A longicorn blood salivary gland multi-component subunit vaccine and a preparation method and application thereof

Abstract

This invention relates to the field of vaccine technology, providing a multi-component subunit vaccine for the salivary glands of *Haemaphysalis longicornis*, its preparation method, and its application. The active ingredients of the vaccine include soluble recombinant proteins rHlPDI-2 and rHlPrx, whose amino acid sequences are shown in SEQ ID NO. 1-2. This invention starts with salivary gland secretion proteins related to tick vitality during the biting process of *Haemaphysalis longicornis*, studies the specific proteins of the tick's salivary glands, and prepares a recombinant protein vaccine that can affect the survival and reproduction of *Haemaphysalis longicornis*. The prepared multi-component subunit vaccine interferes with the biting and passage of *Haemaphysalis longicornis*, exhibiting good anti-tick efficacy. This contributes to the prevention and control of tick-borne diseases and lays the foundation for the development of vaccines for other tick species.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, and in particular relates to a multi-component subunit vaccine for the salivary glands of Haemophilus longicornis, its preparation method, and its application. Background Technology

[0002] The long-horned tick (Haemaphysalis longicornis) is a common ectoparasite whose main hosts include domesticated livestock such as goats, sheep, and cattle, as well as pet dogs, cats, and wild animals such as rodents and hedgehogs. It can also bite humans. As an important vector for zoonotic pathogens, the long-horned tick can transmit various pathogens, including Dabie bandavirus (DBV), tick-borne encephalitis virus (TBEV), Borrelia burgdorferi, Anaplasma phagocytophilum, Ehrlich aspergillus, and spotted fever group Rickettsia (SFGR). Its potential threat has caused serious economic losses to the livestock industry and posed a significant threat to human health. For nearly a century, chemically synthesized insecticides have achieved some success in controlling tick infestations. However, the environmental pollution, drug residues, and tick resistance they cause have increasingly drawn attention. Therefore, vaccination against ticks has become one of the effective tick control strategies due to its friendly and sustainable advantages.

[0003] Currently, many candidate antigen molecules have been reported based on different targets and mechanisms of ticks, leading to the development of various types of vaccines. However, the control efficacy against Haemaphysalis longhorn remains to be studied. Existing commercial vaccines such as Gavac® are developed based on the intestinal glycoprotein BM86 of Haemaphysalis minimus as an immunoreactive antigen, but due to the diversity of tick populations in different regions, their control efficacy against Haemaphysalis longhorn is not ideal. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-component subunit vaccine for the salivary glands of Haemophilus longicornis, aiming to solve the problems mentioned in the background art.

[0005] The present invention is implemented as follows: a multi-component subunit vaccine for the salivary glands of Haemophilus longhorn, wherein the active ingredients of the vaccine include rHlPDI-2 and rHlPrx soluble recombinant proteins, the amino acid sequences of which are shown in SEQ ID NO.1-2.

[0006] Another objective of this invention is to provide a method for preparing a multi-component subunit vaccine for the salivary glands of Haemophilus longhorn, comprising the following steps:

[0007] Recombinant vector construction: Total RNA was extracted from Haemaphysalis longicornis nymphs and reverse transcribed into cDNA using a one-step method. Using the cDNA from Haemaphysalis longicornis as a template, PCR amplification was performed to obtain the target fragment. The target fragment was ligated into the pET-28a(+) vector to obtain recombinant plasmids pET28a-HlPDI-2 and pET28a-rHlPrx.

[0008] Transformation: The recombinant plasmids pET28a-HlPDI-2 and pET28a-rHlPrx2 were successfully transformed into Escherichia coli BL21(DE3) and then cultured on a large scale to obtain a large-scale bacterial culture.

[0009] Expression: IPTG was added to the bacterial culture to induce expression, and the culture was shaken to obtain the protein expression solution;

[0010] Purification: After the culture was completed, the protein expression solution was centrifuged to collect bacteria and obtain a supernatant containing soluble recombinant proteins rHlPDI-2 and rHlPrx. The supernatant was then purified to obtain soluble recombinant proteins rHlPDI-2 and rHlPrx.

