FERM-FSHR-DTT recombinant subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses an FERM-FSHR-DTT recombinant subunit vaccine as well as a preparation method and application of the FERM-FSHR-DTT recombinant subunit vaccine. The amino acid sequence of the FERM-FSHR-DTT recombinant subunit vaccine is shown as SEQ ID NO. 1 in a sequence table. The FERM-FSHR-DTT recombinant subunit vaccine is used for preparing a preparation for inhibiting fat deposition of immune female mammals. The biological activity and targeting efficiency of the FSHR fusion protein are remarkably enhanced through the specific membrane anchoring function and structural stability of the FERM structural domain, and meanwhile, the immunogenicity of the antigen is effectively improved through introduction of the DTT structural domain.

Description

Technical Field

[0001] The present invention relates to the field of molecular vaccinology, and in particular to a FERM-FSHR-DTT recombinant subunit vaccine and a preparation method and application thereof. Background Art

[0002] In the field of animal husbandry, excessive deposition of visceral fat not only leads to a decrease in feed conversion rate, directly affecting the health of livestock and poultry and meat quality, but also affects human health.

[0003] Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone composed of α and β subunits synthesized and secreted by basophils of the anterior pituitary. Traditionally, it is believed to primarily bind to the FSH receptor (FSHR) in the gonads (ovaries and testes), regulating gametogenesis and steroidogenesis. In recent years, with the deepening of research on the intersection of reproductive hormones and metabolic regulation, the role of FSH / FSHR in fat metabolism has gradually attracted attention. Reports indicate that FSHR is widely expressed in non-reproductive organs such as adipose tissue and the liver, and participates in the physiological and pathological processes of fat deposition by regulating lipid synthesis, breakdown, and energy distribution. FSH can activate Pparγ signaling by upregulating Creb expression, which in turn recruits a series of Pparγ target genes, promoting fat accumulation. Vaccines targeting GnRH result in lower FSH concentrations and significantly less fat accumulation compared to surgical castration. Cui et al. found that changes in abdominal fat mass were directly correlated with Fshhr transcript levels, which in turn were directly correlated with tissue FSH levels, further demonstrating the important role of FSH in regulating lipid metabolism.

[0004] Structural vaccines use structural biology and computational simulation techniques to accurately analyze the three-dimensional conformation of antigenic epitopes, and based on this, construct molecular entities with predetermined immunogenicity. Its advantages are: (1) enhancing immunogenicity through epitope optimization; (2) avoiding interference from non-protective antigens. Polyprotein bodies are a design strategy for structural vaccines. They form multimeric proteins with specific structures through cross-linking or site-specific, directional connection of homologous or heterologous proteins. This not only improves the immunogenicity and stability of the vaccine, but also enhances the body's immune response by multivalently displaying antigenic epitopes. Ezrin is a member of the ERM family. Its N-terminal FERM (Four-point-one, Ezrin, Radixin, Moesin) domain is a conserved domain composed of approximately 300 amino acids. It is shaped like a clover leaf and contains multiple B cell epitopes (such as surface-exposed flexible loop regions), forming a natural trimer-like structure.

[0005] Corynebacterium diphtheriae belongs to the phylum Actinobacteria and is characterized by a high cytosine and guanine content in its DNA. DT is an exotoxin protein produced by lysogenized Corynebacterium diphtheriae and encoded by the β-corynephage. The toxin consists of 535 amino acids with a molecular weight of approximately 58 kDa. It can be cleaved by proteases into two functionally distinct fragments: Fragment A, located at the amino terminus of the toxin, causes cell death upon intracellular entry. Fragment B, consisting of the enzymatically active region, the transmembrane domain, and the receptor-binding region, does not contain the toxic elements. The transmembrane domain (DTT domain, amino acids 202-378) consists of nine α-helices and contains two important T cell helper epitopes: DTT271-290 and DTT321-370. DTT is often used as a vaccine carrier protein to enhance the immune efficacy of subunit vaccines.

