CN111848740B - Vaginal secretion derived polypeptide and application thereof in inhibiting candida albicans infection - Google Patents

Abstract

The invention discloses a vaginal secretion derived polypeptide and application thereof in inhibiting candida albicans infection, relates to the technical field of biology, and aims to inhibit candida albicans infection of vaginal epithelial cells by a novel polypeptide PDMAC. The polypeptide amino acid sequence is ATPTSTLRTAPPPKVLT, the theoretical molecular weight is 1751.06Da, the theoretical isoelectric point is 11, the instability coefficient is 49.74, the fat coefficient is 74.71, the hydrophilicity is-0.21, the polypeptide is synthesized by a solid phase method, and the C end and the N end are chemically modified by polyethylene glycol and carbamic acid respectively.

Description

A vaginal secretion-derived polypeptide and its application in inhibiting Candida albicans infection

technical field

The invention relates to the field of biotechnology, in particular to a vaginal secretion-derived polypeptide and its application in inhibiting Candida albicans infection.

Background technique

Vulvovaginal candidiasis (VVC) is one of the most common female vulvovaginal inflammations. Statistics show that about 75% of women have suffered from VVC at least once in their lives, and 45% of women have experienced two or more episodes. Suffering from VVC for a long time will not only bring great troubles to the life of the patient, but also lead to diseases such as cervicitis and cervical erosion, salpingo-oophoritis, pelvic inflammatory disease, secondary infertility, and malignant tumors of the reproductive tract; Risks of premature rupture of membranes, premature birth, fetal intrauterine infection, neonatal thrush, etc. Therefore, the prevention and treatment of VVC is the top priority in the research of female reproductive tract infection.

The main pathogenic bacterium of VVC is Candida albicans, which is a parasite in epithelial cells and has two forms of hyphae and yeast. In the human body, Candida albicans usually exists in the vagina, skin, oral cavity, upper respiratory tract and intestinal tract as a non-pathogenic yeast phase, and is symbiotic with the host. When the composition of normal vaginal flora changes (application of broad-spectrum antibiotics) or systemic and local vaginal immunity is suppressed (pregnancy, diabetes, application of corticosteroids, etc.), Candida albicans transforms into hyphae capable of rapid growth and destruction of host cells Adhesion and colonization of vaginal epithelial cells, and then complete the process of invading vaginal epithelial cells under the action of various enzymes; in addition, Candida albicans can also parasitize in macrophages to escape the body's immune response mechanism, resulting in local Candida albicans infection. Rapid growth and reproduction breaks the vaginal microecological balance, eventually leading to a mixed infection dominated by Candida albicans, accompanied by an increase in Corynebacterium, Bacteroides, and Staphylococcus.

At present, the treatment plan for VVC is mainly to use local short-term antifungal drugs to restore the normal vaginal flora, but its clinical efficacy is not satisfactory, and there are still many patients who have recurrent vulvovaginal candidiasis after receiving standardized antifungal treatment ( Recurrent vulvovaginal candidiasis, RVVC), and prone to drug resistance. In recent years, it has been reported that the development of new drugs for VVC, such as: echinocandin can non-competitively inhibit the biosynthesis of Candida albicans cell wall polysaccharide - β-(1,3)-D-glucan, destroying the cell wall Integrity, so as to achieve the purpose of sterilization; berberine, clove basil and other plant extracts can block Candida albicans from using L-methyl-methionine to synthesize ergosterol, an important component of cell membrane, and can inhibit Candida albicans from yeast phase transition It is the mycelial phase, reducing its pathogenicity; cationic polypeptides such as Hisstatin and BM2 can combine with the acidic phospholipids and lipopolysaccharides outside the phospholipid bilayer of the cell membrane of Candida albicans, and destroy the structure of the lipid bilayer to inhibit the growth of Candida albicans purpose of reproduction.

There are a variety of physiologically active polypeptides in organisms, which are closely related to infection, immune regulation, and tumor lesions. Vaginal secretion examination is currently a routine examination method in the diagnosis of obstetrics and gynecology diseases, which has the advantages of convenience, speed and non-invasiveness. Female vaginal secretions are mainly composed of Bartholin glands, cervical glands, endometrial secretions, vaginal mucosal exudate, vaginal flora, and exfoliated vaginal epithelial cells, which act as physical barriers, flora balance, Antibacterial and protective effects on the body. There are nearly a thousand kinds of antibacterial endogenous polypeptides in vaginal secretions, and they are widely affected by hormone levels (follicular phase, luteal phase, menstrual period, pregnancy, perimenopause, etc.), which provides a basis for understanding the function of vaginal secretions. A certain molecular basis has been established, but the composition and mechanism of vaginal secretions are complex, and it is an effective way to continue to explore new protective components and mechanisms.

