Vaccine for hypolipidemic and hypoglycemic castration of multiple species, preparation method and application thereof

Abstract

The application discloses a novel vaccine suitable for lipid-lowering and blood sugar-lowering castration of multiple species, a preparation method and application thereof, and is related to one or more combinations of a YETVKVPGCAHHADSL fragment of pigs, cattle, sheep, alpacas and horses, a YETVRVPGCARHADSL fragment of dogs and minks, a YPVATECHCGK fragment of pigs, cattle, sheep, alpacas, cats, rabbits and mice, a CDSDSTDCTVRGLGP fragment of humans, pigs, cattle, sheep, alpacas, dogs, giant pandas, rabbits and mice, and a CDSDSTDCTVQGLGP fragment of cats, tigers and lions, and a sequence with a homology of more than 70% to any one of the fragments. The protein vaccine can be effectively applied to treat diseases such as obesity, hyperlipidemia and diabetes caused by ovarian dysfunction, has pet and animal sterilization effects, can treat postmenopausal women or animals and pets that have undergone sterilization or castration surgery, and diseases such as obesity, hyperlipidemia and hyperglycemia caused by ovarian dysfunction, and has a good popularization prospect and medical economic value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine technology, in particular to a vaccine suitable for lipid-lowering, blood glucose-lowering and castration of multiple species, a preparation method and application thereof. BACKGROUND

[0002] Follicle-stimulating hormone (FSH) is a glycoprotein hormone secreted by the pituitary gland, which contains two different subunits: alpha and beta subunits. The human FSH beta subunit consists of 111 amino acids, has specific biological effects, and is responsible for interacting with the FSH receptor (FSHR) located on the surface of target cells, usually testes and ovaries (granulosa cells and Sertoli cells) [1]. Therefore, FSH plays a key role in the reproductive process, stimulating the antral formation of female secondary follicles and the growth and maturation of antral follicles, while male FSH is responsible for the development and maintenance of testes. In mature gonads, FSH acts on Sertoli cells, spermatogonia, primary and secondary spermatocytes, and drives spermatogenesis [2].

[0003] The expression of FSH receptor (FSHR) is not limited to the gonads, and recent studies have found that FSHR is also expressed in adipose tissue and liver outside the gonads, indicating that FSH may play a role in regulating lipid metabolism and fat accumulation [3]. Further studies have shown that FSH plays an important role in promoting fat accumulation in women and men. In particular, the increase in FSH due to ovarian failure in postmenopausal women has been confirmed as a major cause of postmenopausal obesity and hepatic steatosis. Studies using mouse models have also shown that high expression of FSH is a major cause of obesity caused by gonadectomy in mammals.

[0004] In addition to being closely related to obesity, studies have also found that FSH can increase the risk of osteoporosis, atherosclerosis, hypercholesterolemia, prostate cancer, and neurodegenerative diseases [4, 5].

[0005] These findings provide a new perspective for the study of disease control in humans and mammals, and the development of an effective FSH vaccine has broad prospects in controlling obesity, osteoporosis, and neurodegenerative diseases, especially for postmenopausal women and some livestock or pets that need to be castrated before being fattened.

[0006] A new FSH vaccine based on the FSH receptor binding epitope of the FSH beta subunit has been reported. Vaccination with this vaccine was very effective in preventing fat accumulation in ovariectomized mice and also effectively prevented fat accumulation in intact mice fed with normal food [6]. This study designed a new FSH antigen and vaccine by synthesizing a tandem FSH beta 13 AA (LVYKDPARPNIQK) and combining it with ovalbumin (FSH beta 13 AA-T-OVA), and demonstrated its high efficiency in inhibiting fat accumulation using a mouse model. This patent (CN110694056A) also confirms the therapeutic effect of this treatment.

[0007] In addition, early studies have shown that FSH has been studied as a candidate drug for the development of a contraceptive vaccine. After vaccination with sheep FSH (oFSH), the spermatogenesis of adult rhesus monkeys was inhibited, and the testes atrophied. Other research groups have also independently confirmed that oFSH immunization can reduce the fertility of Bonnet monkeys and men [7-10].

[0008] Recent studies have found that a recombinant FSHR subunit vaccine can significantly inhibit testicular development and spermatogenesis in prepubertal and pubertal rats

[11] .