[0006] The porcine FSHR sequence was searched through NCBI, and the amino acid sequence was analyzed online using DNA Star and TMHMM software for hydrophilicity, flexible regions, surface accessibility, antigenic index, and transmembrane region. Predicted and selected sequences were compared with the entire sequence of dog, rat, sheep, goat, and cat using OMIGA software. The leucine-rich regions located in the N-terminal extracellular domain (79-89 and 257-266) were ultimately selected, as they are highly conserved across species. B-cell epitopes of FERM were predicted using BepiPred-3.0, PSIPRED 4.0, and Expasy_ProtScale, and the insertion location of the FSHR fragment was determined.

[0007] Currently, research on immunocastration vaccines targeting GnRH upstream of the hypothalamic-pituitary-gonadal (HPG) axis is intensive, with three commercially approved vaccines available: Improvac®, GonaCon®, and Bopriva®. However, sterilization achieved through the use of downstream reproductive hormones such as FSH, LH, or thyroid-stimulating hormone (TSH) is not always effective. These hormones share a common α-subunit, which can easily generate cross-resistance as antigens. While FSHβ vaccines demonstrate excellent target specificity, the antibodies they induce have orders of magnitude lower binding affinity for FSH than those produced by immunization with intact FSH, and this increased specificity may be accompanied by a loss of affinity. Vaccines targeting FSHR can also block the FSH-FSHR cascade reaction, thereby achieving the purpose of regulating FSH. Receptor vaccines may show more significant immune persistence advantages compared to vaccines using complete gonadotropin or its recombinant β subunit as immunogens. However, there are currently few studies on the role of FSHR in regulating fat metabolism. Summary of the Invention

[0008] The purpose of the present invention is to provide a FERM-FSHR-DTT recombinant subunit vaccine and a preparation method and application thereof, which can inhibit excessive deposition of visceral fat.

[0009] In one aspect of the present invention, the present invention provides a FERM-FSHR-DTT recombinant subunit vaccine, wherein the amino acid sequence thereof is shown in SEQ ID NO. 1 in the sequence listing according to an embodiment of the present invention.

[0010] In another aspect, the present invention provides a method for preparing a FERM-FSHR-DTT recombinant subunit vaccine. According to an embodiment of the present invention, the screened FSHR dimer encoding sequence is replaced with the B cell epitope of FERM, and a DNA sequence encoding FERM-FSHR-DTT containing Nde I and Xho I restriction sites is synthesized. The synthesized DNA sequence is ligated with the enzyme-digested pET28a vector using T4 ligase, and transformed into an Escherichia coli DH5α cloning competent cell. The pET28a-FERM-FSHR-DTT plasmid is screened, and then transformed into an Escherichia coli BL21 expression competent cell to express the recombinant protein, thereby preparing a FERM-FSHR-DTT recombinant subunit vaccine.

[0011] In another aspect, the present invention provides a use of a FERM-FSHR-DTT recombinant subunit vaccine, which is used to prepare a preparation for inhibiting fat deposition in immune female mammals according to an embodiment of the present invention.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1) This invention innovatively fuses the Ezrin-FERM domain with the FSHR and introduces the DTT transmembrane domain. Three-dimensional crystal structure prediction of the fusion protein using SWISS-MODEL revealed that the insertion of FSHR does not disrupt its cloverleaf conformation and actively exposes key FERM epitopes. This design significantly enhances the biological activity and targeting efficiency of the FSHR fusion protein by leveraging the unique membrane anchoring function and structural stability of the FERM domain. Furthermore, the introduction of the DTT domain effectively improves the immunogenicity of the antigen.

[0014] (2) The FSH / FSHR signaling axis is a key target for regulating fat metabolism. Currently, there is limited research on the role of FSHR in regulating fat metabolism. This study expands our understanding of the mechanism of action of FSHR on adipose tissue and the metabolic-reproductive axis, providing a new approach for the prevention and treatment of obesity and related metabolic diseases, as well as for improving lean meat percentage in livestock production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the plasmid map of pET28a-FERM-FSHR-DTT in Example 2 of the present invention;

[0016] Figure 2 is a diagram of the predicted tertiary structure of FERM-FSHR-DTT in Example 2 of the present invention;

[0017] Figure 3 This is a graph showing changes in antibodies after active immunization of FSHR in Example 3 of the present invention. In the graph, ** indicates extremely significant differences between different groups (P < 0.01), completely different lowercase letters indicate significant differences between different periods in the same group (P < 0.05), and completely different uppercase letters indicate extremely significant differences between different periods in the same group (P < 0.01). Arrows indicate the time of vaccination.

