 10 ~ 20 DEG C of storages, directly to vagina when use Administration under the influence of human body temperature, realizes conversion process of the solution to gel, so that medicament is in yin after medicament enters vagina Road and uterine neck interior surface form layer of gel, extend the pharmacy effect time, the lgY in gel in addition to eliminate vagina with And inflammation pathogenic bacteria and the HPV viruse on cervical mucosa surface, mucous membrane is more penetrated under the carrying of targeting peptides enters mucous membrane with lower part Depth virus infection is removed in position targeting, thus thoroughly, efficiently eliminate gynaecological imflammation pathogenic bacteria and HPV viruse.

Preferably, the lgY targeting preparation includes the component of following parts by weight: 5 parts of lgY, 3 parts of targeting peptides, and 50 Part thermo-sensitive gel.

Preferably, it extracts and obtains in the yolk of the birds of the lgY self-infection HPV viruse.

Further, the birds are high immune hen.

Preferably, the targeting peptides are small-molecular peptides, amino acid sequence such as SEQ ID NO.1 or SEQ ID Shown in NO.2 or SEQ ID NO.3.

It preferably, further include parts by weight is 3 ~ 5 parts of ethyl alcohol.

It preferably, further include parts by weight is 0.5 ~ 2 part of motherwort extracting solution.

It preferably, further include parts by weight is 0.5 ~ 2 part of rose extract.

It preferably, further include parts by weight is 0.1 ~ 0.2 part of peppermint extracting solution.

A kind of preparation method of lgY targeting preparation, includes the following steps:

(1) lgY and targeting peptides are added in the container equipped with water, it is spare that ultrasound forms it into mixed solution；

(2) thermo-sensitive gel material is added in the mixed solution prepared to step (1), stirring is so that the dissolution of thermo-sensitive gel material, shape At solution be lgY targeting preparation.

Compared to the prior art, the application has the following advantages:

(1) compared with common vaginal jellies, medicament provided by the invention is solution shape in administration, convenient for medicament is placed in vagina Place, and overcome traditional vagina preparation and lack good spreadability and the low disadvantage of coverage rate；

(2) medicament forms layer of gel in vagina and uterine neck interior surface, extends pharmacy effect time, the lgY in gel Inflammation pathogenic bacteria and HPV viruse in addition to eliminating vagina and cervical mucosa surface, more penetrate mucous membrane under the carrying of targeting peptides It is targeted into mucous membrane with lower portion and removes depth virus infection, to thoroughly, efficiently eliminate gynaecological imflammation pathogenic bacteria and HPV disease Poison；

(3) targeting peptides and the binding force and targeting peptides of lgY are improved using special small molecule targeting peptides through long period of development In conjunction with the potency ratio for eliminating gynaecological imflammation pathogenic bacteria and HPV viruse to the penetration power of skin, is improved after lgY, so that potency is high Up to 1:1024.

Specific embodiment

It, below will be to the skill in the embodiment of the present application in order to make those skilled in the art more fully understand application scheme Art scheme is clearly and completely described, it is clear that and described embodiment is only the embodiment of the application a part, without It is whole embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not before making creative work Every other embodiment obtained is put, shall fall within the protection scope of the present application.

It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.The application is described in detail below in conjunction with embodiment.

Embodiment 1

LgY targeting preparation provided by the invention, each component preparation are as follows:

1, lgY is prepared

U.S.'s breeding bird inlay is raised in the henhouse of no air pollution, non-environmental-pollution, HPV is recombinated using technique for gene engineering Antigen, and using the bird inlay of HPV antigen infection raising, immune egg is picked, prepares aioli with eggbeater and yolk sieve, Extract antiviral specificity composite IgY.

Extracting method is degreasing purification, concrete operations are as follows:

(1) it is slowly added to physiological saline in yolk liquid and is stirred continuously 10min, wherein the volume of yolk liquid and physiological saline Than, with 8000rpm, 6 DEG C of centrifugation 15min, collecting supernatant by mixed liquor after being stored at room temperature 15min for 1:1；

(2) supernatant obtained in step (1) is taken, chloroform is slowly added in supernatant and is stirred, wherein supernatant and chloroform Volume ratio be 1:1, by mixed liquor in -20 DEG C of standings 4h, later with 8000rpm, 6 DEG C of centrifugation 15min, collection supernatant is obtained IgY extracting solution；

