Haemophilus influenzae D protein carrier-based GnRH castration vaccine and preparation method thereof

Abstract

The invention relates to the technical field of vaccine preparation, in particular to a GnRH castration vaccine based on a haemophilus influenzae D protein carrier and a preparation method. The amino acid sequence of the recombinant PD-GnRH fusion protein disclosed by the invention is as shown in SEQ ID NO. 1; the recombinant PD-GnRH fusion protein comprises a haemophilus influenzae D protein and a mammal GnRH sequence. The fusion protein castration vaccine is developed by adopting a PD and GnRH protein recombination mode. Compared with other castration vaccines, the vaccine provided by the invention reduces animal stress, is more convenient to use, has the advantages of safety, convenience, mild action effect and the like, can be directly used for animal intramuscular injection, is simple and easy to operate, achieves a more ideal effect on control of animal reproductive organ development and reproductive function, and can be widely applied to animal intramuscular injection. And the development of an animal castration technology is further promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and particularly relates to a GnRH castration vaccine based on Haemophilus influenzae D protein carrier and a preparation method thereof. Background Art

[0002] The traditional castration method is surgical castration, but surgical castration has risks such as pain, long recovery period, postoperative infection, and urinary incontinence. Immunocastration has the advantages of convenience, high efficiency, reversibility, and safety. Therefore, immunocastration technology is expected to become an alternative to traditional castration.

[0003] Gonadotropin-releasing hormone (GnRH) is synthesized and secreted by the hypothalamus and plays an important role in the regulation of reproduction and endocrine in the body. In the hypothalamic-pituitary-gonadal axis, when GnRH is secreted from the hypothalamus to the anterior pituitary and directly regulates the anterior pituitary to secrete two polypeptide gonadotropins, LH and FSH, these two hormones reach the target organs through the peripheral blood circulation and produce effects, regulating the synthesis and secretion of gonadal steroid hormones, that is, regulating reproductive activities such as estrus and mating of animals. Under normal circumstances, the immune system recognizes GnRH as a self-substance, so vaccines derived from natural GnRH do not produce antibodies. However, if GnRH is conjugated with a macromolecular immunogen that can induce the body to recognize it, for example, it is conjugated with another molecule with many antigenic determinants, it can enable the body to produce an immune response against GnRH. Antibodies neutralize GnRH, thereby inhibiting the secretion of downstream LH and FSH, and the secretion of testosterone and progesterone decreases accordingly, so that animal estrus can be inhibited without surgery.

[0004] Haemophilus influenzae D protein (PD) is a highly conserved 42 kDa surface lipoprotein present in all Haemophilus influenzae. Research shows that PD has strong immunogenicity and stability, can stimulate the body to produce a strong immune response, induce high-titer specific IgG antibodies (including IgG1 and IgG2a subtypes), and enhance the immune memory effect.

[0005] No vaccines prepared based on Haemophilus influenzae D protein have been found in the prior art. Summary of the Invention

[0006] The purpose of the present invention is to provide a GnRH castration vaccine based on Haemophilus influenzae D protein carrier and a preparation method thereof.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a recombinant PD-GnRH fusion protein, and the amino acid sequence of the recombinant PD-GnRH fusion protein is as shown in SEQ ID NO.1; the recombinant PD-GnRH fusion protein includes the Haemophilus influenzae protein D and the mammalian GnRH sequence.

[0009] Preferably, the amino acid sequence of the Haemophilus influenzae protein D is as shown in SEQ ID NO.2;

[0010] the amino acid sequence of the mammalian GnRH sequence is as shown in SEQ ID NO.3.

[0011] Preferably, the mammals include rats, mice, dogs, cats, pigs, cows or sheep.

[0012] The present invention also provides the application of the recombinant PD-GnRH fusion protein in the preparation of mammalian castration products.

[0013] The present invention also provides a recombinant bacterium expressing the recombinant PD-GnRH fusion protein, and the recombinant bacterium is BL21(DE3)-PD-GnRH. The BL21(DE3)-PD-GnRH is Escherichia coli, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2025, and the deposit number is CGMCC No. 34155;

[0014] the amino acid sequence of the recombinant PD-GnRH fusion protein is as shown in SEQ ID NO.1.

[0015] The present invention also provides the application of the recombinant bacterium in the preparation of mammalian castration products.

[0016] The present invention also provides a recombinant PD-GnRH fusion protein castration vaccine, and the recombinant PD-GnRH fusion protein castration vaccine includes the Haemophilus influenzae protein D shown in SEQ ID NO.2.