[0018] Figure 4 This is a graph showing changes in body weight gain in Example 3 of the present invention. The first immunization is marked as 0 w, the arrow indicates the time of vaccination, | represents the start time of high-fat diet feeding, completely different lowercase letters represent significant differences (P < 0.05), and completely different uppercase letters represent extremely significant differences (P < 0.01);

[0019] Figure 53 is a graph showing the effect of FSHR active immunization on VAT in rats in Example 3 of the present invention, wherein A is VAT weight, B is VAT index (VAT index = VAT weight / body weight × %), and C is a rat autopsy diagram. Completely different lowercase letters indicate significant differences (P < 0.05), and completely different uppercase letters indicate extremely significant differences (P < 0.01);

[0020] Figure 6 3 is the VAT HE staining image in Example 3 of the present invention, wherein A is the NC group (400×), B is the HFD group (400×), and C is the FSHR group (400×);

[0021] Figure 7 This is a graph showing the effect of active immunization with FSHR on food intake in rats in Example 3 of the present invention. In the graph, completely different capital letters indicate extremely significant differences (P < 0.01);

[0022] Figure 8 This is a graph showing the effect of active immunization with FSHR on blood lipids in rats in Example 3 of the present invention. In the graph, completely different lowercase letters indicate significant differences (P < 0.05), and completely different uppercase letters indicate extremely significant differences (P < 0.01);

[0023] Figure 9 3 is the HE staining result of the liver in Example 3 of the present invention, wherein A is the NC group (100×), B is the HFD group (100×), C is the FSHR group (100×), a is the NC group (400×), b is the HFD group (400×), and c is the FSHR group (400×). → indicates the central vein, ▭ indicates the hepatic cord, □ indicates the hepatocyte nucleus, ○ indicates the lymphocyte, ★ indicates the hepatic sinusoid, and △ indicates fatty vacuolar degeneration.

[0024] Figure 10 : The HE staining results of muscles in Example 3 of the present invention, wherein A is the NC group (100×), B is the HFD group (100×), and C is the FSHR group (100×). a is the NC group (400×), b is the HFD group (400×), and c is the FSHR group (400×). The scale bars of A, B, and C are 200 μm, and the scale bars of a, b, and c are 50 μm.

[0025] Figure 113. The figures are the transcription level graphs of some fat metabolism-related genes in Example 3 of the present invention, wherein Figure A shows the transcription level changes of some fat metabolism-related genes in VAT; Figure B shows the transcription level changes of some fat metabolism-related genes in liver tissue; C / ebpα is CCAAT / enhancer binding protein α; Creb is cAMP response element binding protein; Pparγ is peroxisome proliferator-activated receptor γ; Lpl is lipoprotein lipase; Perilipin is perilipin; Ucp1 is uncoupling protein 1; Srebf1 is sterol regulatory element binding transcription factor 1; Scarb1 is scavenger receptor class B 1; Ldlr is low-density lipoprotein receptor; Fgf21 is fibroblast growth factor 21; completely different lowercase letters indicate significant differences (P < 0.05); completely different uppercase letters indicate extremely significant differences (P < 0.01). DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] Example 1

[0028] Amino acid sequence of the FERM-FSHR-DTT recombinant subunit vaccine The amino acid sequence of the recombinant subunit vaccine is shown in the sequence listing as SEQ ID NO. 1. It is the amino acid sequence after the modified and repeated FSHR dimer replaces the FERM B cell epitope and the DTT amino acid sequence, wherein the FERM amino acid sequence is shown in the sequence listing as SEQ ID NO. 2, the FSHR amino acid sequence is shown in the sequence listing as SEQ ID NO. 3, and the DTT amino acid sequence is shown in the sequence listing as SEQ ID NO. 4.

