A salivary gland protein of r. turdipes and uses thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SANYA BIOSAFETY CENT OF CHINESE ACAD OF MEDICAL SCI
- Filing Date
- 2026-04-23
- Publication Date
- 2026-06-23
Smart Images
Figure CN122080163B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tick control technology, specifically relating to a salivary gland protein of the tick *Ixodes lahore* and its application. Background Technology
[0002] Ticks, the second leading vector of disease transmission after mosquitoes, parasitize animals and humans, feeding on blood to complete their life and reproductive cycles. Tick salivary glands and their secretions play a crucial role in mediating tick-host interactions. However, tick salivary gland proteins are diverse and exhibit significant genetic and functional redundancy. Traditional single-target vaccines often face the risk of immune evasion through ticks expressing other homologous proteins for functional compensation. Therefore, identifying conserved salivary gland proteins with core functions and using them in monovalent vaccines or in combination with other functional proteins to prepare multivalent vaccines is an urgent need to effectively support the prevention and control of ticks and tick-borne diseases.
[0003] Lahore tick (Lahore tick) Ornithodoros lahorensis, O. lahorensis The Lahore tick (Trichoderma lahore) is a multi-host tick that can infect various animals, including goats, sheep, cattle, and dogs, as well as humans. In sheep flocks, the infection rate can reach over 90%, leading to weakness, dermatitis, and even death. It can also carry pathogens causing animal and human diseases, such as Anaplasma goatii, Brucella, and Xinjiang hemorrhagic fever, posing a serious threat to livestock health and public health safety. Currently, there are no effective drugs or vaccines for controlling the Lahore tick, and related research is still in its early stages. Summary of the Invention
[0004] In view of this, the object of the present invention is to provide a salivary gland protein vaccine candidate antigen of ticks, specifically the tick *Ixodes lahoreensis*, which can be used for the prevention and control of ticks and tick-borne diseases.
[0005] The present invention provides a salivary gland protein of Ornithodoros lahorensis, the amino acid sequence of which is shown in SEQ ID NO.1.
[0006] The present invention provides a gene encoding the above-mentioned protein.
[0007] The present invention provides a recombinant vector containing the above-mentioned genes.
[0008] Further specifying, the launch carriers are the pEASY series and pMAL series.
[0009] The present invention provides a recombinant microbial cell expressing the above-mentioned genes.
[0010] To further specify, the microbial cells are eukaryotic microbial cells or prokaryotic microbial cells.
[0011] The present invention provides the application of the above-mentioned protein, the above-mentioned gene, or the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a vaccine against Lahore tick.
[0012] The present invention provides the use of the above-mentioned protein, the above-mentioned gene, or the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of an anti-Lahore tick drug.
[0013] The present invention provides a vaccine against Lahore ticks, the vaccine comprising the proteins described above.
[0014] Further, it also includes Freund's adjuvant, Montanide ISA 50V, or aluminum salt adjuvant.
[0015] The beneficial effects of this invention are:
[0016] This invention provides a salivary gland protein, Ol-BTSP, and its gene from the tick *O. lahorensis*. In vivo animal immune challenge experiments showed that immunization of mice with the salivary gland protein provided by this invention elicited a strong specific IgG antibody response, significantly increasing the duration of blood-feeding by infected ticks, significantly increasing tick mortality, and significantly reducing tick satiety and molting rates. This indicates that the Ol-BTSP protein provided by this invention effectively interferes with tick blood-feeding and physiological homeostasis, serving as an excellent target for tick control. This protein can not only be independently developed into a monovalent anti-tick vaccine but is also suitable as a core antigenic component of a multivalent (cocktail) broad-spectrum anti-tick vaccine, possessing profound application prospects and extremely high commercial transformation value. The Ol-BTSP protein discovered in this invention has a unique acidic C-terminal tail, playing a crucial role in interfering with tick blood-feeding homeostasis and developmental processes, making it a preferred target for preparing monovalent or multivalent anti-tick vaccines. Attached Figure Description
[0017] Figure 1 The results of RT-PCR amplification of the salivary gland protein Ol-BTSP gene from the tick *Ixodes lahore* are shown. In the image, 1 is a schematic diagram of the bands shown in the marker manual, 2 is the actual marker image, 3 is the negative control, 4 is the blank control, and 5 is Ol-BTSP.
