A recombinant protein and its use in blocking transmission of malaria

Abstract

The application belongs to the field of genetic engineering, and discloses a recombinant protein and application thereof in malaria transmission blocking. In the present application, geap36 is coupled with AP205 by using SpyTag-SpyCatcher reaction. The technology is novel at present, and is different from traditional malaria vaccines which only use proteins expressed in a certain stage of Plasmodium as immunogens. Due to the addition of AP205 protein, the AP205 protein can self-assemble into virus-like particles (VLP) when overexpressed, and can carry antigen fragments, which is beneficial to the uptake and cross-presentation of antigen-presenting cells. In the experiment, geap36 is selected as the coupled antigen, and an experimental group and a control group are established, so as to evaluate whether the VLP vaccine formula can improve the immunogenicity of geap36 and reduce the transmission activity (TRA), and provide a reference for subsequent vaccine research.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and relates to a recombinant protein and its application in blocking malaria transmission. Background Technology

[0002] In 2020, there were approximately 241 million new malaria cases globally, an increase of 14 million cases (6%) from the revised estimate of 227 million in 2019. There were 627,000 deaths, an increase of 69,000 deaths (12%) from the revised estimate of 558,000 in 2019. Although China has eliminated malaria, it still claims hundreds of thousands of lives globally each year, with over 90% of these deaths occurring in Africa and over 60% of deaths occurring in children under the age of five. Malaria accounts for 7.8% of all deaths among children under five globally, almost double the previous estimate, equivalent to one child dying from malaria every minute. As a major global infectious disease, malaria continues to threaten human health. In areas with high malaria prevalence, current antimalarial methods are insufficient to stop transmission, and the increase and spread of drug-resistant mosquitoes and Plasmodium parasites further complicate malaria control.

[0003] Since the discovery in 1967 that inoculation with attenuated sporozoites of Plasmodium berghei irradiated with virus could induce complete protective immunity in mice, researchers have been dedicated to developing effective malaria vaccines. Currently, candidate vaccines are designed for each of the three stages of the Plasmodium life cycle, aiming to block the parasite's life cycle at multiple points. The erythro ... Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a method for conjugating geap36 and AP205 using a SpyTag-SpyCatcher reaction. Due to the addition of the AP205 protein, its overexpression allows it to self-assemble into virus-like particles (VLPs) carrying target antigen fragments, facilitating uptake and cross-presentation by antigen-presenting cells. Mice were immunized with the conjugated protein to obtain antiserum, and its antibody titer and transmission-blocking ability were assessed, providing a reference for subsequent vaccine research.

[0005] To achieve the above objectives, the present invention provides the following technical solution.

[0006] This invention provides a recombinant protein combination, characterized in that it includes AP205-2*SpyTag and GEAP36-Spycatcher.

[0007] Furthermore, the AP205-2*SpyTag is obtained by adding two Spytags to the N-terminus and C-terminus of AP205 at the gene level; the GEAP36-Spycatcher is obtained by adding one Spycatcher to the C-terminus of GEAP36 at the gene level.

[0008] Furthermore, the protein combination utilizes the SpyTag-SpyCatcher reaction to conjugate GEAP36 with AP205, which can display the target antigen on the surface of VLP particles, thereby enhancing the immunogenicity of GEAP36 and reducing its transmissibility.

[0009] Furthermore, the recombinant protein combination described above is used in the preparation of a vaccine to block the transmission of malaria.

[0010] The present invention also provides a recombinant nucleic acid combination, characterized in that it encodes the recombinant protein or recombinant protein combination as described in claims 1-2.

[0011] Furthermore, the nucleic acid is codon-optimized for the host.

[0012] The present invention also provides a recombinant expression vector, characterized in that the expression vector contains the nucleic acid described above.

[0013] The present invention also provides a malaria transmission blocking vaccine, characterized in that the active ingredients in the vaccine include the above-mentioned recombinant protein combination, nucleic acid or vector.

[0014] Furthermore, the vaccine also includes adjuvants, immune enhancers, or immunomodulators.

[0015] The beneficial effects of the present invention compared with the prior art.

[0016] This study utilized the SpyTag-SpyCatcher reaction to conjugate geap36 with AP205. This technique is relatively novel and differs from traditional malaria vaccines that only use proteins expressed at a specific stage of the Plasmodium parasite as immunogens. Due to the addition of the AP205 protein, its overexpression allows it to self-assemble into virus-like particles (VLPs) carrying target antigen fragments, facilitating uptake and cross-presentation by antigen-presenting cells. Geap36 was selected as the conjugated antigen, and experimental and control groups were established to evaluate whether this VLP vaccine formulation can enhance the immunogenicity of geap36 and reduce transmissibility (TRA), providing a reference for future vaccine research. Attached Figure Description

[0017] Figure 1 Schematic diagram of the formation of isopeptide bonds in AP205-2*SpyTag and GEAP36-SpyCatcher.