[0011] Another objective of this invention is to provide the application of a multi-component subunit vaccine of the salivary glands of Haematochezia longicornis in the preparation of drugs for the prevention and treatment of Haematochezia longicornis.

[0012] This invention focuses on the salivary gland secretion proteins of Haemaphysalis longicornis related to tick vitality during the biting process. By studying the special proteins of the tick's salivary glands, a recombinant protein vaccine was prepared. This vaccine can affect the survival and reproduction of Haemaphysalis longicornis. The prepared recombinant protein vaccine has an interfering effect on the biting and transmission of Haemaphysalis longicornis and has a good anti-tick effect. It contributes to the prevention and control of tick-borne diseases and lays the foundation for the development of vaccines related to other tick species. Attached Figure Description

[0013] Figure 1 The above are antibody level graphs of each group of rabbits before and after immunization provided in the embodiments of the present invention;

[0014] Figure 2 The above are antibody titer charts of each group of rabbits after immunization provided in the embodiments of the present invention;

[0015] Figure 3 Statistical chart of the effect of recombinant protein vaccine against Haemaphysalis longicornis infection provided in the embodiments of the present invention (a is the mortality rate of rabbits within 24 hours after adult ticks bite, b is the body weight of recovered adult ticks after engorgement). Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] The *Escherichia coli* BL21(DE3) used in this embodiment of the invention was purchased from Beijing TransGen Biotechnology Co., Ltd.; the New Zealand white rabbits were purchased from Liaoning Changsheng Biotechnology Co., Ltd.; and the *Haemaphysalis longicornis* was a gift from Hebei Normal University.

[0018] All data were statistically analyzed using SPSS 27.0 software, and graphs were created using GraphPadPrism 10.1.2 software. The mean of the experimental results was calculated, and one-way ANOVA was used to determine its significance. All data are expressed as mean ± standard deviation (SD). A p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).

[0019] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0020] Example 1: A method for preparing a multi-component subunit protein of the salivary glands of Haemaphysalis longicornis, comprising the following steps:

[0021] Step 1: Construction of recombinant vector: Using the cDNA of Haemaphysalis longicornis as a template, PCR amplification was performed to obtain the target fragment. The target fragment was then ligated into the pET-28a(+) vector to obtain the recombinant plasmids pET28a-HlPDI-2 and pET28a-rHlPrx.

[0022] The PCR amplification system is shown in Table 1. The nucleotide sequence of the upstream primer F-rH1PDI-2 NdeⅠ is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer R-rH1PDI-2XhoⅠ is shown in SEQ ID NO.4, the nucleotide sequence of the upstream primer F-rHlPrxNdeⅠ is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer R-rHlPrxXhoⅠ is shown in SEQ ID NO.6.

[0023] Table 1

[0024]

[0025] The PCR reaction procedure is shown in Table 2:

[0026] Table 2

[0027]

[0028] The nucleotide sequence of the correctly sequenced target fragment is shown in SEQ ID NO.7-8;

[0029] Step 2, Transformation: The recombinant plasmids pET28a-HlPDI-2 and pET28a-rHlPrx were successfully transformed into Escherichia coli BL21(DE3) and then cultured on a large scale. 10 mL of the cultured bacterial solution and 500 μL of kanamycin at a concentration of 50 μg / mL were added to 500 mL of LB liquid medium and cultured at 37 °C and 220 rpm for 3 h to obtain the expanded bacterial solution.

[0030] Step 3, Expression: Add 250 μL of 1 M IPTG (isopropyl-βD-thiogalactoside) to the expanded bacterial culture obtained in Step 2 to make the IPTG concentration 0.5 mM, and incubate at 25℃ and 150 rpm for 20 h to obtain the protein expression solution.