[0009] This means that FSH vaccines, like FSHR vaccines, are a promising method of immunocastration for animals. It is obviously unrealistic to surgically sterilize a large number of stray cats, stray dogs, and wild animals that need to be sterilized to control breeding populations. There is still a lack of more mature immunocastration products on the market, and the development of FSH vaccines will be beneficial to animal and pet sterilization, avoiding the pain of surgical castration and enhancing animal welfare, and therefore has great commercial value. SUMMARY

[0010] The purpose of the present application is to provide a vaccine suitable for lipid-lowering and blood glucose-lowering castration in multiple species, a preparation method and its application, to solve the problems raised in the background art.

[0011] To solve the above technical problems, the technical scheme provided by the present application is as follows: a vaccine suitable for lipid-lowering and blood glucose-lowering castration in multiple species, designed for specific fragments of FSH beta subunit of multiple species, involving one or more combinations of YETVKVPGCAHHADSL fragments of pigs, cattle, sheep, alpacas, horses, YETVRVPGCARHADSL fragments of dogs and minks, YPVATECHCGK fragments of pigs, cattle, sheep, alpacas, cats, rabbits and mice, CDSDSTDCTVRGLGP fragments of humans, pigs, cattle, sheep, alpacas, dogs, giant pandas, rabbits and mice, and CDSDSTDCTVQGLGP fragments of cats, tigers and lions, and sequences with a homology of more than 70% of any one of them.

[0012] As preferred, the above fragments are connected by a rigid linker A (EAAAK) n The sequences are connected.

[0013] As preferred, to enhance immunogenicity, TEV enzyme cutting sequences ENLYFQSA are added on both sides of the rigid linker sequence, making it a rigid linker A (EAAAK) n After fusion, it has a coiled coil structure that can make the entire antigen form a stable dimer, increases the solubilizing peptide to enhance cell expression, and further enhances the immunogenicity of the antibacterial peptide fused at the C-terminal of the antigen.

[0014] A vaccine preparation method suitable for lipid-lowering and hypoglycemic castration of multiple species and its application, which is used locally in the form of cream, oil, ointment, powder or gel.

[0015] A vaccine preparation method suitable for lipid-lowering and hypoglycemic castration of multiple species and its application, which is administered orally, transdermally, transmucosally, intramuscularly, intravenously or subcutaneously.

[0016] A vaccine preparation method suitable for lipid-lowering and hypoglycemic castration of multiple species, the steps are as follows:

[0017] 1) Synthesize and construct the sequence into the vector pcDNA3.4;

[0018] 2) Cell passage before transfection: when culturing cells, the culture volume should not exceed 1 / 5 of the volume of the shake flask, and the cells should be passaged once every 3-4 days. When passing, first do accurate cell counting, and after confirming the density, there is no need to centrifuge. The cell suspension is prepared in the required proportion into Hi-KDCHO culture medium, so that the cell density after passage is controlled between 0.4-0.6×10 6 cells / ml;

[0019] 3) Prepare transfected cells: place CHOK1-Hi cells in a 5% CO2 constant temperature incubator, shake culture at 37°C, determine the cell density and survival rate, and when the cells grow to a density of 6-15×10 6 cells / ml, calculate and transfer the required volume of cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in fresh Hi-KDCHO culture medium to a density of 20×10 6 cells / ml;

[0020] 4) Transient transfection: transfer the cells to a cell culture shake flask and return them to the shaker for 1 hour of shaking culture, then remove them from the shaker for transfection; when transfecting, add plasmid DNA to the cells at a concentration of 17 μg / m and shake well, then add transfection reagent HiKDCHO-TA at a concentration of 35 μg / ml while mixing.

[0021] 5) Product expression and detection: 2% (2ml / 100ml) of CHO cell protein expression enhancer Hi-KE plus is added 24 hours after transfection; at the same time, nutritional supplement additive HiKDCHO-Feed is added once, and the amount is 2% (2ml / 100ml), and the cells are transferred to a 32℃ shaking incubator for low-temperature expression to obtain high expression effect; the product expression amount is determined on the 6th-8th day after transfection;

[0022] 6) Protein purification: Ni Smart Beads 6FF gravity column (Changzhou Tiandirenhe Biotechnology Co., Ltd.) is used for purifying the protein, and the imidazole is cleaned away by using an ultrafiltration tube, and the obtained protein purity is greater than or equal to 95%.

