Salvary gland apolipoprotein, gene, vector, recombinant bacterium and application of salivary gland apolipoprotein

Abstract

The invention provides salivary gland apolipoprotein, a gene, a carrier, a recombinant bacterium and application of salivary gland apolipoprotein, and belongs to the technical field of tick prevention and control. The amino acid sequence of the salivary gland apolipoprotein of the ligamentosa is as shown in SEQ ID NO.1. The invention further discloses a preparation method of the salivary gland apolipoprotein. After the salivary gland apolipoprotein provided by the invention is used for immunizing mice, the death rate of infected ticks can be increased, the blood sucking time can be prolonged, and the ecdysis rate of the ticks can be reduced, so that the protein or the gene provided by the invention can be used as a prevention and control target of the ticks, can be used for developing anti-tick vaccines, can be used for preventing and controlling the ticks and tick-borne diseases, and has a profound application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of tick prevention and control, and specifically relates to a salivary gland apolipoprotein of Ornithodoros lahorei, a gene, a vector, a recombinant bacterium and applications thereof. Background Art

[0002] Ticks are the second largest disease vector after mosquitoes. They can parasitize on the surface of animals and humans to suck blood to complete their life and reproduction cycles. Tick salivary glands and their secretions play a key role in mediating tick-host interactions. However, the number and types of tick salivary gland proteins are huge and redundant. At present, the discovery of its salivary gland proteins and functions is seriously lagging, making it difficult to effectively support the prevention and control needs of ticks and tick-borne diseases.

[0003] Ornithodoros lahore Ornithodoros lahorensis, O. lahorensis ) is a multi-host tick that can infect goats, sheep, cattle, dogs and humans. The infection rate in sheep can be as high as 90%, which can cause weakness, dermatitis and even death in animals, causing serious impacts on the health of the breeding industry and public health safety. Currently, there is no drug or vaccine that can effectively prevent and control the Lahore Ornithodoros tick. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a salivary gland apolipoprotein of Ornithodoros lahoreensis that can be used for the prevention and treatment of ticks and tick-borne diseases.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a salivary gland apolipoprotein of Ornithodoros lahorei. The amino acid sequence of the salivary gland apolipoprotein is shown in SEQ ID NO.1.

[0006] The present invention also provides a gene encoding the salivary gland apolipoprotein, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0007] The invention also provides a vector carrying the gene.

[0008] Preferably, the vector comprises pEASY-T1 and pET-28a-sumo.

[0009] The invention also provides an engineering bacterium containing the gene.

[0010] Preferably, the engineered bacteria include Escherichia coli DH5α and Escherichia coli BL21 (DE3).

[0011] The present invention also provides the use of the above protein or the above gene as a target in the preparation of tick control products.

[0012] The present invention also provides the use of any one of the following in the preparation of an anti-tick product: (1) the salivary gland apolipoprotein; (2) the gene; (3) the vector; (4) the engineered bacteria.

[0013] Preferably, the product comprises a vaccine.

[0014] The present invention also provides a vaccine against Ornithodoros lahoreensis, which comprises the salivary gland apolipoprotein.

[0015] Beneficial effects of the present invention: The present invention provides a kind of Ornithodoros lahore O. lahorensis ) salivary gland apolipoprotein and its gene. After immunizing mice with the salivary gland apolipoprotein provided by the present invention, the mortality rate and blood sucking time of infected ticks can be increased, and the molting rate of ticks can be reduced, indicating that the protein or gene provided by the present invention can be used as a tick prevention and control target for the development of anti-tick vaccines, and has far-reaching application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The RT-PCR amplification results of the salivary gland apolipoprotein Ol-TSGP1 gene of the Lahore Ornithodoros tick, where 1 is the band diagram on the marker manual, 2 is the actual image of the marker, and 3 is Ol-TSGP1; Figure 2 The protein expression results, where 1 is the band diagram on the marker manual, 2 is the actual picture of the marker, 3 is the supernatant before induction, 4 is the precipitate before induction, 5 is the supernatant expressed at 16°C, 6 is the precipitate expressed at 16°C, 7 is the supernatant expressed at 20°C, and 8 is the precipitate expressed at 20°C; Figure 3 The protein purification results, where 1 is the band diagram on the marker manual, 2 is the actual image of the marker, and 3 is the purified rOl-TSGP1 protein; Figure 4 is the Western blot result; Figure 5 This is the antibody growth and decline process after mice were immunized with rOl-TSGP1 recombinant protein, where the black arrows indicate the three immunization times. DETAILED DESCRIPTION