[0018] Figure 2 Recombinant plasmids AP205-2*SpyTag-pET-20b and GEAP36-Spycatcher-pET-32a were identified by enzyme digestion. M: DNA marker DL5000; 1: Recombinant plasmid AP205-2*SpyTag-pET-20b was digested with BamHI / NotI; 2: Recombinant plasmid GEAP36-Spycatcher-pET-32a was digested with BamHI / NotI.

[0019] Figure 3 Identification of AP205-2*SpyTag. A: Coomassie blue staining of AP205-2*SpyTag in SDS-PAGE, M: protein marker; 1: purified AP205-2*SpyTag protein. B: Western blot identification of AP205-2*SpyTag, M: protein marker; 1: purified AP205-2*SpyTag protein.

[0020] Figure 4 Coomassie blue staining of GEAP36-Spycatcher in SDS-PAGE. M: protein marker; 1: purified GEAP36-Spycatcher protein.

[0021] Figure 5 Identification of VLP-GEAP36. A: Coumarin staining of bound VLP-GEAP36 in SDS-PAGE. M: Protein markers. 1: VLP-subunit with two binding antigens, 2: VLP-subunit with one binding antigen, 3: VLP-subunit.

[0022] Figure 6 Transmission electron microscopy was used to observe the formation of VLP particles.

[0023] Figure 7 ELISA was used to detect antibody titers in mice.

[0024] Figure 8 The effect of antiserum obtained from different immunization groups on the formation of kinetids.

[0025] Figure 9 Evaluation of transmission blocking activity in different immunization groups during mosquito feeding experiments.

[0026] Figure 10 The effect of each immunization group on mosquito infection rate in the direct membrane feeding experiment. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. The following embodiments will help to understand the present invention, but these embodiments are only for illustrative purposes, and the present invention is not limited thereto. The operating methods in the embodiments are all conventional operating methods in this technical field.

[0028] 1. Prokaryotic expressed protein.

[0029] 1.1 Carrier construction.

[0030] The gene sequences of AP205 capsid protein, SpyTag, and Spycatcher were searched in GeneBank. The gene sequence of geap36 (PBANKA_0818200) was obtained from the PlasmoDB database. The SpyTag peptide (AHIVMVDAYKPTK) gene sequence was added to the N-terminus and C-terminus of the AP205 capsid protein gene sequence, respectively. The sequence was then linked using the flexible linkers GSGTAGGGSGS (N-terminus of AP205-2*SpyTag) and GTASGGSGGSG (C-terminus of AP205-2*SpyTag) to obtain the gene sequence of AP205-2*SpyTag. The gene sequence encoding amino acids 45–245 of the predicted protein sequence of geap36 (PBANKA_1119200) was selected. A fusion tag consisting of 6 histidine residues (6*His) was added to its N-terminus, and the gene sequence encoding amino acids 24–139 ​​of Spycatcher was added to its C-terminus. The two were linked together using a flexible linker: GGSGS, resulting in the geap36-Spycatcher gene sequence. Introduced at both ends of the AP205-2*SpyTag and geap36-Spycatcher genes... BamH I and NotThe enzyme cleavage site was optimized, and the codon was optimized without altering the amino acid sequence. The entire genome was then synthesized at Nanjing GenScript Biotech Co., Ltd. AP205-2*SpyTag was subsequently cloned into... pET-20b In the vector, geap36-Spycatcher was cloned into pET-32a The constructed AP205-2*SpyTag- pET-20b and geap36-Spycatcher- pET-32a The expression vector was transformed into E. coli BL21(DE3) competent cells, plasmid was extracted, and enzyme digestion was performed for verification. Figure 2 ).

[0031] 1.2 Prokaryotic expression and purification of two proteins, AP205-2*SpyTag and geap36-Spycatcher.