[0023] The protein vaccine provided by the application can be effectively applied to treat diseases such as obesity, hyperlipidemia and diabetes caused by ovarian dysfunction, and is expected to become a new type of effective drug with defense and treatment functions, has pet and animal sterilization effects, can treat postmenopausal women or animals and pets that have undergone sterilization or castration surgery, and diseases such as obesity, hyperlipidemia, hyperglycemia and the like caused by ovarian dysfunction, and is a new type of protein vaccine with good popularization prospect and medical economic value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a column chart of the influence of ZD9A on intracavity fat.

[0025] Figure 2 is a column chart of the influence of ZD9A on triglyceride.

[0026] Figure 3 is a line chart of the influence of the vaccine after immunization on body weight.

[0027] Figure 4 is a line chart of the influence of the vaccine after immunization on blood glucose.

[0028] Figure 5 is a line chart of the influence of the vaccine after immunization on total cholesterol.

[0029] Figure 6 is a line chart of the influence of the vaccine after immunization on triglyceride Figure 1 .

[0030] Figure 7 is a line chart of the influence of the vaccine after immunization on triglyceride Figure 2 .

[0031] Figure 8 is a line chart of the influence of the vaccine after immunization on high-density lipoprotein.

[0032] Figure 9 is a photo of the effect of the vaccine after immunization on castrated mice.

[0033] Figure 10 Figure 1 is a photograph of a normal mouse ovary after 11 weeks of vaccination. DETAILED DESCRIPTION

[0034] For the purposes of the detailed description below, it is to be understood that the application can assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers

[0035] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0036] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges of the same whole number sub-range. For example, a range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0037] EMBODIMENT

[0038] A vaccine suitable for multi-species lipid-lowering hypoglycemic castration is designed for a specific fragment of multi-species FSHβ subunit, involving one or more combinations of YETVKVPGCAHHADSL fragment of pig, cattle, sheep, alpaca, horse, YETVRVPGCARHADSL fragment of dog, mink, YPVATECHCGK fragment of pig, cattle, sheep, alpaca, cat, rabbit, mouse, CDSDSTDCTVRGLGP fragment of human, pig, cattle, sheep, alpaca, dog, giant panda, rabbit, mouse, and CDSDSTDCTVQGLGP fragment of cat, tiger, lion, and any sequence with a homology of more than 70% to any of the above sequences.

[0039] As a preferred embodiment of the present embodiment, the above fragments are connected by a rigid linker A (EAAAK) n

[0040] ​As a preferred embodiment of the present embodiment, to enhance immunogenicity, TEV enzyme cutting sequence ENLYFQSA is added on both sides of the rigid linker sequence, making it with rigid linker A (EAAAK) n After fusion, it has coiled coil structure that can make the whole antigen form stable dimer, increases solubilizing peptide to enhance cell expression, and fuses antibacterial peptide at the C-terminal of the antigen to further enhance immunogenicity.

[0041] A vaccine preparation method suitable for lipid-lowering and blood glucose-lowering castration of multiple species and its application, which is used topically in the form of cream, oil, ointment, powder or gel.

[0042] A vaccine preparation method suitable for lipid-lowering and blood glucose-lowering castration of multiple species and its application, which is administered orally, transdermally, transmucosally, intramuscularly, intravenously or subcutaneously.

[0043] A vaccine preparation method suitable for lipid-lowering and blood glucose-lowering castration of multiple species, the steps are as follows:

[0044] 1) Synthesize the following sequence and construct it on the plasmid vector pcDNA3.4;

[0045] YETVKVPGCAHHADSLYETVKVPGCAHHADSLYETVRVPGCARHADSLYETVRVPGCARHADSL ENLYF QS AEAAAKE

[0046] AAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKEA ENLYFQS AVQHSNAPLIDLGAEMKKQHKEAAPEGAAP

[0047] AQGKAPAAEAKKEEAPKPKPVV ENLYFQSA YPVATECHCGKCDSDSTDCTVRGLGPCDSDSTDCTVQGLGPCDSDSTD

[0048] CTVRGLGPCDSDSTDCTVRGLGPCDSDSTDCTVRGLGP QCRRLCYKQRCVTYCRGR

[0049] 2) Cell passage before transfection: when culturing cells, the culture liquid volume should not exceed 1 / 5 of the shaking flask volume, and the cells are passaged once every 3-4 days. When passing, first do accurate cell counting, and after confirming the density, do not centrifuge, but prepare the cell suspension in Hi-KDCHO culture liquid according to the required proportion, so that the cell density after passage is controlled between 0.4-0.6 x 10 6 cells / ml;