[0017] The present invention provides a salivary gland apolipoprotein of Ornithodoros lahoreensis, wherein the amino acid sequence of the salivary gland apolipoprotein is MKSVAALALLVILVSVARAQIEGLTKGCPQKRQPPNDAGCMYYCPGKSGDWAYGVYPDKSTCDYQASRDGVCQGGLCYAASNPAPPPHQPSNPGSGSDDDDDWEDK (SEQ ID NO.1). In the present invention, the salivary gland apolipoprotein with the amino acid sequence shown in SEQ ID NO.1 is recorded as Ol-TSGP1.

[0018] The present invention also provides a gene encoding the salivary gland apolipoprotein, the nucleotide sequence of which is ATGAAGTCTGTGGCAGCGCTGGCTCTCCTCGTAATACTTGTGTCGGTGGCGCGAGCTCAGATTGAAGGATTGACCAAGGGTTGCCCGCAAAAACGTCAACCGCCTAACGACGCTGGCTGCATGTACTACTGCCCGGGCAAATCAGGAGACTGGGCGTACGGCGTCTACCCCGACAAAAGCACATGTGACTATCAAGCATCCCGGGACGGCGTATGTCAGGGAGGACTGTGCTACGCGGCGTCAAACCCTGCTCCCCCTCCACACCAGCCTAGCAACCCTGGCAGCGGAAGCGACGATGGTGACGACTGGGAGGACAAGTAG (SEQ ID NO.2).

[0019] The present invention also provides a vector carrying the above gene. In the present invention, the vector preferably includes pEASY-T1 and pET-28a-sumo. The present invention has no special restrictions on the specific sources of the vectors pEASY-T1 and pET-28a-sumo.

[0020] The present invention also provides an engineered bacterium containing the above gene. In the present invention, the engineered bacterium preferably includes Escherichia coli DH5α and Escherichia coli BL21 (DE3). The present invention has no special limitation on the specific sources of Escherichia coli DH5α and Escherichia coli BL21 (DE3).

[0021] The present invention also provides the use of the above protein or the above gene as a target in the preparation of tick control products.

[0022] The present invention also provides the use of any one of the following in the preparation of an anti-tick product: (1) the salivary gland apolipoprotein; (2) the gene; (3) the vector; (4) the engineered bacteria. In the present invention, the product preferably includes a vaccine.

[0023] The present invention also provides a vaccine against Ornithodoros lahore, the vaccine comprising the salivary gland apolipoprotein. In the present invention, the vaccine preferably further comprises an adjuvant, and the present invention has no particular limitation on the specific type of the adjuvant.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1 Cloning and sequence analysis of the salivary gland apolipoprotein Ol-TSGP1 gene of Ornithodoros lahore of the present invention Constructed O. lahorensis Using the salivary gland transcriptome database as a reference, a tick salivary gland apolipoprotein gene was screened and named Ol-TSGP1 gene. Based on the transcriptome gene reference sequence, the upstream and downstream primers of this gene were designed using Primer6.0 software: F: ATGAAGTCTGTGGCAGCGC (SEQ ID NO.3); B: CTACTTGTCCTCCCAGTCG (SEQ ID NO.4). O. lahorensisi The salivary gland RNA was used as a template to amplify the target band using the RT-PCR method. Figure 1 , the PCR product was sequenced to obtain the accurate sequence information of the gene (321 bp), as shown in SEQ ID NO.2.

[0028] The obtained Ol-TSGP1 gene was subjected to bioinformatics analysis to obtain its signal peptide (there is 1 signal peptide) and transmembrane domain (no transmembrane domain) information.

[0029] Construction of cloning vector: Remove the sequence signal peptide sequence to eliminate its effect on protein expression. The gene sequence without the signal peptide was sent to Beijing Qingke Biotechnology Co., Ltd. for gene synthesis, and restriction enzyme sites BamHI and XhoI were added to the 5' and 3' ends of the gene sequence, respectively, and connected to the cloning vector pEASY-T1 to obtain the cloning vector with the Ol-TSGP1 gene inserted.