(3) IgY extracting solution obtained in step (2) is taken, saturated ammonium sulfate solution is slowly added in IgY extracting solution, wherein IgY The volume ratio of extracting solution and saturated ammonium sulfate solution is 2:1, and mixed liquor 1h, 6 DEG C of standing 4h are stirred at room temperature in magnetic stirring apparatus, it Afterwards with 3500rpm, 4 DEG C of centrifugation 20min, supernatant and precipitating are collected respectively；

(4) take supernatant obtained in step (3), saturated ammonium sulfate solution be slowly added in supernatant, wherein supernatant and The volume ratio of saturated ammonium sulfate solution is 1:1, and magnetic stirring apparatus is stirred liquid 1h, 6 DEG C of standing 4h at room temperature, later with 3500rpm, 6 DEG C of centrifugation 20min abandon supernatant until not having Precipitation in mixed liquor, collect precipitating；

(5) it takes and is precipitated obtained in step (3) and step (4), PBS solution resuspension precipitating is added and obtains supernatant, on gained Saturated ammonium sulfate solution is slowly added in clear liquid, wherein the volume ratio of supernatant and saturated ammonium sulfate solution is 2:1, magnetic agitation Device is stirred liquid 1h, 6 DEG C of standing 4h at room temperature, later with 3500rpm, 6 DEG C of centrifugation 20min, abandons supernatant, collects precipitating；

(6) it is precipitated 3 times obtained in PBS solution washing step (5), PBS is added by step (5) Central Plains supernatant volume equivalent Precipitating is resuspended in solution, by the merging of resulting precipitating suspension through in the processed bag filter of EDTA, dialyse 48h in PBS, every It is primary that 12h changes PBS liquid, totally 4 times, is concentrated with PEG20000 to dialysis product after dialysis, obtains IHNV Yolk antibody.It takes suitable It is temporary in -20 DEG C to measure antibody, is stored for future use after Excess antibody freeze-drying in -80 DEG C.Wherein PBS solution used in S4.5 and S4.6 PH=7.4, concentration 0.01mol/L.

2, prepared by targeting peptides

One, targeting peptides shown in SEQ ID NO.1 are prepared

Material: Lys, Ala, Arg, Leu, Glu, Lys with Fmoc (fluorenylmethyloxycarbonyl) protection, TBTU (O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester), HBTU (O- benzotriazole -1- base-N, N, N, N- tetramethyl urinate hexafluorophosphoric acid rouge) With HOBT (1- hydroxy benzo triazole), piperidines, hexahydropyridine, acetic anhydride, methylene chloride；Solvent: DMF (N, N- dimethyl methyl Amide), TFA (trifluoroacetic acid), ACN (acetonitrile), ice ether.

Synthetic method uses the solid-phase synthesis of Fmoc (fluorenylmethyloxycarbonyl) protection, the specific steps are as follows:

(1) coupling of Lys, Ala: claiming Lys 10g, takes off Fmoc 10min with 20% hexahydropyridine solution of 100ml.After suction filtration again It is washed 2 times with 100ml DMF, 2 minutes each time, methanol washs 1 time, and methylene chloride washed once, then be washed with 100ml DMF It washs 2 times, 2 minutes every time, takes a small amount of resin to do ninhydrin inspection after washing, be as a result positive.

Then following raw material is added into reactor:

Ala	12.3g
		TBTU	10.2g
HOBT	6.5g
		HBTU	3.7g
DMF	120ml

After raw material adds, 1h is reacted.It after time, is washed 3 times with 100ml DMF, 2 minutes each time, methanol washing 1 Secondary, 100ml DMF is washed 1 time, the time 2 minutes.Indenes inspection is negative

(2) raw material is converted, step (1) is repeated and is successively coupled Arg, Leu, Glu, Lys, finally closed with acetic anhydride.

(3) methanol shrinks 5min+5min, dry adsorbent.

(4) 2h is cracked using classical lysate, it is SEQ ID NO.1 in sequence table that the sedimentation of ice ether, which obtains amino acid sequence, The thick peptide.

(5) 10-100% that thick peptide obtained above carries out sample is analyzed first, in the retention time for determining main peak Afterwards, linear gradient analyzes sample；

(6) deionized water and acetonitrile (4:1) being capable of more sufficient dissolved samples product polypeptides；

(7) 200mg polypeptide is placed in the dissolution bottle of 10ml, and deionized water is added, and after ultrasonic dissolution, 1 has with 0.45 μm The filtering of machine phase filter, takes supernatant；

(8) it receives peak to be prepared by linear gradient, and determines whether MS is correct；

(9) in order to meet the amount of liquid of purity requirement, the tracking that purity is carried out to the liquid received is fed back；

(10) at 40 DEG C, the solution of collected qualification is concentrated into 40-50ml；

(11) solution being concentrated is placed in 100ml beaker, is placed in refrigerator-freezer and is freezed；

(12) dry；

(13) it weighs；

(14) it instead beats；

(15) it packs.