[0050] 3) Preparation of transfected cells: CHOK1-Hi cells were placed in a 5% CO2 constant temperature incubator, and the cell density and survival rate were determined. When the cells grew to a density of 6-15 x 10 6 cells / ml, the required volume of cell suspension was calculated and transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in fresh Hi-KDCHO culture medium to a density of 20 x 10 6 cells / ml;

[0051] 4) Transient transfection: the cells were transferred to a cell culture flask and placed back in the incubator, shaken for 1 hour, then removed from the incubator for transfection; during transfection, the plasmid DNA was added to the cells at a concentration of 17 μg / ml and shaken well, then the transfection reagent HiKDCHO-TA was added at a concentration of 35 μg / ml and mixed well;

[0052] 5) Product expression and detection: 2% (2 ml / 100 ml) of CHO cell protein expression enhancer Hi-KEplus was added 24 hours after transfection; at the same time, nutritional supplement additive HiKDCHO-Feed was added once, the amount was 2% (2 ml / 100 ml), and the cells were transferred to a 32°C incubator for low-temperature expression to achieve high expression; the product expression was measured 6-8 days after transfection;

[0053] 6) Protein purification: Ni Smart Beads 6FF gravity column (Changzhou Tiandirenhe Biotechnology Co., Ltd.) was used for protein purification, and imidazole was removed using ultrafiltration tube, and the obtained protein purity was ≥95%.

[0054] The use case 1 of the present application is as follows:

[0055] Mice were immunized with 20 micrograms of vaccine as castration post-drug group (ZD9A). The castration group (Castration) and the control group (Control) were injected with the same volume of saline as placebo, and were injected intramuscularly every 21 days for 3 times. After 2 weeks, the intracavity fat and blood lipids were detected. The results showed that compared with the castration group, after injection of FSH vaccine, the abdominal intracavity fat decreased by 68% ( Figure 1 and Figure 9 ), and the triglyceride decreased by 15% ( Figure 2 )

[0056] The use case 2 of the present application is as follows:

[0057] Diabetic rats were immunized with 60 micrograms of vaccine as the immune group (ZD9A). The control group (Control) was injected with the same volume of saline as placebo.

[0058] Results show:

[0059] Body weight: The body weight of the ZD9A group increased at week 1 and week 2, but remained stable at week 3 and week 5. The body weight of the control group continued to increase. Figure 3 ).

[0060] Blood glucose: The blood glucose level of the ZD9A group increased at week 1, but gradually decreased at week 2, week 3, week 4, and week 5, indicating that ZD9A significantly reduced blood glucose levels. The blood glucose level of the control group decreased slightly after reaching a peak at week 2 Figure 4 ).

[0061] TC: The TC level of the ZD9A group increased at week 1, but gradually decreased at week 2 and week 3, indicating that ZD9A may help control TC levels. The TC level of the control group continued to increase Figure 5 ).

[0062] TG: The TG level of the ZD9A group gradually decreased at week 1, week 2, and week 3, indicating that ZD9A may help reduce TG levels. The TG level of the control group remained stable after decreasing at week 1 Figure 6 ).

[0063] HDL: The HDL level of the ZD9A group decreased at week 1, but gradually increased at week 2 and week 3. The HDL level of the control group continued to increase. ZD9A did not limit the increase in HDL Figure 7 ).

[0064] LDL: The LDL level of the ZD9A group increased after week 1, but gradually decreased at week 2 and week 3, indicating that ZD9A may help reduce LDL levels. The LDL level of the control group generally increased Figure 8 ).

[0065] As can be seen above, the vaccine of the present application shows significant effects in reducing LDL, TG, and blood glucose levels, and may help control total cholesterol and body weight. Overall, the vaccine of the present application shows potential therapeutic effects in a diabetic rat model, especially in reducing blood glucose and blood lipids.