[0017] Preferably, the recombinant PD-GnRH fusion protein castration vaccine includes the mammalian GnRH sequence shown in SEQ ID NO.3.

[0018] The present invention also provides a preparation method of a recombinant PD-GnRH fusion protein castration vaccine, including the following steps:

[0019] breaking the recombinant bacterium and purifying to obtain it;

[0020] The purification method is nickel column purification.

[0021] The present invention also provides the application of the recombinant PD-GnRH fusion protein castration vaccine, which can be applied in any of the following situations:

[0022] (1) For male animal castration products;

[0023] (2) Products for inhibiting testicular growth and development of male animals;

[0024] (3) Products for reducing aggressive behavior and / or sexual behavior of male animals.

[0025] The present invention provides a GnRH castration vaccine based on Haemophilus influenzae D protein carrier and its preparation method. The amino acid sequence of the recombinant PD-GnRH fusion protein is shown in SEQ ID NO.1; the recombinant PD-GnRH fusion protein includes Haemophilus influenzae D protein and mammalian GnRH sequence. The amino acid sequence of the Haemophilus influenzae D protein is shown in SEQ ID NO.2; the amino acid sequence of the mammalian GnRH sequence is shown in SEQ ID NO.3.

[0026] When designing the antigen in the present invention, a recombinant method of PD and GnRH proteins was adopted to develop a fusion protein castration vaccine. Only a single copy of GnRH decapeptide molecule was linked, which could stimulate the body to produce a relatively high level of GnRH antibody. It shows that PD introduced into the fusion protein, as an adjuvant and immune enhancer of GnRH, can significantly improve the immunogenicity of low-molecular-weight antigens, enhance the immune effect of antigens, and avoid the unstable immune effect caused by polypeptide instability. Compared with other castration vaccines, it reduces the stress of animals, is more convenient to use, and has the advantages of safety, convenience, mild action effect, etc. It can be directly used for intramuscular injection of animals, with simple and easy operation, and achieves a more ideal effect in controlling the development and reproductive function of animal reproductive organs, further promoting the development of animal castration technology. Brief Description of the Drawings

[0027] Figure 1 is the plasmid map of pET32a-PD-GnRH;

[0028] Figure 2 is the SDS-PAGE gel electrophoresis detection result map of the purified recombinant PD-GnRH fusion protein;

[0029] Figure 3 is the result of detecting the recombinant PD-GnRH fusion protein by WB method;

[0030] Figure 4Results of anti-GnRH antibody levels in cats in the immunization recombinant PD-GnRH fusion protein castration vaccine trial (the abscissa represents time, the ordinate represents OD value, and a larger OD value indicates a higher antibody level).

[0031] Figure 5 Results of the effect of recombinant PD-GnRH fusion protein castration vaccine on cat testosterone concentration (the abscissa represents time, the ordinate represents testosterone concentration).

[0032] Depositing description

[0033] The recombinant bacterium BL21(DE3)-PD-GnRH is Escherichia coli, deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2025, and the deposit number is CGMCC No. 34155. Detailed implementation manners

[0034] The solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Embodiment 1

[0036] Preparation of recombinant PD-GnRH fusion protein castration vaccine

[0037] (1) Construction of pET32a-PD-GnRH plasmid

[0038] The PD protein is a highly conserved surface lipoprotein with a size of 42 kDa and exists in all Haemophilus influenzae. According to the PD sequence and the cat GnRH sequence (including but not limited to the cat sequence, and can also be the GnRH sequences of rats, mice, dogs, cattle, and sheep), the selected recombinant protein sequence is designed.

[0039] Among them, the amino acid sequence of the recombinant PD-GnRH fusion protein is as shown in SEQ ID NO.1, specifically as follows:

[0040] CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQ

[0041] DLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQS

[0042] LEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGK

[0043] KVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDF

[0044] NELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYD

[0045] WMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQ

[0046] YNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKGGGGSGGGGSGGGGSEHWSYGLRPG。

[0047] Among them, the PD sequence is as shown in SEQ ID NO.2, specifically as follows: CSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK。

[0048] Among them, the cat GnRH sequence is as shown in SEQ ID NO.3, specifically as follows: EHWSYGLRPG。

[0049] Method for constructing plasmid of recombinant PD-GnRH fusion protein containing the sequence shown in SEQ ID NO.1: Design and synthesize a nucleotide sequence (SEQ ID NO.4) encoding the amino acid sequence shown in SEQ ID NO.1 using a codon optimization system. This nucleotide sequence was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. and was constructed into the pET-32a vector by homologous recombination to obtain a recombinant plasmid. The recombinant plasmid was ligated into Escherichia coli DH5α for resistance screening. The positive plasmid was selected to obtain the plasmid pET32a-PD-GnRH encoding the GnRH recombinant protein of the sequence shown in SEQ ID NO.1( Figure 1 ).