After purification, to get targeting peptides small molecule of the present invention, sequence is SEQ ID in sequence table to HPLC for freeze-drying Described in NO.1.

Two, targeting peptides shown in SEQ ID NO.2 and 3 are prepared

According to the preparation step of SEQ ID NO.1, raw material are replaced, synthesize other two targeting peptides small molecules of the present invention , sequence is described in SEQ ID NO.2 and SEQ ID NO.3 in sequence table.

3, lgY targeting preparation is prepared

LgY made from the present embodiment is weighed 6 parts, every part of 5g, is added in 500ml water, 6 are assembled with the container difference label of lgY For A, B, C, D, E, F, SEQ ID NO.1/ SEQ ID NO.1/SEQ ID NO.2/ is wherein mixed into A, B, C, D, E respectively SEQ ID NO.3/ outsourcing targeting peptides (holding high biotechnology purchased from Nanjing Lay) mix, and do not add any one targeting peptides in F, ultrasound After dissolution, B, C, D, E, F, which are added, to be dissolved purchased from the temperature sensing material 50g of BASF AG's production up to lgY targeting preparation, and A is made Ordinary gel agent.

The character situation of final 6 groups of lgY targeting preparations is as follows:

Product	Potency ratio
		A	1:105
B	1:1024
		C	1:1020
D	1:1021
		E	1:565
F	1:57
		Pure lgY	1:1

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for those skilled in the art For member, various changes and changes are possible in this application.Within the spirit and principles of this application, it is made it is any modification, Equivalent replacement, improvement etc., should be included within the scope of protection of this application.

Sequence table

<110>Hua Yi Cisco (Chengdu) Biotechnology Co., Ltd

<120>a kind of elimination gynaecological imflammation pathogenic bacteria and the lgY targeting preparation of HPV viruse and preparation method thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Lys Ala Arg Leu Glu Lys

1 5

<210> 2

<211> 6

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Lys Asp Ala Leu Glu Lys

1 5

<210> 3

<211> 6

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

Lys Glu Leu Leu Glu Lys

1 5

Claims (10)

1. a kind of lgY targeting preparation for eliminating gynaecological imflammation pathogenic bacteria and HPV viruse, which is characterized in that including following parts by weight Component: 4 ~ 6 parts of lgY, 2 ~ 4 parts of targeting peptides, 30 ~ 60 parts of thermo-sensitive gels.

2. lgY targeting preparation as described in claim 1, which is characterized in that the component including following parts by weight: 5 parts of lgY, 3 Part targeting peptides, 50 parts of thermo-sensitive gels.

3. lgY targeting preparation as claimed in claim 1 or 2, which is characterized in that the birds of the lgY self-infection HPV viruse It extracts and obtains in yolk.

4. lgY targeting preparation as claimed in claim 3, which is characterized in that the birds are high immune hen.

5. lgY targeting preparation as claimed in claim 1 or 2, which is characterized in that the targeting peptides are small-molecular peptides, amino Acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3.

6. lgY targeting preparation as described in claim 1, which is characterized in that further include the ethyl alcohol that parts by weight are 3 ~ 5 parts.

7. lgY targeting preparation as described in claim 1, which is characterized in that further include that the motherwort that parts by weight are 0.5 ~ 2 part mentions Take liquid.

8. lgY targeting preparation as described in claim 1, which is characterized in that further include that the rose that parts by weight are 0.5 ~ 2 part mentions Take liquid.

9. lgY targeting preparation as described in claim 1, which is characterized in that further include that the peppermint that parts by weight are 0.1 ~ 0.2 part mentions Take liquid.

10. a kind of preparation method of lgY targeting preparation as claimed in claim 1 or 2, which comprises the steps of:

(1) lgY and targeting peptides are added in the container equipped with water, it is spare that ultrasound forms it into mixed solution；

(2) thermo-sensitive gel material is added in the mixed solution prepared to step (1), stirring is so that the dissolution of thermo-sensitive gel material, shape At solution be lgY targeting preparation.