A combination of haemonchus contortus antigen proteins and its use in the preparation of an immunoprotective haemonchus contortus subunit vaccine

Abstract

The application discloses a Haemonchus contortus antigen protein combination and application thereof in preparation of a Haemonchus contortus immunoprotective subunit vaccine, and belongs to the field of veterinary vaccines. The subunit vaccine comprises galactose and fucose modified antigen proteins MEP1, MEP3, MEP4, PEP2 and CP1, the fucose modification comprises proximal and distal alpha 1,3 fucose modification, the antigen proteins in the subunit vaccine are closer to natural proteins in terms of N glycosylation modification, the subunit vaccine can induce goats to generate protective antibodies after mixed immunization, and can significantly reduce the parasitic quantity of Haemonchus contortus in the goats and the oviposition quantity of the worms. The subunit vaccine can be used for preparing a drug for preventing and treating Haemonchus contortus disease. The application lays a foundation for development of a Haemonchus contortus subunit vaccine, provides an effective technical means for prevention and treatment of the disease, and has important application value and popularization prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary vaccines, specifically relating to a combination of Haemaphysalis contortus antigen proteins and its application in the preparation of Haemaphysalis contortus immunoprotective subunit vaccines. Background Technology

[0002] Haemonchus contortus is a blood-sucking parasitic nematode that infests the abomasum of ruminants such as sheep and cattle. Haemonchus contortus disease is one of the most serious parasitic diseases affecting small ruminants. Affected animals exhibit emaciation, anemia, and weakness; severe cases can lead to death.

[0003] Currently, the control of Haemaphysema contortus disease relies heavily on chemical anti-helmintic drugs such as benzimidazoles, imidazothiazides, macrolides, and aminoethyl cyanide derivatives. Long-term irrational use of these drugs has led to severe drug resistance in this nematode worldwide, and existing chemical control methods are at risk of becoming ineffective.

[0004] Currently, the only commercially available vaccine against Haemophilus contortus globally is Barbervax®, whose active ingredients are the H11 protein and H-gal-GP protein derived from the natural intestinal worm. However, because Haemophilus contortus cannot be cultured in vitro, the yield of natural protein antigens is extremely low, and the production cost is high, making large-scale production and widespread application difficult. Various forms of recombinant proteins have failed to provide adequate protection after immunization. When goats were immunized with a mixture of recombinantly expressed H11 subtypes (H11, H11-1, H11-2, and H11-4) in Hi5 insect cells, the protective effect against egg reduction was 66.29%, with no significant difference. When Sf9 insect cells were recombinantly expressed with MEP1, MEP3, and MEP4 components of H-gal-GP, and another component, PEP1, was expressed in E. coli, the protective effect against egg reduction in goats was only 2.5%. Other recombinant protein forms also failed to induce good immune protection in goats.

[0005] The applicant's prior patent, "A Subunit Vaccine of Haemaphysema contortus" (Publication No. CN120241990A), discloses a Haemaphysema contortus antigen-protein combination that provides effective immune protection in goats infected with Haemaphysema contortus, reducing egg production by 81.43%. However, when this antigen-protein combination was used for immunization and challenge according to the described protocol, and the goats were euthanized on day 28 post-challenge, their abomasums were examined, and the adult worm load was calculated, the results showed that the reduction rate of female worms in the experimental group was 27.9%, the reduction rate of male worms was 23.68%, and the total reduction rate was 25.90%. This result indicates that the antigen-protein combination has limitations in reducing adult worm load (reduction rate), and it may be missing some key immune protective components. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a combination of Haemaphysalis contortus antigen proteins and its application in the preparation of Haemaphysalis contortus immunoprotective subunit vaccines, thereby solving the problems mentioned in the background art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a *Haemaphysalis contortus* antigen protein assembly comprising galactose-modified and fucose-modified antigen proteins MEP1, MEP3, MEP4, PEP2, and CP1, wherein the fucose modification includes proximal α1,3-fucose modification and distal α1,3-fucose modification. Preferably, the galactose and fucose modifications of the antigen proteins are achieved by co-expressing one or more of the *Haemaphysalis contortus* β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the aforementioned antigen proteins in eukaryotic cells. The amino acid sequence of the β1,4-galactosyltransferase HcGALT is shown in SEQ ID NO.1; the amino acid sequence of the proximal α1,3-fucosyltransferase HcFUT1 is shown in SEQ ID NO.3; the amino acid sequence of the distal α1,3-fucosyltransferase HcFUT6 is shown in SEQ ID NO.5; the amino acid sequence of the antigen protein MEP1 is shown in SEQ ID NO.7; the amino acid sequence of the antigen protein MEP3 is shown in SEQ ID NO.9; the amino acid sequence of the antigen protein MEP4 is shown in SEQ ID NO.11; the amino acid sequence of the antigen protein PEP2 is shown in SEQ ID NO.13; and the amino acid sequence of the antigen protein CP1 is shown in SEQ ID NO.15.

[0009] In some embodiments, the eukaryotic cells include mammalian cells, insect cells, plant cells, etc., wherein the mammalian cells include human embryonic (HEK) 293 cells, Chinese hamster ovary (CHO) cells, etc., and the insect cells include Sf9 cells, Hi5 cells, Sf21 cells, etc. Further, the eukaryotic cells are insect cells.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned *Haemaphysalis contortus* antigen protein combination, comprising the following steps: constructing one or more of the following nucleotide sequences into the same eukaryotic expression vector or multiple eukaryotic expression vectors: encoding β1,4-galactosyltransferase HcGALT, α1,3-fucosyltransferase HcFUT1, α1,3-fucosyltransferase HcFUT6, and antigen protein; transfecting the constructed eukaryotic expression vector into eukaryotic cells; culturing the cells to express β1,4-galactosyltransferase HcGALT, α1,3-fucosyltransferase HcFUT1, α1,3-fucosyltransferase HcFUT6, and antigen protein; and modifying the antigen protein with galactose and fucose; and purifying the antigen protein to obtain galactose-modified and fucose-modified antigen protein. Preferably, a signal peptide is attached to the N-terminus of one or more of the β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, or the antigen protein, and a tag is preferably attached to the N-terminus or C-terminus of the antigen protein to facilitate the purification of the antigen protein; more preferably, the N-terminus of the antigen protein is attached to a signal peptide or a tag.