[0031] Step 4, Purification: Centrifuge the protein expression solution at 6000 rpm for 10 min to collect the bacteria, discard the supernatant, wash the collected bacterial cells three times with PBS buffer, sonicate and centrifuge at 6000 rpm for 10 min, aspirate the supernatant into a clean centrifuge tube and centrifuge at 12000 rpm for 20 min to obtain the supernatant containing soluble recombinant proteins rHlPDI-2 and rHlPrx and the precipitate containing inclusion body proteins;

[0032] Add 1 mL of nickel affinity packing material to the chromatography column, add equilibration buffer to equilibrate the packing material, equilibrate 3-5 times, add supernatant containing soluble recombinant proteins rHlPDI-2 and rHlPrx, wash 4-6 times with washing buffer, add elution buffer to elute the target protein, concentrate and replace the target protein with ultrafiltration tube to obtain purified soluble recombinant proteins rHlPDI-2 and rHlPrx, the amino acid sequence of which is shown in SEQ ID NO.1-2;

[0033] The replaced protein was aspirated into a centrifuge tube and sealed with a sealing film at -80°C.

[0034] Performance testing:

[0035] The steps for detecting Haemaphysalis longicornis infection and statistically analyzing its physiological parameters are as follows:

[0036] Step 1: Randomly divide New Zealand White rabbits into 3 groups of 3 rabbits each, labeled as the low-dose combined immunization group, the high-dose combined immunization group, and the control group. Immunize according to Table 3, with each immunization 14 days apart, for a total of 3 immunizations.

[0037] Table 3

[0038]

[0039] Step 2: After each immunization, blood was collected from the low-dose combined immunization group, the high-dose combined immunization group, and the control group to detect antibody levels. The collected serum samples were analyzed using an enzyme-linked immunosorbent assay (ELISA) to detect IgG antibodies against the recombinant protein vaccine in rabbit serum. The specific method is as follows:

[0040] S1. Coating: Coat a 96-well plate with the optimal concentration of 3 μg / ml of protein using coating buffer. Add 100 μL of coating buffer to each well and leave overnight at 4°C.

[0041] S2. Washing: Remove the 96-well plate, shake off the contents, and rinse 3 times with 200 μL PBST solution using a pipette, 3 min each time. Discard the washing solution and pat dry with absorbent paper.

[0042] S3, Blocking: Add 100 μL of blocking solution to each well and incubate at 37°C for 2 hours in a constant temperature incubator;

[0043] S4. Washing: Repeat step S2;

[0044] S5. Use of test sample: Dilute the test sample (1:1600 with 1% BSA solution, transfer 100μL per well), add it to the sealed test plate, and incubate at 37℃ for 30min.

[0045] S6. Washing: Repeat washing 5 times;

[0046] S7, Secondary antibody: Dilute with 1% BSA solution 1:5000, add 100μL of IgG secondary antibody to each well, and incubate at 37℃ for 30min;

[0047] S8. Washing: Repeat washing 3 times;

[0048] S9. Color development: Add 100 μL of color development solution to each well and wait 15 min in a dark environment, then add 50 μL of stop solution to each well.

[0049] S10, Reading: Place the 96-well plate into the microplate reader and measure at a wavelength of 450 nm;

[0050] The results are as follows Figure 1 and Figure 2As shown, antibody levels began to rise after the first immunization and remained stable until the end of the third immunization, with an OD value of around 2.0, which was significantly higher than that of the control group (p < 0.05). The antibody titer of the combined low-dose immunization group was 1:256000, and the antibody titer of the combined high-dose immunization group was 1:2048000. The antibody level of the control group did not show a statistically significant difference before and after immunization and can be used for subsequent insect challenge experiments.

[0051] Step 3: After the immune titers of the three groups of rabbits reach a certain level, the challenge experiment can be carried out. Blood from the longhorn ticks is fed to the rabbits' backs. The specific operation is as follows:

[0052] First, sew a straight tube made of white cotton cloth with openings at both ends, large enough to cover the rabbit's back. Heat and melt animal glue and apply it to the edge of the cloth bag or the rabbit's skin. After attaching, let it dry to prevent the long-horned ticks from crawling out. Then, put an Elizabethan collar over the rabbit's head to prevent it from biting the cloth bag. Each rabbit has 25 adult hermaphroditic ticks on its back.