[0032] The constructed AP205-2*SpyTag- pET-20b The expression vector was transformed into *E. coli* BL21(DE3) competent cells. After identifying the correct single colonies, the cells were cultured at 37°C until the OD value of the bacterial culture was between 0.4 and 0.6. 1 mM IPTG was added and the cells were induced at 20°C for 16 h. Protein purification was performed using a Ni-NTA His•Bind Superflow (Novagen) column with nitrilotriacetic acid (NTA) agarose. The purified recombinant protein was dialyzed sequentially at 4 M, 2 M, 1 M, and 0.5 M PB concentrations at 4°C, and finally dialyzed overnight at 4°C in phosphate-buffered saline (PBS, pH 7.4). Figure 3 AB). The initial purification process of the geap36-Spycatcher protein is similar to this. Figure 4 The difference lies in the fact that the protein was induced at 19°C for 12 hours with 0.5 mM IPTG, and the purified protein was directly dialyzed overnight in phosphate-buffered saline (PBS, pH 7.4) at 4°C. Both purified proteins were analyzed by 10% SDS-PAGE gel electrophoresis.

[0033] 2. Observation of protein coupling and coupling effect.

[0034] 2.1 Protein coupling.

[0035] The purified AP205-2*SpyTag protein and geap36-Spycatcher protein were incubated overnight at 4°C in a 1:3 molar ratio. The two were then mixed in standard phosphate buffer with 0.2% polysorbate 80 added to pH 7.2.

[0036] 2.2 SDS-PAGE was used to verify protein coupling, and transmission electron microscopy was used to observe the coupling of the two proteins.

[0037] Take 20 μL of the conjugated protein, add 5 μL of protein loading buffer, prepare a 10% SDS-PAGE gel, load the sample, stack the gel and perform electrophoresis at 60 V, then separate the gel at 80 V. After electrophoresis, place the gel in Coomassie Brilliant Blue R-250 staining solution for 30 min, then place it in Coomassie Brilliant Blue destaining solution and destain on a shaker for 5-6 h. Afterwards, observe the gel on a gel imaging analyzer and analyze the protein conjugation based on the molecular weight of the protein bands. Figure 5 The conjugated protein was diluted to 0.2 mg / mL in PBS. The diluted conjugated protein was adsorbed onto carbon and negatively stained with 2% phosphotungstic acid (pH=7.0) for 1 min. The formation of VLP particles was observed by transmission electron microscopy. Figure 6 ).

[0038] 3. Detection of antibody titers in immunized mice.

[0039] 3.1 Establishment of control and experimental groups, and immunization of BALB / c mice to obtain antiserum. To study the immunogenicity of the conjugated protein, mice were immunized according to the following protocol: 5 mice were selected in each group, for a total of 4 groups. Group 1 was the conjugated protein immunization group, i.e., immunized with VLP-geap36 protein, which was formed by conjugating geap36 and AP205 through the SpyTag-SpyCatcher reaction, which was the experimental group; Group 2 was the unconjugated protein immunization group, which served as the control vaccine, i.e., immunized with a mixture of AP205 protein without the SpyTag base sequence and r geap36 protein without the Spycatcher base sequence; Group 3 was the r geap36 protein immunization group, immunized with r geap36 antigen alone, which served as the negative control; Group 4 was the r geap36 protein + alum adjuvant immunization group, which served as the positive control. For the initial immunization, groups 1, 3, and 4 each received 50 μg of the corresponding protein in 200 μL of serum. Group 4 received an alum adjuvant. Group 2 received 50 μg of r geap36 protein subcutaneously. Booster immunizations were administered on days 14 and 28. For booster immunizations, groups 1, 3, and 4 each received 25 μg of the corresponding protein in 200 μL of serum. Group 4 received an alum adjuvant. Group 2 received 25 μg of r geap36 protein subcutaneously. At 14, 28, and 38 days after the first immunization, 100 μL of venous blood was collected from the tail of each mouse. Serum was collected after agglutination. The r geap36 protein (gene sequence without Spycatcher bases, 45–245 aa) was obtained in our laboratory previously. The AP205 protein without SpyTag bases was constructed using AP205- pET-32aThe expression vector was transformed into Escherichia coli BL21(DE3) competent cells, and then expressed and purified in prokaryotes.

[0040] 3.2 ELISA was used to detect antibody titers in four groups of mice.