[0011] In some embodiments, the method for preparing the *Haemaphysalis contortus* antigen protein combination includes the following steps: constructing the nucleotide sequence encoding the proximal α1,3-fucosyltransferase HcFUT1 and the nucleotide sequence encoding the distal α1,3-fucosyltransferase HcFUT6 into one or more different eukaryotic expression vectors, transfecting them into eukaryotic cells to obtain glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6; and converting the nucleotide sequence encoding the β1,4-galactosyltransferase HcFUT6 into a glycoengineered cell. One or more of the nucleotide sequences of GALT and the nucleotide sequences encoding the antigen protein are constructed into the same eukaryotic expression vector or multiple eukaryotic expression vectors. The constructed eukaryotic expression vectors are transfected into the above-mentioned glycoengineered cells. The cells are cultured to express β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the antigen protein. The antigen protein is then modified with galactose and fucose. The galactose-modified and fucose-modified antigen proteins are obtained after purification.

[0012] In some embodiments, the method for preparing the *Haemaphysalis contortus* antigen protein combination includes the following steps: constructing an optimized nucleotide sequence encoding the β1,4-galactosyltransferase HcGALT onto pFastBacDual to obtain the recombinant plasmid pFastBacDual-Hcgalt; constructing an optimized nucleotide sequence encoding the antigen protein onto pFastBacDual-Hcgalt to obtain the recombinant plasmids pFastBacDual-Hcgalt-MEP1, pFastBacDual-Hcgalt-MEP3, pFastBacDual-Hcgalt-MEP4, pFastBacDual-Hcgalt-PEP2, or pFastBacDual-Hcgalt-CP1; and transposing the plasmid to obtain the recombinant rod mid-Hc The recombinant baculoviruses rBV-Hcgalt-MEP1, rBV-Hcgalt-MEP3, rBV-Hcgalt-MEP4, rBV-Hcgalt-PEP2, or rBV-Hcgalt-CP1 were transfected into insect cells and packaged to obtain recombinant baculoviruses rBV-Hcgalt-MEP1, rBV-Hcgalt-MEP3, rBV-Hcgalt-MEP4, rBV-Hcgalt-CP1, and rBV-Hcgalt-PEP2. The target proteins were expressed in glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6 via infection with the recombinant baculoviruses. The galactose-modified and fucose-modified antigen proteins MEP1, MEP3, MEP4, PEP2, or CP1 were purified. The insect cells used were preferably Sf9 cells. The glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6 are preferably obtained by a method comprising the following steps: constructing optimized nucleotide sequences encoding proximal α1,3-fucosyltransferase HcFUT1 and optimized nucleotide sequences encoding distal α1,3-fucosyltransferase HcFUT6 into an insect overexpression vector, and transforming the constructed insect overexpression vector into Sf9 cells to obtain the glycoengineered cells; wherein the overexpression vector is preferably a pIEx vector.

[0013] In the above-mentioned method for preparing the *Haemaphysalis contortus* antigen protein combination, the optimized nucleotide sequence encoding β1,4-galactosyltransferase HcGALT is shown in SEQ ID NO.2, the optimized nucleotide sequence encoding proximal α1,3-fucosyltransferase HcFUT1 is shown in SEQ ID NO.4, the optimized nucleotide sequence encoding distal α1,3-fucosyltransferase HcFUT6 is shown in SEQ ID NO.6, the optimized nucleotide sequence encoding antigen protein MEP1 is shown in SEQ ID NO.8, the optimized nucleotide sequence encoding antigen protein MEP3 is shown in SEQ ID NO.10, the optimized nucleotide sequence encoding antigen protein MEP4 is shown in SEQ ID NO.12, the optimized nucleotide sequence encoding antigen protein PEP2 is shown in SEQ ID NO.14, and the optimized nucleotide sequence encoding antigen protein CP1 is shown in SEQ ID NO.16.

[0014] In the above-mentioned method for preparing the *Haemaphysalis contortus* antigen protein combination, the N-terminus of one or more of the following: β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, or the antigen protein, may be linked to one or more of the following: a Kozak sequence, a signal peptide sequence, or a tag sequence. The signal peptide sequence is preferably MKTIIALSYIFCLVFAAG (SEQ ID NO.17), and the tag is preferably one or more of the following: a Flag tag, a 10×His tag, or a HA tag. The signal peptide, the tag, and the antigen protein are preferably linked by a flexible linker (preferably GS).

[0015] In the above-mentioned method for preparing the antigen protein combination of Haemaphysalis contortus, the galactose-modified and fucose-modified antigen proteins are purified by tagging during protein purification, without retaining β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6.

[0016] Thirdly, this invention provides the application of the above-mentioned Haemaphysalis contortus antigen protein combination in the preparation of Haemaphysalis contortus vaccines. The vaccine is a subunit vaccine.

[0017] Fourthly, the present invention provides a protective subunit vaccine against Haemonchus contortus, comprising the above-mentioned Haemonchus contortus antigen protein combination, and may further comprise an adjuvant. The adjuvant is preferably a saponin adjuvant.

[0018] In some embodiments, the Haemonchus contortus immunoprotective subunit vaccine may also contain other antigenic proteins or combinations of antigenic proteins from Haemonchus contortus, such as one or more of the antigenic protein combinations in the applicant's prior patent "A Haemonchus contortus Subunit Vaccine" (Publication No. CN120241990A): antigenic protein MEP3, antigenic protein AP1, antigenic protein AP8, antigenic protein H11-2, antigenic protein H11-4, and antigenic protein AP5.