Contents of the invention

The purpose of the present invention is to provide a novel vaginal secretion-derived polypeptide PDMAC, the amino acid sequence of which is ATPTSTLRTAPPPKVLT, which has the advantages of low molecular weight, good lipophilicity, and easy entry into cells and nuclei.

Another object of the present invention is to provide the application of polypeptide PDMAC in inhibiting the infection of vaginal epithelial cells by Candida albicans, and affect the autophagy function of vaginal epithelial cells by regulating the expression of Beclin-1, thereby enhancing its removal of invading intracellular Candida albicans role.

To achieve the above object, the present invention provides the following technical solution: a vaginal secretion-derived polypeptide, the amino acid sequence of which is ATPTSTLRTAPPPKVLT.

Preferably, the polypeptide has a theoretical molecular weight of 1751.06 Da, a theoretical isoelectric point of 11, an instability coefficient of 49.74, a fat coefficient of 74.71, and a hydrophilicity of -0.218.

Preferably, the polypeptide is synthesized by solid phase method alanine-threonine-proline-threonine-serine-threonine-leucine-arginine-threonine-alanine- Amino acid sequence of Proline-Proline-Proline-Lysine-Valine-Leucine-Threonine.

Preferably, the C-terminal and N-terminal of the polypeptide are chemically modified by polyethylene glycol and carbamic acid, respectively.

Application of a vaginal secretion-derived polypeptide in inhibiting Candida albicans infection.

Preferably, the polypeptide affects the autophagy function of vaginal epithelial cells by regulating the expression of Beclin-1.

Preferably, the polypeptide enhances its ability to clear the invading intracellular Candida albicans by affecting the autophagy function of vaginal epithelial cells.

Compared with prior art, the beneficial effect of the present invention is:

1. In this invention, the theoretical molecular weight of the polypeptide PDMAC is 1751.06Da, the theoretical isoelectric point is 11, the instability coefficient is 49.74, the fat coefficient is 74.71, and the hydrophilicity is -0.218. It has low molecular weight, good lipophilicity, and is easy to enter cells and Nuclear and other advantages, it has the advantage of being developed as a drug or daily chemical additive;

2. In this invention, the polypeptide PDMAC has the function of regulating the autophagy function of vaginal epithelial cells, enhancing the ability of vaginal epithelial cells to eliminate the invading intracellular Candida albicans, and inhibiting the infection of vaginal Candida albicans. It has the ability to be developed as an antifungal infection drug or The ability of daily chemical additives;

3. In this invention, the polypeptide PDMAC has the potential to become a substitute for antibiotics, and has a certain probability of overcoming the problem of fungal drug resistance;

4. In this invention, the polypeptide PDMAC is an endogenous substance secreted by the human body and has good immune compatibility.

Description of drawings

Fig. 1 is the quantity of control group Candida albicans invasion vaginal epithelial cells of the present invention;

Fig. 2 is the quantity that Candida albicans invades vaginal epithelial cells after the effect of polypeptide PDMAC of the present invention;

Fig. 3 is the HE staining contrast figure of control group Candida albicans infection mouse vaginal tissue and PDMAC effect of the present invention behind Candida albicans infection mouse vaginal tissue;

Fig. 4 is the secondary structure prediction figure of polypeptide PDMAC of the present invention;

Fig. 5 is the protein molecule that the pull-down lysate of the present invention PAGE gel separates and screens the protein molecule that interacts with PDMAC polypeptide;

Fig. 6 is the colocalization analysis of polypeptide PDMAC of the present invention and Beclin-1 in vaginal epithelial cell VK2/E6E7;

Fig. 7 shows the effect of the polypeptide PDMAC of the present invention on the expression of autophagy-related proteins in vaginal epithelial cells VK2/E6E7.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention.

A vaginal secretion-derived polypeptide, the amino acid sequence of which is ATPTSTLRTAPPPKVLT.

Further, the theoretical molecular weight of the polypeptide is 1751.06Da, the theoretical isoelectric point is 11, the instability coefficient is 49.74, the fat coefficient is 74.71, and the hydrophilicity is -0.218.

Example 1

The amino acid sequence synthesized by solid phase method is alanine-threonine-proline-threonine-serine-threonine-leucine-arginine-threonine-alanine-proline- Polypeptide PDMAC of Proline-Proline-Lysine-Valine-Leucine-Threonine (ATPTSTLRTAPPPKVLT).