[0041] Serum IgG levels were measured using a standard enzyme-linked immunosorbent assay (ELISA). Recombinant protein rgeap36 (5 μg / mL) was diluted with carbonate buffer (pH 9.6) and coated onto 96-well ELISA plates, then incubated overnight at 4°C. After washing three times with 200 μL PBST (0.1 mol / L PBS, pH 7.4, 0.02% Tween 20), the plates were blocked with PBS blocking buffer containing 1% bovine serum albumin (BSA) at 37°C for 1 h. After washing three times with PBST, 100 μL of mouse serum diluted with blocking buffer (dilution ratios from 1:200 to 1:25600) was added to each well, and the plates were incubated at 37°C for 2 h. After washing three times with PBST, HRP-labeled goat anti-mouse IgG (1:5000) was added, and the plates were incubated at 37°C for 2 h. After washing the plate 7 times with PBST, o-phenylenediamine and hydrogen peroxide were added for color development. The reaction was terminated with 100 μL of dilute H2SO4, and the OD value at 450 nm was measured using a microplate reader. Figure 7 ELISA results showed that the VLP-GEAP36 group had better immunogenicity compared to other control groups.

[0042] 4. Evaluate the transmission blocking activity of the SpyTag-SpyCatcher-mediated geap36 conjugate AP205 VLP vaccine.

[0043] 4.1 In vitro transmission blocking experiment.

[0044] Female BALB / c mice were injected intraperitoneally with 5 × 10 6 Three WT-type Plasmodium berghei strains were infected. On day 3 post-infection, 10 μL of mouse tail blood was placed in 90 μL of zygote culture medium diluted with immune serum (serum dilutions of 1:5, 1:10, and 1:50), and cultured at 19-20°C for 24 h. The culture was labeled with anti-Pbs21 antibody, and the zygote formation rate was observed under a fluorescence microscope. Figure 8 Compared to other control groups, the VLP-GEAP36 group showed a significantly enhanced ability of antiserum to inhibit kinetic zygote formation.

[0045] 4.2 In vivo transmission blocking experiment.

[0046] Antiserum conjugated to AP205 VLP via SpyTag-SpyCatcher-mediated geap36 was mixed 1:1 with blood from infected mice and incubated at 37°C for 15 min. The mixed blood sample was then added to a membrane feeder and used to feed mosquitoes for 30 min. Unfeeded mosquitoes were removed after feeding. The midgut was dissected 8-10 days post-feeding, stained, and the oocysts were counted using an optical microscope. Figure 9 ) and Anopheles infection rate ( Figure 10 TBA = 1 - (average number of ovarian cysts in the experimental group / average number of ovarian cysts in the control group) × 100. For example... Figure 9 and Figure 10 As shown, the VLP-GEAP36 group significantly reduced the number of midgut oocysts and the mosquito infection rate.

[0047] This study utilized the SpyTag-SpyCatcher reaction to conjugate geap36 with AP205. This technique is relatively novel and differs from traditional malaria vaccines that only use proteins expressed at a specific stage of the Plasmodium parasite as immunogens. Due to the addition of the AP205 protein, its overexpression allows it to self-assemble into virus-like particles (VLPs) carrying target antigen fragments, facilitating uptake and cross-presentation by antigen-presenting cells. Geap36 was selected as the conjugated antigen, and experimental and control groups were established to evaluate whether this VLP vaccine formulation can enhance the immunogenicity of geap36 and reduce transmissibility (TRA), providing a reference for future vaccine research.

Claims

1. A recombinant protein combination, characterized in that, The recombinant protein includes AP205-2*SpyTag and GEAP36-Spycatcher; AP205-2*SpyTag is obtained by adding two Spytags to the N-terminus and C-terminus of AP205 at the gene level; GEAP36-Spycatcher is obtained by adding one Spycatcher to the C-terminus of GEAP36 at the gene level; the amino acid sequence of GEAP36 is 45-245 of PBANKA_1119200.

2. The recombinant protein combination according to claim 1, characterized in that, The protein combination uses the SpyTag-SpyCatcher reaction to conjugate GEAP36 with AP205, which can display the target antigen on the surface of VLP particles, thereby enhancing the immunogenicity of GEAP36 and reducing its transmissibility.

3. The use of the recombinant protein combination as described in claim 1 in the preparation of a vaccine for blocking the transmission of Plasmodium berghei.

4. A recombinant nucleic acid combination, characterized in that, It encodes the recombinant protein combination described in claim 1 or 2.

5. The recombinant nucleic acid combination according to claim 4, characterized in that, The recombinant nucleic acid combination is codon-optimized for its host.

6. A recombinant expression vector, characterized in that, The expression vector contains the recombinant nucleic acid combination as described in claim 4 or 5.

7. A malaria transmission blocking vaccine, characterized in that the active ingredient in the vaccine comprises the recombinant protein combination of claim 1 or 2, the recombinant nucleic acid combination of claim 4 or 5, or the recombinant expression vector of claim 6.

8. The vaccine according to claim 7, characterized in that, The vaccine may also include adjuvants, immune enhancers, or immunomodulators.

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