[0019] Fifthly, the present invention provides the use of the above-mentioned Haemaphysalis contortus antigen protein combination or the above-mentioned Haemaphysalis contortus immunoprotective subunit vaccine in the preparation of a drug for the prevention and treatment of Haemaphysalis contortus disease.

[0020] In a sixth aspect, the present invention provides a drug for preventing and treating Haemonchus contortus disease, comprising the above-mentioned Haemonchus contortus antigen protein combination or the above-mentioned Haemonchus contortus immunoprotective subunit vaccine.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] (1) The subunit vaccine made from the antigen protein combination of the present invention can provide effective immune protection in goats infected with Haemaphysema contortus, with a parasite reduction rate of 51.28% and an egg reduction rate of 47.3%.

[0023] (2) Currently, the commercial vaccine Barbervax® for Haemonchus contortus is derived from the natural intestinal tract of the parasite. However, since the parasite cannot be cultured in vitro, the yield of natural protein antigens is low. Recombinant subunit vaccines can be expressed and purified in large quantities in vitro, reducing production costs.

[0024] (3) This invention lays the foundation for the development of a highly efficient Haemaphysema contortus subunit vaccine, provides an effective technical means for the prevention and control of the disease, and has important application value and promotion prospects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the proximal and distal α1,3-fucosyltransferase overexpression plasmids pIEx-HcFUT1 and pIEx-HcFUT6.

[0026] Figure 2 The expression levels of HcFUT1 and HcFUT6 in Sf9 cells after double plasmid transfection were analyzed by qPCR and Western blot. In the figure, A represents the qPCR results and B represents the Western blot results.

[0027] Figure 3 A schematic diagram illustrating the construction of the recombinant donor plasmid.

[0028] Figure 4The results are from the PCR identification of Bacmid.

[0029] Figure 5 The results are Western-bolt images of the viral supernatants. In the figure, R indicates the protein sample treated under reducing conditions.

[0030] Figure 6 This is an SDS-PAGE electrophoresis image of the recombinant protein.

[0031] Figure 7 The mass spectrum of N-glycans released from recombinant proteins cleaved by PNGase F.

[0032] Figure 8 This is a mass spectrum of N-glycans released from recombinant proteins cleaved by PNGase A.

[0033] Figure 9 Immunization and challenge procedures for animal experiments.

[0034] Figure 10 This refers to the egg count and adult load in goats during a subunit vaccine immunization trial.

[0035] Figure 11 The antibody levels in the goat serum of each experimental group are shown.

[0036] Figure 12 Western blot plots of serum antibodies from the immunized goat group and the control group against recombinant protein and native H-gal-GP protein, respectively. In the plots, NR indicates protein samples treated under non-reducing conditions. Detailed Implementation

[0037] The applicant discovered that the natural protein has proximal and distal α1,3-fucose and Galβ1,4-Fuc modifications in its N-glycosylation, but the recombinant protein expressed by Sf9 cells does not have these glycan modifications. Using a glycoengineering strategy, a glycoengineered Sf9 cell line capable of expressing dual α1,3-fucosyltransferases (HcFUT1 and HcFUT6) was constructed. Based on this, using a dual-gene expression vector, the target gene and β1,4-galactosyltransferase HcGALT were simultaneously expressed in the glycoengineered Sf9 cells. Proximal and distal α1,3-fucose modifications and Galβ1,4Fuc modifications were introduced into the N-glycan core of the recombinant H-gal-GP protein, resulting in the recombinant expression of five components of H-gal-GP (MEP1, MEP3, MEP4, PEP2, and CP1). This subunit vaccine is closer to the N-glycosylation modification of the natural protein in terms of N-glycosylation modification, and after mixed immunization, it induces protective antibodies in goats, which can significantly reduce the number of Haemaphysema contortus parasites and the number of eggs laid by the parasites in goats.

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] Example 1: Construction of Sf9 cells engineered from proximal and distal α1,3-fucosyltransferases

[0040] Based on the sequence information of the proximal core α1,3-fucosyltransferase CeFUT1 (GenBank: CAG32977.1, Uniprot: G5EDR5) of *C. elegans*, the homologous protein CDJ96721.1 in *Haemaphysalis contortus* was identified and named HcFUT1; based on the sequence information of the distal core α1,3-fucosyltransferase CeFUT6 (GenBank: CAG32980.1, Uniprot: G5EEE1) of *C. elegans*, the homologous protein CDJ84058.1 in *Haemaphysalis contortus* was identified and named HcFUT6. The coding sequences of HcFUT1 and HcFUT6 were optimized based on insect cell codon preferences, and the optimized gene fragments were cloned into the pIEx insect cell overexpression vector (this vector utilizes the IE1 (Immediately Early 1) promoter to drive continuous and stable high-level expression of exogenous genes without relying on a baculovirus very late initiation system), resulting in pIEx-HcFUT1 and pIEx-HcFUT6. A schematic diagram of plasmid construction is shown below. Figure 1 As shown in SEQ ID NO.3, the amino acid sequence of the proximal α1,3-fucosyltransferase HcFUT1 is shown in SEQ ID NO.4, and the nucleotide sequence encoding α1,3-fucosyltransferase HcFUT6 is shown in SEQ ID NO.5. The amino acid sequence of the distal α1,3-fucosyltransferase HcFUT6 is shown in SEQ ID NO.6.