[0018] Figure 2 The results show the protein expression results, where 1 is a schematic diagram of the bands in the marker manual, 2 is the actual marker image, 3 is the expression supernatant at 16℃, 4 is the expression precipitate at 16℃, 5 is the expression supernatant at 30℃, 6 is the expression precipitate at 30℃, 7 is the supernatant before induction, 8 is the precipitate before induction, 9 is the supernatant of the empty vector, and 10 is the precipitate of the empty vector.
[0019] Figure 3The results show the protein purification results, where 1 is a schematic diagram of the bands in the marker manual, 2 is the actual marker image, and 3 is the purified rOl-BTSP protein.
[0020] Figure 4 For Western blot results;
[0021] Figure 5 This describes the antibody fluctuation process after mice are immunized with rOl-BTSP recombinant protein.
[0022] Figure 6 This is a diagram showing the alignment results of the target sequence sequencing in the cloning vector.
[0023] Figure 7 The image shows the alignment results of the target sequence sequencing in the expression vector. Detailed Implementation
[0024] Expression vector information: pMAL-c5X, catalog number: V012449, purchased from: Shanghai Newp Biotechnology Co., Ltd.
[0025] Cloning vector information: pEASY®-T1 Cloning Kit T1 gene cloning kit (double antibiotic) catalog number: CT101-01 purchased from Beijing TransGen Biotech Co., Ltd.
[0026] Example 1.
[0027] This invention relates to the cloning and sequence analysis of the Ol-BTSP gene, a salivary gland protein from the tick *Ixodes lahore*, for the construction of... O. lahorensis Using the salivary gland transcriptome database as a reference, a tick salivary gland protein gene was screened and named Ol-BTSP. Based on the transcriptome gene reference sequence, upstream and downstream primers for this gene were designed using Primer 6.0 software: F: ATGATTGTGTGTGCTGATC (SEQ ID NO.3); B: TTAATCCCAGTCTTCGTCC (SEQ ID NO.4). The extracted... O.lahorensisi Using salivary gland RNA as a template, the target band was amplified by RT-PCR. (See below) Figure 1 The PCR product was sequenced to obtain the accurate sequence information of the gene (321bp), as shown in SEQ ID NO.2.
[0028] Bioinformatics analysis was performed on the obtained Ol-BTSP gene to obtain its signal peptide (one signal peptide) and transmembrane domain (no transmembrane domain). A salivary gland protein from the tick *Ixodes lahore*, wherein the amino acid sequence of the salivary gland protein contains one signal peptide (…). MMCVLILALLTALVSG, SEQ ID NO.5);
[0029] Other areas:
[0030] APEDSEQDMVKDCPEKPQPPGDKDCMYYCGKDDSGRWKWGPYTNGTPCDYNGETEGVCKGGLCHFKGPDTDTKGQPPHEEQPQEEGPQDEDTEGEVPTEDGDEDEDWD (SEQ ID NO.1). In this invention, the salivary gland protein with the amino acid sequence shown in SEQ ID NO.1 is designated as Ol-BTSP.