[0053] Observe the physiological indicators of Haemaphysalis longicornis every day, and record the number of Haemaphysalis longicornis ticks adhering to each rabbit, the number of ticks shed after engorgement, the engorgement cycle, and the weight after engorgement (engorgement cycle: the time from adhering to engorgement and shedding of Haemaphysalis longicornis ticks; tick 24-hour mortality rate: the proportion of ticks that die during the blood-feeding process; engorgement weight: the average weight of ticks after blood-feeding).

[0054] The harvested, engorged Haemaphysalis longicornis ticks were weighed and recorded, and then placed in a 25°C constant temperature incubator with sufficient humidity and in the dark for incubation.

[0055] Step 4: Organize the collected data of Haemaphysalis longicornis and use GraphpadPrism software and Oneway ANOVA to perform biostatistical analysis on various physiological indicators.

[0056] The results are as follows Figure 3 As shown, there was no significant difference in the mortality rate of adult ticks between the low-dose combined immunization group and the control group. However, the 24-hour mortality rate of adult ticks in the high-dose combined immunization group reached 54%, which was significantly different from the control group (*p< 0.05). This result indicates that this multi-component subunit vaccine is related to the survival of Haemaphysalis longicornis (e.g., Figure 3 As shown in Figure a), the hydrated body weight of ticks in the low-dose combined immunization group was 216.7 (±34.78) mg, while that in the high-dose combined immunization group was 188.0 (±63.46) mg. The hydrated body weight of the control group was 251.5 (±11.79) mg, showing a significant difference compared to the control group (*p< 0.05). Figure 3 (as shown in b)

[0057] In summary, the multi-component subunit vaccine for salivary glands of Haemaphysalis longicornis prepared according to the embodiments of the present invention has a good effect on the prevention and control of Haemaphysalis longicornis.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-component subunit vaccine for the salivary glands of Haematococcus longicornis, characterized in that, The active ingredients of the vaccine include rHlPDI-2 and rHlPrx soluble recombinant proteins, the amino acid sequences of which are shown in SEQ ID NO.1-2; The concentration of the rHlPDI-2 soluble recombinant protein was 5.0 mg / mL, and the concentration of the rHlPrx soluble recombinant protein was 5.0 mg / mL.

2. A method for preparing a multi-component subunit vaccine for the salivary glands of Haematococcus pluvialis as described in claim 1, characterized in that, Includes the following steps: Recombinant vector construction: Total RNA was extracted from Haemaphysalis longicornis and reverse transcribed into cDNA in one step. Using the cDNA from Haemaphysalis longicornis as a template, PCR amplification was performed to obtain the target fragment. The target fragment was ligated into the pET-28a(+) vector to obtain recombinant plasmids pET28a-HlPDI-2 and pET28a-rHlPrx. Transformation: The recombinant plasmids pET28a-rHlPrx and pET28a-HlPDI-2 were successfully transformed into Escherichia coli BL21(DE3) and then cultured on a large scale to obtain a large-scale bacterial culture. Expression: IPTG was added to the bacterial culture to induce expression, and the culture was shaken to obtain the protein expression solution; Purification: After the culture was completed, the protein expression solution was centrifuged to collect bacteria and obtain a supernatant containing soluble recombinant proteins rHlPDI-2 and rHlPrx. The supernatant was then purified to obtain soluble recombinant proteins rHlPDI-2 and rHlPrx.

3. The method for preparing the multi-component subunit vaccine for the salivary glands of Haemaphysalis longicornis according to claim 2, characterized in that, The purification process is as follows: nickel affinity packing material is added to a chromatography column, equilibration buffer is added to equilibrate the packing material, and after equilibration 3-5 times, supernatant containing soluble recombinant proteins rHlPDI-2 and rHlPrx is added, and the column is washed 4-6 times with washing buffer. The target protein is eluted with elution buffer, and the target protein is concentrated and replaced using an ultrafiltration tube to obtain purified soluble recombinant proteins rHlPDI-2 and rHlPrx.

4. The use of the multi-component subunit vaccine of salivary glands of Haematochezia as described in claim 1 in the preparation of Haematochezia prevention and treatment drugs.

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