[0029] Wherein, the sequence table SEQ ID NO.1 is as follows:

[0030] MPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWYFGLQYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRARFYPEDVSEELIQDITQKLFFLQVKDGILSDEIYCPPETAVLLGSYQVAAKFSKVTEIPPDLPGGSIHNCAFNGTQGGGGSSKVTEIPPDLPGGSIHNCAFNGTQGQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDSAMLEYLKIAQDLEMYGYINFEIKSKVTEIPPDLPGGSIHNCAFNGTQGGGGSSKVTEIPPDLPGGSIHNCAFNGTQGDLWLGVDALGLNIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLCMGNHELYMRRRINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP

[0031] The sequence table SEQ ID NO. 2 is as follows:

[0032] MPKPINVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWYFGLQYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRARFYPEDVSEELIQDITQKLFFLQVKDGILSDEIYCPPETAVLLGSYQVAAKFQRVMDQHKLTRDQWEDRIQVWHAEHRGMLKDSAMLEYLKIAQDLEMYGYINFEIKDLWLGVDALGLNIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILQLCMGNHELYMRRR

[0033] The sequence table SEQ ID NO. 3 is as follows:

[0034] SKVTEIPPDLPGGSIHNCAFNGTQGGGGSSKVTEIPPDLPGGSIHNCAFNGTQG

[0035] The sequence table SEQ ID NO.4 is as follows:

[0036] INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP

[0037] Example 2

[0038] The preparation method of the FERM-FSHR-DTT recombinant subunit vaccine comprises the following steps:

[0039] The screened FSHR dimer encoding sequences were replaced with the B cell epitopes of FERM, and a DNA sequence encoding FERM-FSHR-DTT containing Nde I and Xho I restriction sites was synthesized. The synthesized DNA sequence was ligated with the enzyme-digested pET28a vector using T4 ligase, transformed into Escherichia coli DH5α cloning competent cells, and the pET28a-FERM-FSHR-DTT plasmid was screened. The DNA sequence was then transformed into Escherichia coli BL21 expression competent cells to express the recombinant protein, and the FERM-FSHR-DTT recombinant subunit vaccine was prepared.

[0040] Among them, the pET28a-FERM-FSHR-DTT plasmid map is as follows Figure 1 shown.

[0041] The porcine FSHR sequence was searched through NCBI, and the amino acid sequence was analyzed online for hydrophilicity, flexibility, surface accessibility, antigenic index, and transmembrane region using DNA Star and TMHMM software. The gene sequence was designed using SnapGene 7.1.0 software, and the three-dimensional crystal structure of the fusion protein was predicted using SWISS-MODEL.

[0042] The protein three-dimensional structure was predicted by SWISS-MODEL. The results are as follows Figure 2 As shown, the insertion of FSHR dimer does not disrupt the FERM cloverleaf spatial conformation.

[0043] Example 3

[0044] The rat vaccination test includes the following steps:

[0045] a. Experimental Design: Healthy female Sprague Dawley (SD) rats of similar body weight were randomly divided into three groups: a conventional diet control group (NC group), a high-fat diet control group (HFD group), and a FSHR immunization group (FSHR group, subcutaneously inoculated with 200 μg of FERM-FSHR-DTT protein). Rats in the immunized groups received their first vaccination at 8 weeks of age (denoted as week 0), followed by booster vaccinations at 4 and 8 weeks after the first vaccination, for a total of three vaccinations. Rats in the NC and HFD groups received equal injections of the same amount of immune adjuvant plus saline at the same time and site.

[0046] b. Establishment of an Obese Rat Model: To highlight the effects of immunization on fat deposition, an obese rat model was established. Except for the NC group, which was fed a standard maintenance diet throughout, all other groups were fed a standard maintenance diet until 16 weeks of age, after which they were switched to a high-fat diet (60% fat).

[0047] c. Sample Collection: Blood was collected from the infraorbital venous plexus of rats at 0, 4, 8, 11, and 19 weeks after the first immunization. The collected blood samples were allowed to rest at room temperature for at least 3 hours, then centrifuged at 3,000 g / min at 4°C for 10 minutes. The supernatant was transferred to a fresh tube and stored in an ultra-low-temperature freezer for analysis of serum antibody titers and levels of triglycerides, TC, HDL-C, and LDL-C. Starting from the first immunization, all rats were weighed regularly. After the end of the experiment (19 weeks), inguinal, perirenal, and periovarian adipose tissue, as well as liver and muscle, were collected. Visceral adipose tissue (VAT) and liver were rinsed with saline, then excess liquid was removed with filter paper and weighed. A portion of the collected tissues was fixed with a dedicated fixative for subsequent histological analysis, while a portion was frozen in liquid nitrogen and stored in an ultra-low-temperature freezer until further use.