[0031] The full-length amino acid sequence of salivary gland protein is as follows:
[0032] MMCVLILALLTALVSG APEDSEQDMVKDCPEKPQPPGDKDCMYYCGKDDSGRWKWGPYTNGTPCDYNGETEGVCKGGLCHFKGPDTDTKGQPPHEEQPQEEGPQDEDTEGEVPTEDGDEDEDWD (SEQ ID NO.6)
[0033] The present invention also provides a gene encoding the above-mentioned salivary gland protein, the nucleotide sequence of which is as follows: ATG ATGTGTGTGCTGATCTTGGCTCTTCTCACTGCTCTTGTTTCAGGC (signal peptide, SEQ ID NO.7 ）；
[0034] GCGCCAGAAGATTCCGAGCAAGATATGGTAAAGGACTGCCCAGAGAAACCTCAACCTCCTGGTGACAAAGACTGCATGTACTACTGCGGGAAGGATGATTCGGGAAGATGGAAGTGGGGCCCATACACTAATGGAACCCCGTGCGATTACAACGGAGAAACAGAA GGCGTATGCAAGGGAGGCTTGTGTCACTTCAAGGGACCGGACACCGACACCAAGGGGCAACCACCGCACGAGGAGCAGCCGCAAGAGGAAGGACCGCAGGATGAAGACACCGAAGGCGAAGTGCCAACTGAGGACGGCGATGAGGACGAAGACTGGGATTAA (SEQ ID NO.2).
[0035] Construction of the cloning vector: The signal peptide sequence was removed to eliminate its influence on subsequent protein expression. PCR amplification primers with the signal peptide sequence removed were designed, and restriction enzyme sites BamHI and XhoI were added to the upstream and downstream primers, respectively (the italicized and bolded parts are the restriction enzyme site sequences): Upstream primer: 5'-CGCGGATCCGCGCCAGAAGATTCCGAGCA-3' (SEQ ID NO. 8); Downstream primer: 5'-CCGCTCGAGTTAATCCCAGTCTTCGTCCTC-3' (SEQ ID NO. 9). The amplification product was recovered by gel electrophoresis, double-digested, and ligated into the cloning vector pEASY-T1 to obtain the recombinant cloning vector rpEASY-T1-OlBTSP containing the Ol-BTSP gene. The identification results are as follows: Figure 6 As shown.
[0036] Example 2.
[0037] Expression and purification of Ol-BTSP protein
[0038] 1. Constructing recombinant expression vectors
[0039] (1) The cloning vector containing the Ol-BTSP gene obtained in Example 1 was transformed into Escherichia coli DH5α competent cells, plated on LB plates with 100 μg / mL ampicillin resistance, and incubated upside down at 37°C overnight.
[0040] (2) Single clones were selected for PCR identification. Positive bacteria were sent to Beijing Qingke for cloning vector target sequence sequencing. The sequencing results showed that the target sequence was 100% identical to the nucleotide sequence of SEQ ID NO.2 after removing the signal peptide. Figure 6 Take a single clone of bacteria and add it to 100 μg / mL ampicillin-resistant LB broth. Incubate overnight at 37°C and 220 r / min, and then extract the plasmid.
[0041] (3) The extracted plasmid and expression vector pMAL-c5x were ligated after double digestion with BamHI and XhoI to construct the recombinant expression vector pMAL-c5x-OlBTSP. The identification results are as follows: Figure 7 As shown.
[0042] (4) Add 1 μL of the recombinant expression vector pMAL-c5x-OlBTSP plasmid to 100 μL of Escherichia coli BL21-DE3 competent cells and place on ice for 20 min.
[0043] (5) Heat shock at 42℃ for 90 s, then quickly place on ice for 5 min, and add 600 μL LB culture medium.
[0044] (6) Shake at 220 r / min for 1 h at 37℃, then spread the entire mixture onto LB plates containing 50 μg / mL Kan+ and incubate upside down at 37℃ overnight.
[0045] 2. Identification of IPTG-induced expression of recombinant bacterial fusion protein
[0046] (1) Single clones of bacteria were picked from the transformation plate for PCR identification. Positive bacteria were sent to Beijing Qingke for further sequencing of the expression vector. The sequencing results showed that the target sequence was 100% identical to the nucleotide sequence of SEQ ID NO.2 after removing the signal peptide. Figure 7 The culture medium was inoculated into a test tube containing 4 mL of LB medium containing 50 μg / mL Kan+ and incubated overnight at 37°C with shaking at 220 r / min.
[0047] (2) The next day, the bacteria were inoculated at a ratio of 1:100 into 100 mL of LB medium containing 50 μg / mL Amp, and shaken at 37°C and 220 r / min until the bacterial cell OD was reached. 600 It is 0.5-0.8.