Chemically modify the main chain group and side chain group of the polypeptide, and use polyethylene glycol (Polyethyleneglycol, PEG) and carbamic acid to modify the C-terminal and N-terminal of the polypeptide PDMAC respectively, so as to improve the activity and stability of PDMAC, inhibit Enzymatic degradation reduces antigenicity and prolongs half-life in vivo.

The synthetic polypeptide PDMAC was added to the culture medium of vaginal epithelial cells VK2/E6E7 to create the polypeptide action group and the control group. After incubation for 24 hours, Candida albicans was added to the polypeptide action group, and PI/SYTO9 was used for staining and flow cytometry. Cytometer technique was used to detect the invasion of VK2/E6E7 cells by Candida albicans in the two groups.

Combining Figure 1 and Figure 2, the results show that the invading Candida albicans in VK2/E6E7 cells was significantly reduced after the action of the polypeptide PDMAC.

Application of a vaginal secretion-derived polypeptide in inhibiting Candida albicans infection.

Example 2

Purchase female BALB/c mice aged 6-8 weeks at SPF level, keep a light and dark environment for 12 hours a day, control the temperature at 25°C, and feed them with food and water randomly.

9 days before vaginal infection and 2 days before infection, 50 μg of estradiol (dissolved in 100 μL filtered sterile sesame oil) was subcutaneously pretreated, and 20 μL of Candida albicans (10 ⁹ CFU/mL) was inoculated into small Into the vagina of rats, the insertion depth is about 1-1.5cm, and the bacterial solution is stored for about 2 hours after injection, once a day. After 7 days, the vaginal secretions of the mice were taken for microscopic examination and biochemical pathological examination.

The results showed that after the inoculation of Candida albicans, the vulva of the mice was swollen and the secretions increased; combined with the HE staining in Figure 3 a, the interstitium of the vaginal tissues of the mice inoculated with Candida albicans was thickened and loosened, and even ruptured. Symptoms are obvious. It shows that the mouse vaginal infection candida albicans mycotic vaginitis model was established successfully.

At the same time, 30 μM polypeptide PDMAC was injected vaginally once a day for a week.

The results showed that, compared with the control model group, combined with the HE staining in Figure 3b, the symptoms of vulvar congestion and redness of the mice in the polypeptide PDMAC intervention group were significantly improved, and the symptoms of vaginal inflammation were alleviated; the vaginal secretions were diluted and coated on plates for Colony counts showed that the number of Candida albicans was significantly reduced.

Example 3

Using the PredictProtein (www.predictprotein.org) website to analyze the secondary structure of PDMAC, the results show that there are multiple amino acids with protein binding ability and multiple protein-interacting sites in the polypeptide (as shown in Figure 4), suggesting that PDMAC The function may be realized by interacting with the target protein.

Use the pull-down method to screen for protein molecules that interact with the PDMAC polypeptide (as shown in Figure 5). After biotin labeling, co-incubation with cell protein lysate, eluent elution, SDS-PAGE gradient gel separation, and silver staining, differential bands were taken for mass spectrometry analysis, and three peptides with large differences were found—— β-Tubulin, SLP-2, Beclin-1, and the mass spectrometry signal is strong.

Then PDMAC was transferred into VK2/E6E7 cells, and the localization of PDMAC and Beclin-1 was observed by immunofluorescence experiment.

Combined with the results in Figure 6, it shows that PDMAC and the autophagy-related protein Beclin-1 have a large degree of co-localization, suggesting that Beclin-1 is the target of PDMAC.

The expression levels of autophagy-related proteins p62, LC3-I and LC3-II in VK2/E6E7 cells were detected by Western method after PDMAC intervention.

Combined with the results in Figure 7, compared with the control group, the expression level of LC3-II increased after PDMAC intervention, while the expression levels of p62 and LC3-I were significantly reduced, thus proving that autophagy is the mechanism by which PDMAC inhibits vaginal Candida albicans infection, Beclin -1 is its potential target gene.

Therefore, PDMAC affects the autophagy function of vaginal epithelial cells by regulating the expression of Beclin-1, thereby enhancing its ability to clear the invading intracellular Candida albicans.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (3)

1. A vaginal secretion-derived polypeptide, characterized in that: the amino acid sequence of the polypeptide is ATPTSTLRTAPPPKVLT. 2. A vaginal secretion-derived polypeptide according to claim 1, characterized in that: the polypeptide is synthesized by a solid-phase method from alanine-threonine-proline-threonine-serine-threonine Amino acid sequence of acid-leucine-arginine-threonine-alanine-proline-proline-proline-lysine-valine-leucine-threonine. 3. A vaginal secretion-derived polypeptide according to claim 2, characterized in that: the C-terminal and N-terminal of the polypeptide are chemically modified by polyethylene glycol and carbamic acid, respectively.

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