[0048] The following tests were performed on the experiment:

[0049] (1) Antibody level detection (to assess the immune effect after vaccination):

[0050] a. Dilute GST-FSHR protein of known concentration to 2 μg / mL using PBS. Add 100 μL of the diluted protein solution to each well of the ELISA plate and coat overnight at 4°C.

[0051] b. The next day, discard the coated protein solution, wash the plate three times with 200 μL PBST, pat dry on paper, and block the plate with 3% skim milk powder at 37°C for 1 h.

[0052] c. Serum samples were diluted 200-fold and added sequentially to ELISA plates, with 100 μL added to each well in triplicate. Six wells of each ELISA plate were reserved for blank controls, with only 100 μL of blocking buffer added. Incubate at 37°C for 1 h. Wash the plates three times with PBST and pat dry on paper.

[0053] d. Dilute goat anti-rat IgG (HRP) antibody (commercial secondary antibody, Cat. No. A21040; Yacoin Biotechnology Co., Ltd.) and blocking buffer at a volume ratio of 1:10,000. After thorough vortexing, add 100 μL / well to the ELISA plate and incubate at 37°C for 1 h. Wash the plate three times with PBST and pat dry on paper.

[0054] e. Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 15 minutes. Then add 50 μL of stop solution. When the liquid in the well completely changes from blue to yellow, remove the bubbles and measure the OD within 15 minutes. 450 value.

[0055] like Figure 3 As shown, 4 weeks after the first immunization, rats in the FSHR group produced high levels of antibodies. Antibody levels increased after booster immunizations, reaching a peak 3 weeks after the third immunization. Antibody levels in the FSHR group were significantly higher than those in the NC and HFD groups at 4, 8, 11, and 19 weeks (P < 0.01).

[0056] (2) Recording the food intake of rats

[0057] Food intake is a key factor influencing obesity. Obesity is essentially a chronic imbalance of energy intake and energy expenditure, leading to excess energy storage as fat. High-fat diets induce energy excess and promote fat accumulation. Food intake is also a key factor influencing livestock health, production performance, and economic profitability. Food intake was recorded for rats: Each group of female rats was given a fixed amount of food between 9:00 and 10:00 daily. After 24 hours, the remaining feed weight and cage food scraps were collected. The amount of feed provided and the amount of remaining feed were recorded to calculate the rats' food intake.

[0058] like Figure 4 As shown in the data, the body weight of rats in the FSHR group was significantly lower than that of the NC group and the HFD group 4 weeks after the first immunization (P < 0.05), significantly lower than that of the HFD group at the 8th week (P < 0.05), and extremely significantly lower than that of the HFD group at the 12th and 19th weeks (P < 0.01).

[0059] like Figure 7 As shown in the figure, the average daily food intake of rats in the NC group was significantly higher than that in the HFD group and the FSHR group (P<0.01), while there was no significant difference in food intake between the HFD group and the FSHR group.

[0060] (3) Histological analysis

[0061] The external characteristic of obesity is weight gain, while the internal characteristic is adipocyte hypertrophy or increased number. The relationship between adipocyte size and number and fat mass is a dynamic equilibrium, and adipocyte hypertrophy is the primary way in which short-term caloric excess leads to adipocyte growth. The liver, a key endocrine organ, undergoes significant pathological changes in obesity and serves as an important assessment indicator. In animal husbandry, muscle fiber diameter is closely linked to meat tenderness. Enlarged muscle fibers may lead to increased muscle firmness, decreased meat tenderness, and increased connective tissue shear stress. Therefore, histological analysis was performed: fixed liver, muscle, and VAT were dehydrated and paraffin-embedded sections were prepared for hematoxylin and eosin staining. The stained muscle and adipose tissue sections were photographed under an Olympus BX-53 microscope, with five different fields of view selected for each section. Myofiber cross-sectional area and adipocyte area were measured using Image J biological image analysis software.