[0048] (3) Take out 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet with 100 μL TBS buffer.
[0049] (4) Add IPTG to the remaining culture to a final concentration of 0.2 mM, shake at 30℃ and 220 r / min for 10 h, and set up parallel controls at 16℃ and 20℃ and shake at 220 r / min for 18 h to induce fusion protein expression.
[0050] (5) Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL PBS. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in TBS.
[0051] (6) Take the pre-induction bacterial culture, the bacterial culture induced at 16℃, and the bacterial culture induced at 20℃, add PMSF to a final concentration of 1mM, and sonicate. After ultrasonic disruption, take the supernatant and precipitate respectively, add them to TBS for resuspending, and perform 12% SDS-PAGE detection and analysis. Coomassie brilliant blue staining is used for banding. The results are shown in the figure. Figure 2 It can be seen that when expressed at 20℃, the target protein was expressed in large quantities in the supernatant.
[0052] (7) The protein was expressed in large quantities using 1000 mL of LB culture medium according to the optimized expression conditions. After sonication, the supernatant was taken for protein purification.
[0053] 3. Amylose Resin affinity purification (MBP fusion protein)
[0054] (1) Equilibrium and sample loading
[0055] Using a low-pressure chromatography system, the processed fragmented supernatant was loaded at a flow rate of 1 mL / min into an Amylose Resin affinity chromatography column pre-equilibrated with Amylose Binding-Buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4) to ensure that the MBP fusion protein was fully bound to the resin.
[0056] (2) Washing away impurities
[0057] The column was continuously flushed with Binding-Buffer at a flow rate of 1 mL / min to remove unbound host proteins until the OD280 value of the effluent returned to baseline and remained stable.
[0058] (3) Elution of target protein
[0059] The target protein bound to the column was eluted with Amylose Elution-Buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM maltose, pH 7.4) at a flow rate of 1 mL / min, and the elution peak was collected in separate tubes.
[0060] (4) SDS-PAGE analysis
[0061] The collected solutions from each stage were subjected to SDS-PAGE protein electrophoresis analysis. Since the MBP tag is approximately 42 kDa and rOl-BTSP is approximately 13 kDa, it is necessary to confirm the appearance of a clear target band around 55 kDa (see Figure 3) to verify the purification effect.
[0062] (5) Western Blot Validation
[0063] The purified rOl-BTSP recombinant protein was electrotransferred to a PVDF membrane for Western blot analysis. O. lahorensis-positive mouse serum diluted 1:100 was used as the primary antibody, and HRP-labeled goat anti-mouse IgG diluted 1:2000 was used as the secondary antibody. The results are shown in Figure 4. The experimental group showed a specific chromogenic band at the expected molecular weight, demonstrating that the rOl-BTSP recombinant protein can be specifically recognized by positive serum and exhibits good immunoreactivity.
[0064] Example 3.
[0065] Antibody changes in mice immunized with the recombinant protein rOl-BTSP obtained in Example 2.
[0066] 1. Immunity
[0067] (1) Thirty 8-week-old BALB / c mice were randomly divided into two groups: the rOl-BTSP immunization group and the blank control group. Each group was further divided into three parallel control groups, with five mice in each parallel control group.
[0068] (2) For the first immunization of the immunization group, 200 μg of recombinant protein per mouse was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of the experimental mice. For the second immunization, 100 μg of recombinant protein per mouse was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the back of the experimental mice. The third immunization was the same as the second immunization. The control group used an equal volume of sterile PBS instead of recombinant protein, and all other aspects were the same as the immunization group. The interval between each immunization was 14 days.
[0069] 2. Monitoring changes in antibody levels
[0070] (1) The purified recombinant protein rOl-BTSP was used as the coating antigen, the polyclonal antibody diluted 2 times was used as the primary antibody, the serum of the control group mice was used as the negative control, and HRP-labeled goat anti-mouse IgG H&L (1:10000) was used as the secondary antibody. The serum antibody level was detected by indirect ELISA.