[0050] SEQ ID NO.4

[0051] tgcagcagccatagcagcaacatggcgaacacccagatgaaaagcgataaaattattattgcgcatcgcggcgcg

[0052] agcggctatctgccggaacataccctggaaagcaaagcgctggcgtttgcgcagcaggcggattatctggaacag

[0053] gatctggcgatgaccaaagatggccgcctggtggtgattcatgatcattttctggatggcctgaccgatgtggcgaaa

[0054] aaatttccgcatcgccatcgcaaagatggccgctattatgtgattgattttaccctgaaagaaattcagagcctggaaat

[0055] gaccgaaaactttgaaaccaaagatggcaaacaggcgcaggtgtatccgaaccgctttccgctgtggaaaagccat

[0056] tttcgcattcatacctttgaagatgaaattgaatttattcagggcctggaaaaaagcaccggcaaaaaagtgggcattt

[0057] atccggaaattaaagcgccgtggtttcatcatcagaacggcaaagatattgcggcggaaaccctgaaagtgctgaa

[0058] aaaatatggctatgataaaaaaaccgatatggtgtatctgcagacctttgattttaacgaactgaaacgcattaaaacc

[0059] gaactgctgccgcagatgggcatggatctgaaactggtgcagctgattgcgtataccgattggaaagaaacccagg

[0060] aaaaagatccgaaaggctattgggtgaactataactatgattggatgtttaaaccgggcgcgatggcggaagtggtg

[0061] aaatatgcggatggcgtgggcccgggctggtatatgctggtgaacaaagaagaaagcaaaccggataacattgtgt

[0062] ataccccgctggtgaaagaactggcgcagtataacgtggaagtgcatccgtataccgtgcgcaaagatgcgctgcc

[0063] ggaattttttaccgatgtgaaccagatgtatgatgcgctgctgaacaaaagcggcgcgaccggcgtgtttaccgatttt

[0064] ccggataccggcgtggaatttctgaaaggcattaaaggtggtggtggttctggtggtggtggttctggtggtggtggttctgaacattggagctatggcctgcgcccgggctaa。

[0065] (2) Induced Expression and Purification of Mammalian Castration Vaccine

[0066] The above plasmid was transformed into Escherichia coli BL21(DE3) by conventional methods and cultured on LB solid medium containing Amp+(100 μg / mL) for 14 h. One transformant was picked and inoculated into LB liquid medium (5 mL) containing Amp + (100 μg / mL) and cultured with shaking at 37 °C at 220 rpm for 5 h; 4 mL of the bacterial liquid after 16 h of culture was taken and added to 300 mL of fresh LB liquid medium containing Amp +In LB liquid medium (100 μg / mL), shake-culture at 160 rpm at 16 °C for 3 h until OD 600 reaches 0.7, add IPTG with a final concentration of 0.1 mmol / L, continue to shake-culture at 16 °C at 160 rpm for 24 h, centrifuge at 4000 rpm and 4 °C for 15 min, collect the bacterial precipitate to obtain the recombinant bacterium BL21(DE3)-PD-GnRH, Escherichia coli, which is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2025, and the deposit number is CGMCC No. 34155.

[0067] Take 200 mL of the bacterial precipitate (recombinant bacterium) and resuspend it in 40 mL of PBS solution. Add lysozyme with a final concentration of 0.4 mg / mL to the resuspended bacteria and incubate for 45 min; the ultrasonic power is 400 W, ultrasonic for 4 s, intermittent for 6 s, and ultrasonic for 60 min; after the ultrasonic treatment, centrifuge the bacterial solution at 12,000 rpm and 4 °C for 20 min, and purify the supernatant with a nickel column to obtain the PD-GnRH recombinant protein castration vaccine.

[0068] Use SDS-PAGE electrophoresis to detect the molecular weight of the PD-GnRH recombinant protein. When detecting, first electrophorese at 80 V for 20 min, and then electrophorese at 140 V for 60 min. The detection results are as Figure 2 shown.

[0069] Use GnRH antibody (purchased from Beijing Protein Innovation Co., Ltd.) to perform WB analysis on the PD-GnRH recombinant protein to detect whether the recombinant protein is correctly expressed. The results are as Figure 3 shown. After WB detection, the molecular weight of the recombinant protein is between 35 kDa and 48 kDa, which is consistent with the theoretical value of the recombinant protein of 43.2 kDa. WB analysis shows that the recombinant protein contains the amino acid sequence shown in SEQ ID NO. 1.