[0062] The results are as follows Figure 5 As shown in the data, the weight of visceral adipose tissue (VAT) of rats in the FSHR group was significantly lower than that in the NC group (P < 0.05), and extremely significantly lower than that in the HFD group (P < 0.01); the VAT index of rats in the HFD group was extremely significantly higher than that in the FSHR group (P < 0.01).

[0063] The collected rat fat tissue was further stained with HE (specific instructions are given in the experimental design). The results are shown in Table 1 and Figure 6 As shown, the average cross-section of VAT cells in the FSHR group was significantly lower than that in the HFD group (P < 0.01) and significantly lower than that in the NC group (P < 0.05).

[0064] Table 1 Analysis of average fat cell area

[0065] Group <![CDATA[ Average adipocyte area in VAT (μm 2 )]]> 2 )]]> NC group <![CDATA[3343.24±155.74 Ba ]]> HFD group <![CDATA[6323.80±405.90 A ]]> FSHR group <![CDATA[2720.46±188.12 Bb ]]>

[0066] Note: In the same column of data, completely different superscript lowercase letters indicate significant differences (P < 0.05); completely different superscript capital letters indicate extremely significant differences (P < 0.01).

[0067] like Figure 8 As shown in the data, the TG level in the HFD group was significantly higher than that in the NC group (P < 0.01); the TC level in the NC group was significantly higher than that in the FSHR group (P < 0.05); and the HDL-C level in the NC group was significantly higher than that in the HFD group and the FSHR group (P < 0.01).

[0068] Tissue sections showed that the liver plates in the NC group were neatly arranged, with clear structures of the central vein and portal area, and no inflammatory cell infiltration or fibrous tissue proliferation was observed. In the HFD group, the liver plates were disordered, the sinusoids were significantly enlarged, and lymphocyte inflammatory infiltration was observed in the sinusoids. Some hepatocytes were swollen; the cytoplasm was loose, and fatty vacuolar degeneration was observed in the cytoplasm. FSHR active immunization alleviated the enlargement of the sinusoids, inflammatory cell infiltration, hepatocyte swelling, and fatty vacuolar degeneration caused by the high-fat diet to a certain extent. ).

[0069] (4) RT-qPCR

[0070] The liver and VAT were placed in a mortar and pestle and ground into a powder using liquid nitrogen. Tissue RNA was extracted and reverse transcribed using the SPARKeasy Tissµe RNA Isolation Kit for liver tissue and the FastPure Complex Tissue / Cell Total RNA Isolation Kit for adipose tissue according to the manufacturer's instructions. The resulting cDNA was used as a template for quantitative PCR. See Table 2 for primer information for quantitative PCR. (Measuring the expression of specific genes under different conditions and comparing gene expression differences between groups; Signaling pathway studies: Analyzing changes in the expression of key regulatory genes to reveal molecular mechanisms.)

[0071] Table 2 RT-qPCR primer sequences and product sizes

[0072] Gene Downstream primer Product length (bp) β-Actin F:CGTGACATCAAGGAGAAGR:GAAGGAAGGCTGGAAGAG 171 C / ebpα F: GCGCAAGAGCCGAGATAAAGR: CAGCTTGGCGGAAGATACCC 148 Creb F:CATTGCCCCTGGAGTTGTTATGR:TTTCCTTGCTGCCTCCCTGTT 115 Pparγ F:GTCCTTCCCGCTGACCAAAGR:AGCAAACCTGGGCGGTTG 80 Lpl F:TCCAGAGTTTGACCGCCTTCR:CTCAGCTGTGTCTTCAGGGG 110 Perillipin F:CTGTGTGCAATGCCTATGAGAR:CTGGAGGGTATTGAAGAGCCG 171 Ucp1 F:GATCTCGGCTGGCTTGATGAR:AGTGTAGCGGGGTTTGATCC 118 Srebf1 F: GCTCTTGACCGACATCGAAGAR: CCAGCATAGGGGGCATCAAA 87 Scarb1 F:ACCCTGAACACGTTCTACACG R:AATAAAAAGCATTTCTCCTGGCTG 143 Ldlr F:CAGTGCGATGGCCCTAACAA R:CACTCGTTGGTCTTGCACTC 131 Fgf21 F:CAGATGACGACCAGGACACC R:GGAGACTTTCTGGACTGCGG 90

[0073] RT-qPCR amplification conditions: 95°C for 30 seconds; 95°C for 10 seconds, 60°C for 30 seconds, for 40 cycles.