[0041] Sf9 cells were co-transfected with pIEx-HcFUT1 and pIEx-HcFUT6 expression plasmids and the G418 selection plasmid (pIE1-NEO). A control group (pIEx empty vector plasmid + pIE1-NEO) and a blank control group were also established. Stably transfected polyclonal cell lines were established using a G418 resistance selection strategy. Forty-eight hours after transfection, the cell culture medium was replaced with G418 selection medium at a final concentration of 0.3 mg / mL for further selection of positive clones. During the selection process, untransfected cells exhibited significant apoptosis on day 7 and completely lost proliferative activity by day 12, with a cell death rate of 100%. In contrast, cells in the experimental group and the empty vector control group showed good viability, exhibiting a stable G418 resistance phenotype, and surviving cells showed typical adherent growth characteristics. The selection process was then complete. The resistant clones were further expanded and passaged multiple times, then cryopreserved and subsequently thawed. In this study, the glycoengineered Sf9 cells screened were successfully revived and resumed normal growth. Protein expression was identified by qPCR and Western blotting, and the results are as follows: Figure 2 The RNA and protein levels of HcFUT1 and HcFUT6 in the glycoengineered Sf9 cell line were significantly higher than those in the control group, indicating that the cell line can stably express exogenous genes.

[0042] Example 2: Expression of N-glycosylated modified antigen proteins MEP1, MEP3, MEP4, PEP2, and CP1 from Haemaphysalis contortus.

[0043] 1. Construction of recombinant donor plasmid

[0044] The antigen proteins in this invention are truncated protein fragments designed based on the sequences published in Uniprot (MEP1: A0A7I4Y5P2, MEP3: O76751, MEP4: Q9Y1I4, PEP2: Q70JE2, CP1: A0A7I4XSM3): MEP1 protein 30-823 AAs (antigen protein MEP1), MEP3 protein 30-837 AAs (antigen protein MEP3), MEP4 protein 30-938 AAs (antigen protein MEP4), PEP2 protein 17-427 AAs (antigen protein PEP2), and CP1 protein 21-339 AAs (antigen protein CP1). The nucleotide sequences encoding each protein fragment were codon-optimized. The amino acid sequences of MEP1 protein 30-823AAs are shown in SEQ ID NO.7, and the optimized nucleotide sequences encoding MEP1 protein 30-823AAs are shown in SEQ ID NO.8; the amino acid sequences of MEP3 protein 30-837AAs are shown in SEQ ID NO.9, and the optimized nucleotide sequences encoding MEP3 protein 30-837AAs are shown in SEQ ID NO.10; the amino acid sequences of MEP4 protein 30-938AAs are shown in SEQ ID NO.11, and the optimized nucleotide sequences encoding MEP4 protein 30-938AAs are shown in SEQ ID NO.12; the amino acid sequences of PEP2 protein 17-427AAs are shown in SEQ ID NO.13, and the optimized nucleotide sequences encoding PEP2 protein 17-427AAs are shown in SEQ ID NO.14; the amino acid sequences of CP1 protein 21-339AAs are shown in SEQ ID NO.15, and the optimized nucleotide sequences encoding CP1 protein 21-339AAs are shown in SEQ ID NO.15. As shown in NO.16, a kozak sequence, a SIP signal peptide, a Flag tag, and a 10×His tag were inserted at the N-terminus of MEP1 protein 30-823 AAs, MEP3 protein 30-837 AAs, MEP4 protein 30-938 AAs, and CP1 protein 21-339 AAs, respectively. Similarly, a kozak sequence, a SIP signal peptide, a HA tag, and a 10×His tag were inserted at the N-terminus of PEP2 protein 17-427 AAs. The coding sequences for these fragments were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The kozak sequence is gccgccacc; the signal peptide sequence is MKTIIALSYIFCLVFAAG (SEQ ID NO.17); and the signal peptide, tag, and antigen protein are linked by a flexible linker (GS).Each synthesized sequence was inserted into the middle of the BamHI and EcoRI sites of the pFastBacDual-Hcgalt plasmid, respectively, to construct the pFastBacDual-Hcgalt-MEP1, pFastBacDual-Hcgalt-MEP3, pFastBacDual-Hcgalt-MEP4, pFastBacDual-Hcgalt-CP1, and pFastBacDual-Hcgalt-PEP2 plasmids. The schematic diagram of the construction of each recombinant plasmid is shown below. Figure 3 As shown.

[0045] The aforementioned pFastBacDual-Hcgalt plasmid is a plasmid in which "Kozak sequence - SIP signal peptide coding sequence - Flag tag coding sequence - optimized HcGALT protein 27-442AAs coding sequence" is inserted between the XhoI and KpnI sites of pFastBacDual. Its construction is described in the applicant's prior patent, "A Subunit Vaccine of Haemaphysalis contortus" (Publication No. CN120241990A). The amino acid sequence of HcGALT protein 27-442AAs is shown in SEQ ID NO.1, and the optimized HcGALT protein 27-442AAs coding sequence is shown in SEQ ID NO.2.

[0046] 2. Construction of recombinant baculovirus plasmid (Bacmid)

[0047] (1) Thawing DH10Bac competent cells on ice.

[0048] (2) 200 ng of pFastBacDual-Hcgalt-MEP1, pFastBacDual-Hcgalt-MEP3, pFastBacDual-Hcgalt-MEP4, pFastBacDual-Hcgalt-CP1, and pFastBacDual-Hcgalt-PEP2 plasmids were added to competent cells, and after mixing by gently tapping the tube wall, the cells were placed on ice for 30 min and then heat-shocked in a 42℃ water bath for 60 s.

[0049] (3) Quickly transfer to an ice bath, cool for 2 min, add 500 μL of sterile LB medium without antibiotics to the EP tube, mix well and place in a 37℃, 220 rpm shaking culture for 4 h.

[0050] (4) Dilute LB medium 10 times, take 100 μL of the 100-fold diluted bacterial solution and spread it on an LB plate containing 50 mg / mL kanamycin, 7 mg / mL gentamicin, 10 mg / mL tetracycline, 100 mg / mL X-gal, and 40 mg / mL IPTG. Invert the plate and place it in a 37°C incubator for 48 h.