[0070] Escherichia coli BL21(DE3) was purchased from Nanjing Novozymes Biotech Co., Ltd.

[0071] Example 2

[0072] Eight adult male cats of any breed, sexually mature (>9 months old), with a weight difference of no more than 1 kg, were selected, with 4 cats in each of the control group and the experimental group. Cats in the control group were subcutaneously injected with 0.5 mL of PBS each time, while cats in the experimental group were subcutaneously injected with 800 μg of the PD-GnRH recombinant protein castration vaccine prepared in Example 1 each time. Strengthening immunization was carried out according to the above method at an interval of 2 weeks, for a total of 3 times. Blood was collected every 2 weeks, and GnRH antibodies ( Figure 4 ) and testosterone concentrations ( Figure 5 ) were detected by indirect ELISA method.

[0073] Figure 4 It was shown that after the second immunization, the GnRH antibody titer increased significantly, significantly higher than that of the control group (p < 0.05).

[0074] Figure 5 It was shown that 6 weeks after immunization, the testosterone concentration in the blood of cats in the experimental group was significantly lower than that of the control group (p < 0.05). This indicates that the PD-GnRH recombinant protein castration vaccine can inhibit the testosterone concentration in cats, thus achieving the purpose of castration.

[0075] As can be seen from the above examples, the present invention provides a GnRH castration vaccine based on the Haemophilus influenzae D protein carrier and a preparation method thereof. When designing the antigen, the present invention adopted the method of recombining PD and GnRH proteins to develop a fusion protein castration vaccine, which only connects a single copy of GnRH decapeptide molecule and can stimulate the body to produce a relatively high level of GnRH antibodies. It shows that PD introduced into the fusion protein, as an adjuvant and immune enhancer of GnRH, can significantly improve the immunogenicity of low-molecular-weight antigens, enhance the immune effect of antigens, and avoid the unstable immune effect caused by polypeptide instability. Compared with other castration vaccines, it reduces the stress of animals, is more convenient to use, and has the advantages of safety, convenience, and mild action effect. It can be directly used for intramuscular injection of animals, with simple and easy operation, and achieves a more ideal effect in controlling the development and reproductive function of animal reproductive organs, further promoting the development of animal castration technology.

[0076] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant PD-GnRH fusion protein, characterized in that, The amino acid sequence of the recombinant PD-GnRH fusion protein is shown in SEQ ID NO.1; the recombinant PD-GnRH fusion protein includes the Haemophilus influenzae D protein and the mammalian GnRH sequence.

2. The recombinant PD-GnRH fusion protein according to claim 1, wherein, The amino acid sequence of the Haemophilus influenzae D protein is shown in SEQ ID NO.2; The amino acid sequence of the mammalian GnRH sequence is shown in SEQ ID NO.

3.

3. The recombinant PD-GnRH fusion protein according to claim 2, wherein The mammals include rats, mice, dogs, cats, pigs, cows or sheep.

4. Use of the recombinant PD-GnRH fusion protein according to any one of claims 1 to 3 in the preparation of mammalian castration products.

5. A recombinant bacterium expressing a recombinant PD-GnRH fusion protein, characterized in that, The recombinant bacterium is BL21(DE3)-PD-GnRH, and the BL21(DE3)-PD-GnRH is Escherichia coli, deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 10, 2025, and the deposit number is CGMCC No. 34155; The amino acid sequence of the recombinant PD-GnRH fusion protein is shown in SEQ ID NO.

1.

6. Use of the recombinant bacterium according to claim 5 in the preparation of mammalian castration products.

7. A recombinant PD-GnRH fusion protein castration vaccine, characterized in that, The castration vaccine of the recombinant PD-GnRH fusion protein includes the Haemophilus influenzae D protein shown in SEQ ID NO.

2.

8. The recombinant PD-GnRH fusion protein castration vaccine according to claim 7, wherein The castration vaccine of the recombinant PD-GnRH fusion protein includes the mammalian GnRH sequence shown in SEQ ID NO.

3.

9. A preparation method of a recombinant PD-GnRH fusion protein castration vaccine, characterized in that, Comprising the following steps: Obtained by lysing and purifying the recombinant bacterium according to claim 5; The purification method is nickel column purification.

10. Use of the recombinant PD-GnRH fusion protein castration vaccine according to any one of claims 7 to 8, characterized in that, It can be applied in any of the following situations: (1) For male animal castration products; (2) Products for inhibiting the testicular growth and development of male animals; (3) Products for reducing the aggressive behavior and / or sexual behavior of male animals.

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