[0071] (2) Blood was collected from the tail tip of mice before immunization and weekly after immunization. Serum was separated and the changes in the level of rOl-BTSP antibody in mice were detected by indirect ELISA.
[0072] (3) The results are as follows Figure 5 As shown, antibody changes went through four stages:
[0073] Slow start-up period (W0-W2): Initial immunization using Freund's complete adjuvant (CFA) establishes immune memory. During this period, antibody production is slower, the curve slope is gentler, and antibody titers are at a low level of 1:400.
[0074] Logarithmic growth phase (W2-W5): Secondary and tertiary immunizations use Freund's incomplete adjuvant (IFA) to elicit a secondary immune response. The curve shows an extremely steep rise here, with titers jumping rapidly from the thousands to the tens of thousands.
[0075] Peak plateau period (W6-W8): Antibody titers reach their highest point 1-2 weeks after the third immunization. At this time, plasma cell secretion activity in mice is at its peak, which is also the optimal window for protection.
[0076] Slow decay period (W9-W12): As free antigens are cleared, antibody titers begin to decline physiologically. By week 12 (approximately 3 months), they drop back to around 1:5,000.
[0077] The core role of Montanide ISA 50V or aluminum salt adjuvant in preparing vaccines against Lahore ticks is to enhance the immune efficacy of the vaccine, with effects similar to those of Freund's adjuvant. Those skilled in the art can expect that vaccines prepared using Montanide ISA 50V or aluminum salt adjuvant instead of Freund's adjuvant will have the same effect as those in Example 3 of this application.
[0078] Example 4.
[0079] Immunological effects of the recombinant protein rOl-BTSP obtained in Example 2 on mice
[0080] After immunizing the 30 mice described in Example 3, a one-week interval was observed, and then... O. lahorensis Mice were infected with tick larvae, with 20 larvae infecting each mouse. After engorgement, the ticks were weighed, and infection status was observed daily. Shed ticks were collected and placed in a constant temperature and humidity incubator (26±1℃; 90±5% relative humidity; 12h light / 12h dark) to observe molting. The duration of tick feeding and mortality were recorded. All data are expressed as mean ± standard deviation (SD). One-way ANOVA was used to determine the significance of results between the experimental and control groups; P < 0.05 was considered statistically significant.
[0081] The differences in saturation rate, molting rate, mortality rate, feeding time, and saturated body weight between the experimental and control groups of juvenile ticks are shown in Table 1. It can be seen that after immunizing mice with recombinant protein rOl-BTSP, the mortality rate and saturation time were significantly increased (after immunization, the host developed resistance to ticks, inhibiting the anticoagulant and anti-inflammatory functions of salivary gland proteins, resulting in prolonged saturation time) (P<0.05), while the molting rate was significantly decreased (P<0.05). This indicates that the antibody of this protein has an inhibitory effect on juvenile ticks and can be used as a candidate target in the clinical research of anti-tick vaccines.
[0082] Table 1. Effects of recombinant protein rOl-BTSP immunization on tick larvae in mice.
[0083]
[0084] For those skilled in the art, various improvements and modifications can be made without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.
Claims
1. A salivary gland protein of the tick Ornithodoros lahorensis, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.
1.
2. The gene encoding the protein of claim 1.
3. A recombinant vector containing the gene described in claim 2.
4. The recombinant vector according to claim 3, characterized in that, The launch vehicles are pEASY series and pMAL series.
5. A recombinant microbial cell expressing the gene of claim 2.
6. The recombinant microbial cell according to claim 5, characterized in that, The microbial cells are either eukaryotic or prokaryotic microbial cells.
7. The use of the protein of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the preparation of a vaccine against Lahore tick.
8. The use of the protein of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the preparation of an anti-Lahore tick drug.
9. A vaccine against Lahore ticks, characterized in that, The vaccine comprises the protein as described in claim 1.
10. The vaccine according to claim 9, characterized in that, It also includes Freund's adjuvant, Montanide ISA 50V, or aluminum salt adjuvant.