[0051] (5) Select white single colonies and inoculate them onto LB plates containing 50 mg / mL kanamycin, 7 mg / mL gentamicin, 10 mg / mL tetracycline, 100 mg / mL X-gal, and 40 mg / mL IPTG. Streak the plates upside down in a 37°C incubator and incubate for 24 hours. The colonies that grow are all white colonies.

[0052] 3. Extraction and identification of recombinant Bacmid

[0053] The above-mentioned white single colonies were inoculated into LB liquid medium containing 50 mg / mL kanamycin, 7 mg / mL gentamicin, and 10 mg / mL tetracycline, and incubated at 37°C and 220 rpm for 12-16 h. The bacterial culture was collected, and bacmids were extracted using the Beyotime baculovirus shuttle vector bacmid mini-extraction kit. PCR amplification was performed according to the primer sequences in Table 1. The electrophoresis results of the products are shown below. Figure 4 As shown, the fragment matching the target fragment size was successfully amplified, and the sequencing was correct, proving that the recombinant rod midacids rbacmid-Hcgalt-MEP1, rbacmid-Hcgalt-MEP3, rbacmid-Hcgalt-MEP4, rbacmid-Hcgalt-CP1, or rbacmid-Hcgalt-PEP2 were successfully constructed.

[0054] Table 1. PCR primers for recombinant plasmid identification

[0055] M13-Forward 5'-TGTAAAACGGACGGCCAGT-3' (SEQ ID NO.18) M13-Reserve 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO.19)

[0056] 4. Recombinant bacmid transfected Sf9 cells for viral packaging.

[0057] (1) Culture of Sf9 insect cells: Sf9 cells are a semi-adherent cell line that can be cultured in suspension. The optimal culture temperature is 27-28℃. They do not require CO2 during growth, have a relatively fast growth rate, and a passage time of about 3 days.

[0058] (2) Count the cultured Sf9 cells and take approximately 1×10⁻⁶ cells. 6 One Sf9 cell was placed in a six-well plate and kept still in a cell culture incubator for 20-40 minutes to allow the cells to adhere.

[0059] (3) Preparation of plasmid and transfection agent: Take two sterile EP centrifuge tubes, add 100 μL of cell culture medium Sf-900TM SFM and 8 μL of transfection agent Cellfectin to one tube, and add 100 μL of cell culture medium Sf-900TM SFM and 3 μg of baculovirus plasmid (about 2 μL in volume) to the other tube. Let stand for 5 min, mix well, and then let stand for 30 min.

[0060] (4) After the cells have completely adhered to the well, tilt the six-well plate, aspirate the culture medium along the well wall, add 800 μL of plate culture medium or Sf-900TM SFM culture medium along the well wall, and then add 210 μL of well-mixed Bacmid and transfection agent.

[0061] (5) After covering the six-well plate, the experimental group, negative control and blank control were marked and placed in the incubator for 4 hours to successfully transfect Sf9 insect cells with baculovirus.

[0062] (6) After 4 hours, tilt the six-well plate, aspirate the culture medium along the well wall, add complete culture medium along the well wall, seal the plate, and incubate in an incubator for 96 hours until the cells show typical signs of viral infection, such as increased cell diameter, brightening and rounding, indicating successful transfection. The P0 generation recombinant baculoviruses P0-rBV-Hcgalt-MEP1, P0-rBV-Hcgalt-MEP3, P0-rBV-Hcgalt-MEP4, P0-rBV-Hcgalt-CP1, and P0-rBV-Hcgalt-PEP2 are packaged. The supernatant is collected as the viral solution containing the P0 generation recombinant baculovirus.

[0063] 5. Viral amplification in generations P1, P2, and P3

[0064] Sf9 cells in the exponential growth phase are prepared at a density of 2 × 10⁻⁶. 6 Cells / mL, P0 generation recombinant baculovirus solution was inoculated at 1:30, cultured for 4-5 days, centrifuged to collect the virus, and the supernatant was P1 generation recombinant baculovirus. Similarly, P2 and P3 generation recombinant baculoviruses were amplified.

[0065] 6. Protein expression verification

[0066] The proximal and distal α1,3-fucosyltransferase-engineered Sf9 cells constructed in Example 1 were cultured. The culture of the glycoengineered Sf9 insect cells was consistent with that of wild-type Sf9 insect cells. Glycoengineered Sf9 cells in the exponential growth phase were prepared at a density of 2 × 10⁻⁶ cells / year. 6Cells / mL, P3 generation recombinant baculovirus was used to infect glycoengineered Sf9 cells at a multipilicity of infection (MOI) of 0.1, and the cells were cultured at 28℃ and 120 rpm for 96 h. The supernatant was collected by centrifugation. Protein expression was detected by Western blot. The primary antibody used was anti-flag mouse monoclonal antibody / anti-His mouse monoclonal antibody, and the secondary antibody was HRP-goat anti-mouse secondary antibody. Results are as follows: Figure 5 As shown, all proteins are expressed in Sf9 cells.

[0067] 7. Protein purification

[0068] The purification of recombinant proteins mainly involves the His tag on the target protein. The β1,4-galactosyltransferase Hcgalt functions in the viral solution to transfer galactose residues to the N-glycosylation modification of the target protein. Because it does not carry a His tag, it does not bind to the packing material and does not appear in the eluent. Meanwhile, the proximal and distal α1,3-fucosyltransferases expressed in the cytoplasm are not secreted into the viral solution. Finally, the N-glycosylated directed modified target proteins GE-rMEP1 (Glycoengineered-rMEP1), GE-rMEP3 (Glycoengineered-rMEP3), GE-rMEP4 (Glycoengineered-rMEP4), GE-rCP1 (Glycoengineered-rCP1), and GE-rPEP2 (Glycoengineered-rPEP2) are obtained through purification. The specific purification steps are as follows:

[0069] (1) Mix 2 mL of Ni SepHarose 6FF His packing material and load it into the purification column. After the ethanol solution in the packing material flows out under gravity, start the protein purification work. All solutions used in the purification process are sterilized by passing through a 0.22 μm filter.