[0066] The vaccine of the present application has an animal sterilization effect. As an example, 8-week-old ICR female mice were used, and were injected once every 21 days, and after 3 injections, ovulation was induced 2 weeks later. Pituitary follicle stimulating hormone (Ningbo Second Hormone Factory) 10 IU was injected daily for 3 days, and hCG (Ningbo Second Hormone Factory) 5 IU was injected once on the 3rd day, and ovulation was observed on the 4th day. The results showed that the mice did not ovulate, and no obvious developing follicles were observed in the ovaries, such as Figure 10The results show that the present application can be used as a castration vaccine for sterilization of animals.

[0067] The present application relates to vaccine sequences

[0068] YETVKVPGCAHHADSLYETVKVPGCAHHADSLYETVRVPGCARHADSLYETVRVPGCARHADSL ENLYF QS AEAAAKEAAAK

[0069] EAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKEA ENLYFQS AVQHSNAPLIDLGAEMKKQHKEAAPEGAAPAQGKAPAA

[0070] EAKKEEAPKPKPVV ENLYFQSA YPVATECHCGKCDSDSTDCTVRGLGPCDSDSTDCTVQGLGPCDSDSTDCTVRGLGPCDSD

[0071] STDCTVRGLGPCDSDSTDCTVRGLGP QCRRLCYKQRCVTYCRGR

[0072] The above description is only a preferred embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

[0073] 1. Bhartiya, D. and H. Patel, An overview of FSH-FSHR biology and explaining the existing conundrums. J Ovarian Res, 2021. 14(1): p. 144.

[0074] 2. Rannikko, A., et al., Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium. J Endocrinol, 1996. 151(1): p. 29-35.

[0075] 3. Cui, H., et al., FSH stimulates lipid biosynthesis in chicken adipose tissue by upregulating the expression of its receptor FSHR. J Lipid Res, 2012. 53(5): p. 909-917.

[0076] 4. Spaziani, M., et al., Extra-Gonadal and Non-Canonical Effects of FSH in Males. Pharmaceuticals (Basel), 2023. 16(6).

[0077] 5. Frolinger, T., et al., Gene-Dose-Dependent Reduction Fshr Expression Improves Spatial Memory Deficits in Alzheimer's Mice. Res Sq, 2024.

[0078] 6. Han, X., et al., A novel follicle-stimulating hormone vaccine for controlling fat accumulation. Theriogenology, 2020. 148: p. 103-111.

[0079] 7. Wickings, E.J. and E. Nieschlag, Suppression of spermatogenesis over two years in rhesus monkeys actively immunized with follicle-stimulating hormone. Fertil Steril, 1980. 34(3): p. 269-74.

[0080] 8. Moudgal, N.R., et al., Long-term contraceptive efficacy of vaccine of ovine follicle-stimulating hormone in male bonnet monkeys (Macaca radiata). J Reprod Fertil, 1992. 96(1): p. 91-102.

[0081] 9. Moudgal, N.R., et al., Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: results of a pilot study. Hum Reprod, 1997. 12(3): p. 457-63.

[0082] 10. Srivastav, A. and R.P. Das, Sperm production and fertility of bonnet monkeys (Macaca radiata) following immunization with ovine follicle stimulating hormone. Indian J Exp Biol, 1992. 30(7): p. 574-7.

[0083] 11. Pan, F., et al., Effects of Vaccination against Recombinant FSH or LH Receptor Subunits on Gonadal Development and Functioning Male Rats. Vet Sci, 2024. 11(4).

Claims

1. A vaccine, characterized in that: The sequence of the vaccine is YETVKVPGCAHHADSLYETVKVPGCAHHADSLYETVRVPGCARHADSLYETVRVPGCARHADSLENLYFQSAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKEAENLYFQSAVQHSNAPLIDLGAEMKKQHKEAAPEGAAPAQGKAPAAEAKKEEAPKPKPVVENLYFQSAYPVATECHCGKCDSDSTDCTVRGLGPCDSDSTDCTVQGLGPCDSDSTDCTVRGLGPCDSDSTDCTVRGLGPCDSDSTDCTVRGLGPQCRRLCYKQRCVTYCRGR.

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