[0074] The formula for calculating the relative expression of target genes is F = 2 -ΔΔCT , where ΔΔCt = change in cycle threshold (Ct) value of target gene - ΔCt value of reference gene.

[0075] like Figure 11As shown, the transcription levels of C / ebpα, Creb and Srebf1 in the HFD group were extremely significantly higher than those in the NC group and FSHR group (P < 0.01), and the transcription levels of Pparγ and Lpl were significantly higher than those in the NC group and FSHR group (P < 0.05); the transcription level of Perilipin in the HFD group was significantly higher than that in the FSHR group (P < 0.05); the transcription level of Scare1 in the FSHR group was significantly higher than that in the NC group (P < 0.05), and the transcription level of Ldlr was significantly lower than that in the NC group (P < 0.05); the transcription level of Fgf21 in the HFD group was significantly higher than that in the NC group (P < 0.05) and extremely significantly higher than that in the NC and FSHR groups (P < 0.01).

[0076] (5) In order to observe the effect of immunization on muscle development, rat leg muscles were collected, sliced ​​perpendicular to the direction of muscle fibers, and stained with HE.

[0077] The results showed that the cross-sectional area of ​​muscle fibers in the HFD group was significantly larger than that in the NC group and the FSHR group (P < 0.01); the cross-sectional area of ​​muscle fibers in the FSHR group was significantly smaller than that in the NC group (P < 0.01); in addition, the lean body mass of rats in the HFD group was significantly higher than that in the NC group (P < 0.01) and significantly higher than that in the FSHR group (P < 0.05); the increase in lean body mass in rats in the HFD group was significantly higher than that in the NC group and the FSHR group (P < 0.01) (Table 3, Figure 10 ).

[0078] Table 3 Average values ​​of muscle fiber cross-sectional area and lean body mass analysis

[0079] Group <![CDATA[Muscle cross-sectional area (μm 2 )]]> 2 > Lean body mass (g) Lean body mass gain (g) NC group <![CDATA[3449.03±126.08 B ]]> <![CDATA[310.20±3.86 B ]]> <![CDATA[107.41±4.70 B ]]> HFD group <![CDATA[5743.21±270.70 A ]]> <![CDATA[346.95±7.05 Aa ]]> <![CDATA[144.16±6.11 A ]]> FSHR group <![CDATA[2407.56±96.52 C ]]> <![CDATA[318.82±8.78 ABb ]]> <![CDATA[114.79±8.61 B ]]>

[0080] Note: In the same column of data, completely different superscript lowercase letters indicate significant differences (P < 0.05); completely different superscript capital letters indicate extremely significant differences (P < 0.01).

[0081] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall fall within the scope of protection of the present invention.

Claims

1. FERM-FSHR-DTT recombinant subunit vaccine, characterized by: Its amino acid sequence is shown in the sequence listing SEQ ID NO.

1.

2. A method for preparing the FERM-FSHR-DTT recombinant subunit vaccine according to claim 1, characterized in that: The screened FSHR dimer encoding sequences were replaced with the B cell epitopes of FERM, and a DNA sequence encoding FERM-FSHR-DTT containing Nde I and Xho I restriction sites was synthesized. The synthesized DNA sequence was ligated with the enzyme-digested pET28a vector using T4 ligase, transformed into Escherichia coli DH5α cloning competent cells, and the pET28a-FERM-FSHR-DTT plasmid was screened. The DNA sequence was then transformed into Escherichia coli BL21 expression competent cells to express the recombinant protein, and the FERM-FSHR-DTT recombinant subunit vaccine was prepared.

3. The use of the FERM-FSHR-DTT recombinant subunit vaccine according to claim 1, characterized in that: Used for preparing preparations for inhibiting fat deposition in immune female mammals.

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