[0070] (2) Wash away any residual ethanol solution in the packing material with 5-10 times the volume of deionized water. Equilibrate the packing material with 10 column volumes of Binding Buffer A.

[0071] (3) Mix the supernatant protein (through a 0.45μm filter) with the filler evenly. To improve the binding efficiency with the protein, incubate at 4℃ for 1h.

[0072] (4) After passing the supernatant protein through the column three times, use 15 column volumes of Binding Buffer A to wash away any extraneous proteins that do not bind to the packing material.

[0073] (5) Use 5 column volumes of different concentrations of imidazole solution (20mM, 40mM, 60mM, 80mM, 100mM, 200mM, 500mM) to perform gradient elution of the target protein, collect the effluent, and determine the optimal elution gradient.

[0074] (6) Equilibrate with 10 column volumes of Binding Buffer A and deionized water respectively. Clean the packing material and finally store it in 20% ethanol solution at 4°C.

[0075] (7) Cut a dialysis bag of appropriate length, soak it in boiling water for 10 minutes, and check for leaks. After identification by SDS-PAGE, add the protein elution buffer into the dialysis bag, fix it with a clamp, and place it in pre-cooled PBS solution. Dialyze at 4°C for 24 hours, stirring frequently and changing the solution 3-4 times during the process.

[0076] (8) Cover the surface of the dialysis bag with sucrose. After 2-3 hours, liquid will be clearly precipitated out.

[0077] (9) Transfer the concentrated target protein from the dialysis bag to centrifuge tubes, aliquot and store at -80℃.

[0078] (10) The SDS-PAGE results of all recombinant proteins are as follows: Figure 6 As shown in the figure. The GE-PEP2 protein purification effect was poor, and the presence of the target protein in the eluent was confirmed by Western blot. The concentration of the purified target protein was determined by the BCA method.

[0079] 8. Preparation of N-glycans from protein samples and MALDI-ToF analysis

[0080] The workflow for N-glycan preparation includes enzymatic digestion, enrichment, and full methylation derivatization. The specific steps for processing each protein sample are as follows:

[0081] (1) Protein sample preparation: Take 200 μg of protein and add methanol, sterile water and chloroform in the ratio of methanol: protein + water: chloroform = 600 μL: 600 μL: 150 μL. Mix well by pipetting and vortexing. At this time, the sample solution is milky white. Centrifuge the sample solution at 10000 r / min for 3 min. A white flocculent film appears in the middle of the solution. Discard the supernatant. Add 500 μL of methanol to wash the protein film. Mix well by pipetting and vortexing. Centrifuge at 12000 r / min for 3 min. Discard the supernatant and keep the precipitate. Put the precipitate sample into a vacuum rotary evaporator for about 30 min.

[0082] (2) PNGase F digestion and enrichment of polysaccharides: Using Beyotime's PNGase F deglycosylation kit (P2318S), 2 μL of 10× Denaturing Buffer and 18 μL of ddH2O were added to the evaporated protein sample. After thorough vortexing, the sample was boiled at 100℃ for 10 min to denature. After denaturation, the sample was immediately placed on ice for 1 min to pre-cool. Then, 4 μL of 10× Reaction Buffer, 4 μL of 10× Renaturing Buffer and 12 μL of ddH2O were added in sequence. After thorough vortexing, 2 μL of PNGase F enzyme was added and thoroughly vortexed. The sample was then incubated overnight at 37℃ and 1500 rpm using an enzyme digester.

[0083] (3) The polysaccharides released from PNGase F were desalted and purified using a porous graphite carbon column (PGC column). The specific steps are as follows: First, the PGC column was activated with 3 mL of acetonitrile and 3 mL of washing solution, and then the PGC column was equilibrated with 3 mL of equilibration buffer. Next, the sample diluted with equilibration buffer was loaded into the PGC column. After the sample in the PGC column settled naturally by gravity, it was washed three times with 3 mL of equilibration buffer to remove impurities such as proteins, peptides, and salts. Finally, the sample was eluted with 1 mL of elution buffer and dried using a vacuum rotary evaporator.

[0084] (4) PNGase A digestion of glycans: After collecting the glycan samples digested with PNGase F, 1 mL of methanol solution was added to the remaining protein sample, centrifuged at 12000 r / min for 5 min, and the supernatant was discarded. This process was repeated 3 times. The protein precipitate was then dried completely in a clean bench. Then, using the NEB PNGase A deglycosylation kit (P07070S), 2 μL of 10× Denaturing buffer and 18 μL of ddH2O were added to the dried protein sample. After thorough vortexing, the sample was denatured at 100℃ for 10 min. After denaturation, the sample was immediately pre-cooled on ice for 1 min. Then, 4 μL of 10× Reaction buffer, 4 μL of 10× Renaturing buffer and 12 μL of ddH2O were added in sequence. After thorough vortexing, 2 μL of PNGase A enzyme was added and thoroughly vortexed. The sample was then digested in a metal bath at 37℃ and 1500 r / min for 5 h.

[0085] (5) Enrichment of PNGase A-digested glycans: After digestion, the sample was centrifuged at 12000 r / min for 5 min, and the supernatant glycan solution was collected. The obtained glycan solution was desalted and purified by a PGC column, and the specific steps were the same as those for the enrichment of PNGase F-digested glycans.