[0030] Example 2 Expression and purification of Ol-TSGP1 protein 1. Construction of recombinant expression vector (1) The cloning vector obtained in Example 1 and inserted with the Ol-TSGP1 gene was transformed into competent E. coli DH5α bacteria, spread on a 100 μg / mL ampicillin-resistant LB plate, and cultured inverted at 37° C. overnight.

[0031] (2) Select monoclonal bacteria for PCR identification and gene sequencing. Take the positively identified monoclonal bacteria and add them to 100 μg / mL ampicillin-resistant LB culture medium. Incubate the culture overnight at 37°C and 220 r / min, and extract the plasmid.

[0032] (3) The extracted plasmid and expression vector pET-28a-sumo were double-digested with BamHI and XhoI and then ligated to successfully construct the recombinant expression vector rpET-28a-sumo-OlTSGP1.

[0033] (4) Add 1 μL of the recombinant expression vector rpET-28a-sumo-OlTSGP1 plasmid into 100 μL of Escherichia coli BL21-DE3 competent bacteria and place on ice for 20 min.

[0034] (5) Heat shock at 42°C for 90 s, quickly place on ice for 5 min, and add 600 μL of LB culture medium.

[0035] (6) Shake at 220 rpm for 1 h at 37°C. After centrifugation, spread the entire plate onto an LB plate containing 50 μg / mL Kan+ and incubate inverted at 37°C overnight.

[0036] (1) Pick a single clone from the transformation plate and inoculate it into a test tube containing 4 mL LB culture medium containing 50 μg / mL Kan+, and shake it at 37℃ and 220 r / min overnight.

[0037] (2) The next day, the cells were inoculated at a ratio of 1:100 into 100 mL LB culture medium containing 50 μg / mL Amp and shaken at 37°C and 220 rpm until the OD 600 It is 0.5-0.8.

[0038] (3) Take out 1 mL of the culture, centrifuge at 10,000 rpm for 2 min at room temperature, discard the supernatant, and resuspend the bacterial pellet in 100 μL of TBS buffer.

[0039] (4) Add IPTG to the remaining culture to a final concentration of 0.2 mM and shake at 30°C and 220 r / min for 10 h. Set up parallel controls at 16°C and 20°C, shaking at 220 r / min for 18 h, to induce the expression of the fusion protein.

[0040] (5) Take out 1 mL of culture respectively, centrifuge at 10,000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet with 100 μL PBS. Centrifuge the remaining culture at 4,000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet with TBS.

[0041] (6) Take the pre-induction bacterial solution, 16℃ induced expression bacterial solution and 20℃ induced expression bacterial solution, add PMSF with a final concentration of 1mM, and perform ultrasonic disruption. Then take the supernatant and precipitate, add TBS to resuspend, perform 12% SDS-PAGE detection and analysis, and stain with Coomassie Brilliant Blue to visualize the bands. The results are shown in Figure 2 It can be seen that when expressed at 20°C, a large amount of target protein was expressed in the supernatant.

[0042] (7) According to the optimized expression conditions, use 1000mL LB culture medium to express the protein in large quantities. After sonication, take the supernatant for protein purification.

[0043] 3. Ni column affinity purification (1) Using a low-pressure chromatography system, the supernatant solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (20 mM Tris-HCl, 0 mM imidazole, 0.5 M NaCl, pH 8.0) at a flow rate of 1 mL / min.

[0044] (2) Rinse with Binding-Buffer at a flow rate of 1 mL / min until the OD of the effluent reaches 280 value reaches the baseline.

[0045] (3) Elute the target protein with Ni-NTA Elution-Buffer (20 mM Tris-HCl, 50 mM imidazole, 0.5 M NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the effluent.

[0046] (4) Elute the target protein with Ni-NTA Elution-Buffer (20 mM Tris-HCl, 500 mM imidazole, 0.5 M NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the effluent.

[0047] (5) SDS-PAGE analysis was performed. The purified rOl-TSGP1 protein was successfully obtained (see Figure 3 ).

[0048] (6) The rOl-TSGP1 recombinant protein was subjected to Western blot analysis at a dilution of 1:100. O. lahorensis Positive mouse serum was used as the primary antibody, and HRP-labeled goat anti-mouse IgG diluted 1:2000 was used as the secondary antibody. Figure 4 As shown in Figure 2, it can be seen that the rOl-TSGP1 recombinant protein can recognize positive serum and has good immunoreactivity.