[0086] (6) Full methylation derivatization of polysaccharides: The polysaccharide sample obtained by the above treatment was dissolved in 50 μL DMSO, and then added to 100 μL DMSO-NaOH homogenate and mixed thoroughly. Then, 50 μL iodomethane was added in a fume hood and vortexed at room temperature for 15 min until the solution turned milky white. Then, 500 µL ultrapure water was added to terminate the reaction, and then 250 µL chloroform was added for extraction. After the liquid phases separated, the upper layer was gently removed, and the lower organic phase containing polysaccharides was thoroughly washed with 1 mL ultrapure water. The washing was repeated 5 times. Finally, the two organic phases were transferred to a new centrifuge tube and dried by vacuum rotary evaporator.

[0087] MALDI-ToF analysis:

[0088] 1) Dissolve the evaporated fully methylated derivatized polysaccharide in 15 μL of methanol solution.

[0089] 2) Take 1 μL of polysaccharide solution and mix it thoroughly with 1 μL of freshly prepared matrix DHB (2,5-dihydroxybenzoic acid).

[0090] 3) Take 1 μL of the sample to be tested and spot it on the MALDI metal plate, and let it stand at room temperature to allow it to crystallize.

[0091] 4) Then, the sample was detected using the SCIEX MALDI 5800 ToF / ToF instrument. The relevant parameters of the instrument were set as follows: detection mode, positive ion reflection mode; accelerating voltage 20kV; laser intensity 5000; using 2kV collision energy, with air as the CID gas for MS / MS analysis; each spectrum consists of 1000 shots.

[0092] 5) The final data were analyzed using Data Explorer 4.0, and the inferred N-glycan structure was obtained using GlycoWorkbench 2.1. Galactose-fucose modification was identified in the N-glycans of all glycosyl-engineered recombinant proteins. Figure 7 and Figure 8 (indicated by the red arrow) and proximal α1,3-fucose ( Figure 8 (Indicated by the blue arrow); distal α1,3-fucose was identified in the N-glycans of GE-rPEP2 and GE-rMEP3. Figure 7 (As indicated by the green arrow).

[0093] Example 3: Immunoprotective Experiment of Haematococcus contortus Antigen Protein Combination

[0094] Five purified recombinant antigen proteins were mixed for a goat immunoprotection experiment, with an adjuvant control group and an immunization group, each containing eight goats. Five fecal examinations were performed on the animals before the experiment, and no parasite eggs were found. Each goat in the experimental group was immunized with 70 μg each of recombinant proteins GE-MEP1, GE-MEP3, GE-MEP4, GE-CP1, and GE-PEP2, combined with 500 μg of Quil-A (saponin) adjuvant. Each goat in the control group was immunized with only 500 μg of Quil-A adjuvant. All immunizations were administered via subcutaneous injection at multiple sites on the neck and back, with immunizations every three weeks for a total of three immunizations. At the third immunization, all goats were orally infected with 7000 infective third-stage larvae of *Haemaphysema contortus*. Blood samples were collected every 7 days from the first immunization, for a total of 12 serum samples. Changes in goat serum antibody levels were detected by indirect ELISA. The overall animal experimental protocol is as follows: Figure 9 As shown. Starting from day 18 (Day 60) after the challenge, fecal samples were collected rectally every one day for fecal egg count (FEC), for a total of 9 collections. Three goats in the adjuvant control group died unexpectedly. To exclude the interference of abnormal individuals on statistical inference and to ensure the scientific validity and accuracy of the data between groups, these deceased samples were excluded according to the pre-set exclusion criteria and not included in the final protective efficacy evaluation. The remaining goats were euthanized on day 35 (Day 77) after the challenge, and the adult worm load in the abomasum was counted.

[0095] The results of worm reduction rate, egg reduction rate, and fecal egg count are as follows: Figure 10 As shown. Regarding oviposition inhibition, the experimental group of this antigen-protein combination vaccine showed a cumulative oviposition reduction rate of 51.28% compared to the control group, indicating that the vaccine-induced immune response weakened the reproductive potential of adult worms to some extent. In terms of worm reduction, necropsy results showed a female worm reduction rate of 51.02% and a male worm reduction rate of 43.89% in the experimental group, with a final total worm reduction rate of 47.3%. Compared to the antigen-protein combination in the applicant's prior patent application "A Subunit Vaccine of Haemaphysema contortus" (Publication No. CN120241990A), the antigen-protein combination of this invention exhibits better efficacy in directly eliminating adult worms (i.e., the worm reduction rate indicator).

[0096] Results of antibody level changes after immunization Figure 11As shown in the diagram, indirect ELISA results indicated that after the initial immunization, serum IgG levels in the experimental group of goats rapidly seroconverted, with antibody titers significantly increasing and far exceeding those in the adjuvant control group. As the immunization program progressed, antibody levels reached a steady-state plateau one week after the second immunization. Crucially, this high level of specific immune response demonstrated excellent persistence throughout the subsequent experiments, maintaining its effectiveness until the end of the animal studies, providing a robust humoral immune barrier against Haemonchus contortus attack.

[0097] Western blot results of serum antibodies from the immunized goat group and the control group against recombinant antigen protein and native H-gal-GP protein are as follows: Figure 12 As shown, the antigen protein of the present invention has good immunogenicity.

[0098] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A composition of Haemaphysalis contortus antigen protein, characterized in that: Antigen proteins MEP1, MEP3, MEP4, PEP2, and CP1, which contain galactose-modified and fucose-modified proteins, wherein the fucose modification includes proximal α1,3-fucose modification and distal α1,3-fucose modification. The galactose and fucose modifications of the antigen protein are achieved by expressing the β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the aforementioned antigen protein together in eukaryotic cells. The amino acid sequence of the β1,4-galactosyltransferase HcGALT is shown in SEQ ID NO.1; the amino acid sequence of the proximal α1,3-fucosyltransferase HcFUT1 is shown in SEQ ID NO.3; the amino acid sequence of the distal α1,3-fucosyltransferase HcFUT6 is shown in SEQ ID NO.5; the amino acid sequence of the antigen protein MEP1 is shown in SEQ ID NO.7; the amino acid sequence of the antigen protein MEP3 is shown in SEQ ID NO.9; the amino acid sequence of the antigen protein MEP4 is shown in SEQ ID NO.11; the amino acid sequence of the antigen protein PEP2 is shown in SEQ ID NO.13; and the amino acid sequence of the antigen protein CP1 is shown in SEQ ID NO.

15.

2. The method for preparing the *Haemaphysalis contortus* antigen protein composition according to claim 1, characterized in that, Includes the following steps: The nucleotide sequences encoding β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the antigen protein were constructed into the same or multiple eukaryotic expression vectors. The constructed eukaryotic expression vectors were transfected into eukaryotic cells, and the cells were cultured to express β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the antigen protein. The antigen protein was then modified with galactose and fucose, and the galactose-modified and fucose-modified antigen proteins were obtained after purification.

3. The method for preparing the *Haemaphysalis contortus* antigen protein composition according to claim 2, characterized in that: The β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, or one or more of the antigen protein have an N-terminal signal peptide attached, and the N-terminus or C-terminus of the antigen protein has a tag attached.

4. The method for preparing the *Haemaphysalis contortus* antigen protein composition according to claim 2, characterized in that, The process includes the following steps: constructing the nucleotide sequences encoding proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6 into one or more different eukaryotic expression vectors, transfecting them into eukaryotic cells to obtain glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6; and constructing the nucleotide sequences encoding β1,4-galactosyltransferase HcGALT... The nucleotide sequence encoding the antigen protein is constructed into the same eukaryotic expression vector or multiple eukaryotic expression vectors. The constructed eukaryotic expression vector is transfected into the above-mentioned glycoengineered cells. The cells are cultured to express β1,4-galactosyltransferase HcGALT, proximal α1,3-fucosyltransferase HcFUT1, distal α1,3-fucosyltransferase HcFUT6, and the antigen protein. The antigen protein is then modified with galactose and fucose, and purified to obtain the galactose-modified and fucose-modified antigen proteins.

5. The method for preparing the *Haemaphysalis contortus* antigen protein composition according to claim 2, characterized in that, The process includes the following steps: constructing the optimized nucleotide sequence encoding the β1,4-galactosyltransferase HcGALT onto pFastBacDual to obtain the recombinant plasmid pFastBacDual-Hcgalt; constructing the optimized nucleotide sequence encoding the antigen protein onto pFastBacDual-Hcgalt to obtain the recombinant plasmids pFastBacDual-Hcgalt-MEP1, pFastBacDual-Hcgalt-MEP3, pFastBacDual-Hcgalt-MEP4, pFastBacDual-Hcgalt-PEP2, and pFastBacDual-Hcgalt-CP1; and transposing these plasmids to obtain the recombinant rod mid-Hcgalt-MEP1 and pFastBacDual-Hcgalt-CP1. mid-Hcgalt-MEP3, rbacmid-Hcgalt-MEP4, rbacmid-Hcgalt-PEP2, and rbacmid-Hcgalt-CP1 were obtained. Recombinant baculoviruses rBV-Hcgalt-MEP1, rBV-Hcgalt-MEP3, rBV-Hcgalt-MEP4, rBV-Hcgalt-PEP2, and rBV-Hcgalt-CP1 were obtained after transfection of insect cells with recombinant baculoviruses and packaging. Glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6 were infected with the recombinant baculoviruses to express the target proteins. The galactose-modified and fucose-modified antigen proteins MEP1, MEP3, MEP4, PEP2, and CP1 were purified. The glycoengineered cells stably expressing proximal α1,3-fucosyltransferase HcFUT1 and distal α1,3-fucosyltransferase HcFUT6 were obtained by a method comprising the following steps: constructing optimized nucleotide sequences encoding proximal α1,3-fucosyltransferase HcFUT1 and optimized nucleotide sequences encoding distal α1,3-fucosyltransferase HcFUT6 into an insect overexpression vector, and then transforming the constructed insect overexpression vector into insect cells to obtain the glycoengineered cells.

6. The method for preparing the *Haemaphysalis contortus* antigen protein composition according to claim 5, characterized in that: The optimized nucleotide sequences encoding β1,4-galactosyltransferase HcGALT are shown in SEQ ID NO.2; the optimized nucleotide sequences encoding proximal α1,3-fucosyltransferase HcFUT1 are shown in SEQ ID NO.4; the optimized nucleotide sequences encoding distal α1,3-fucosyltransferase HcFUT6 are shown in SEQ ID NO.6; the optimized nucleotide sequences encoding antigen protein MEP1 are shown in SEQ ID NO.8; the optimized nucleotide sequences encoding antigen protein MEP3 are shown in SEQ ID NO.10; the optimized nucleotide sequences encoding antigen protein MEP4 are shown in SEQ ID NO.12; the optimized nucleotide sequences encoding antigen protein PEP2 are shown in SEQ ID NO.14; and the optimized nucleotide sequences encoding antigen protein CP1 are shown in SEQ ID NO.

16.

7. The use of the Haemaphysalis contortus antigen protein composition according to claim 1 in the preparation of Haemaphysalis contortus vaccine.

8. A protective subunit vaccine against Haemonchus contortus, characterized in that: The composition comprising the Haemaphysalis contortus antigen protein as described in claim 1.

9. The use of the Haemaphysema contortus antigen protein composition of claim 1 or the Haemaphysema contortus immunoprotective subunit vaccine of claim 8 in the preparation of a drug for the prevention and treatment of Haemaphysema contortus disease.

10. A drug for preventing and treating Haemonchus contortus infection, characterized in that: The vaccine comprises the Haemaphysalis contortus antigen protein composition of claim 1 or the Haemaphysalis contortus immunoprotective subunit vaccine of claim 8.

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