[0049] Example 3 Example 2 Antibody changes after mice were immunized with the recombinant protein rO1-TSGP1 1. Immunization (1) Thirty 8-week-old BALB / c mice were randomly divided into two groups: rOl-TSGP1 immunization group and blank control group; each group was further divided into three parallel control groups, each with 5 mice.

[0050] (2) For the first immunization of the immunization group, 200 μg / mouse recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points on the back to immunize the experimental mice; for the second immunization, 100 μg / mouse recombinant protein was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points on the back to immunize 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 the rest was the same as the immunization group; each immunization was separated by 14 days.

[0051] 2. Monitoring of changes in antibody levels (1) The purified recombinant protein rOl-TSGP1 was used as the coating antigen, the polyclonal antibody diluted 2 times was used as the primary antibody, the control group mouse serum 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.

[0052] (2) Before immunization and every week after the start of immunization, blood was collected from the tail tip of mice, and the serum was separated. The changes in the levels of rOl-TSGP1 antibodies in mice were detected by indirect ELISA.

[0053] (3) The results are as follows Figure 5 As shown in the figure, the average IgG antibody level in the serum of the control group was maintained at a low level before and after immunization, while the average IgG antibody level in the serum of the rOl-TSGP1 immunization group increased significantly after immunization, which was significantly higher than that of the control group (P < 0.05). It reached a peak at 7 weeks and then slowly declined, but the antibody titer remained at a high level before 14 weeks.

[0054] Example 4 Example 2 Immune effect of the obtained recombinant protein rO1-TSGP1 on mice After immunization of the 30 mice described in Example 3, one week later, O. lahorensis The mice were infected with 20 larvae per mouse. The mice were weighed after being full of blood. The infection was observed and the full-blooded larvae were collected every day. The collected larvae were placed in a constant temperature and humidity incubator (26±1℃; 90±5 relative humidity; 12h light / 12h dark), the molting of the larvae was observed, and the blood sucking time and death of the ticks were recorded. All data are expressed as mean±standard deviation (SD), and the significance of the results of the experimental group and the control group was determined by one-way analysis of variance. When P<0.05, it was considered to be significantly different.

[0055] The differences in mortality, blood-sucking time, average body weight and molting rate of tick larvae between the experimental group and the control group are shown in Table 1. It can be seen that after mice were immunized with the recombinant protein rOl-TSGP1, the mortality rate and blood saturation time increased significantly (after immunization, the host developed resistance to ticks, inhibiting the anticoagulant and anti-inflammatory functions of salivary gland proteins, resulting in prolonged blood saturation time) (P < 0.05), and the molting rate decreased significantly (P < 0.05), indicating that the protein antibody has an inhibitory effect on tick larvae and can be used as a candidate target in clinical research on anti-tick vaccines.

[0056] Table 1 Effects of mice immunized with recombinant protein rOl-TSGP1 on larval ticks

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A salivary gland apolipoprotein of Ornithodoros lahoreensis, characterized in that The amino acid sequence of the salivary gland apolipoprotein is shown in SEQ ID NO.

1.

2. A gene encoding the salivary gland apolipoprotein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A vector carrying the gene according to claim 2.

4. The carrier according to claim 3, characterized in that The vectors include pEASY-T1 and pET-28a-sumo.

5. An engineered bacterium containing the gene according to claim 2.

6. The engineered bacteria according to claim 5, characterized in that The engineered bacteria include Escherichia coli DH5α and Escherichia coli BL21 (DE3).

7. Use of the protein according to claim 1 or the gene according to claim 2 as a target in the preparation of a tick control product.

8. Use of any one of the following in the preparation of an anti-tick product, characterized in that: (1) the salivary gland apolipoprotein described in claim 1; (2) the gene described in claim 2; (3) the vector described in claim 3 or 4; (4) the engineered bacteria described in claim 5 or 6.

9. The use according to claim 8, characterized in that: The products include vaccines.

10. A vaccine against Ornithodoros lahoreensis, characterized in that The vaccine comprises the salivary gland apolipoprotein of claim 1.

Images