Expression plasmid system of antigen-loaded PHA (polyhydroxyalkanoate) nanoparticles as well as preparation and application of expression plasmid system

Abstract

The invention discloses an antigen-loaded PHA (polyhydroxyalkanoate) nano-particle expression plasmid system as well as preparation and application of the antigen-loaded PHA nano-particle expression plasmid system. Comprising a pACYC-PhaA-PhaB plasmid and a pET28a-PhaC-Target plasmid, and the nucleotide sequences of the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Target plasmid are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5; the pACYC-PhaA-PhaB plasmid is characterized in that a PhaA protein expression cassette and a PhaB protein expression cassette, which are respectively started by two T7 promoters, are inserted into multiple cloning sites of a vector pACYCDuet-1; the pET28a-Phac-Target plasmid is characterized in that a PhaC-vaccine antigen fusion protein expression cassette is inserted into a plurality of cloning sites of a vector pET-28a. The invention lays a good foundation for preparation of PHA nanoparticle vaccines and related research.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an expression plasmid system of antigen-loaded PHA nanoparticles and its preparation and application. Background Art

[0002] PHA particles are a general term for renewable polyhydroxyalkanoate (Polyhydroxyalkanoate), which are widely used in the fields of biotechnology and biomedicine due to their excellent biocompatibility and easy modification of the microsphere surface.

[0003] Both Gram-negative and Gram-positive bacteria produce PHA particles, which are used as a carbon and energy storage material when there is environmental stress, such as when there is an excess of carbon and a limited supply of important growth nutrients such as nitrogen or phosphate. Some bacteria can even collect up to 90% of their dry cell weight in PHA particles. PHA particles are deposited in the bacterial cells as insoluble spherical inclusions, and the size of the particles is generally around 500 nm.

[0004] The synthesis of PHA requires three important enzymes, β-pyruvylase (PhaA), acetoacetyl-CoA reductase (PhaB) and polyester synthase (PhaC). Studies have found that the fusion expression of exogenous proteins and PhaC synthase can express nanoparticles with exogenous protein activity, which has promoted the vigorous development of PHA in the medical field. At the same time, PHA particles themselves have adjuvant properties, so that they may not require additional adjuvants in their applications, which makes the PHA particle system promising to become a flexible and effective particle vaccine platform.

[0005] In recent years, PHA nanoparticles with antigens presented on their surfaces have been gradually confirmed as a reliable new vaccine. There are many microorganisms that can be used to produce PHA, among which the E. coli expression system is considered to be an ideal choice for producing PHA, because E. coli has a clear genetic background and a mature and simple culture process, and E. coli does not contain enzymes that degrade PHA. Compared with other microorganisms, E. coli can produce more PHA, and its higher conversion rate can significantly reduce production costs.

[0006] At present, the problems faced by PHA nanoparticle vaccine production mainly include the following aspects. First, the assembly efficiency of PHA nanoparticles is low and the protein yield is low, which requires precise control of nutritional conditions, temperature and other parameters during the fermentation process to ensure high assembly efficiency and high protein yield; secondly, PHA extracted from Escherichia coli needs to remove cell walls and other impurities, and the purification process is complicated and costly. Summary of the invention

[0007] The object of the present invention is to overcome the deficiencies of the prior art and provide an expression plasmid system of antigen-loaded PHA nanoparticles, as well as its preparation and application. In the present invention, plasmids capable of expressing PhaA and PhaB proteins and a plasmid expressing a PhaC-vaccine antigen fusion protein are constructed. The expression conditions are optimized in Rosetta strains, and a large amount of antigen-loaded PHA nanoparticles are obtained. The antigen-loaded PHA nanoparticles are purified using three different purification buffers to remove the contaminating proteins, and finally pure antigen-loaded PHA nanoparticles are obtained.

[0008] To achieve the above object, the technical solutions designed by the present invention are as follows: The present invention provides an expression plasmid system of antigen-loaded PHA nanoparticles, and the expression plasmid system includes a pACYC-PhaA-PhaB plasmid and a pET28a-PhaC-Target plasmid; wherein, the nucleotide sequences of the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Target plasmid are respectively shown in SEQ ID NO: 4 and SEQ ID NO: 5; the pACYC-PhaA-PhaB plasmid is a PhaA and PhaB protein expression cassette in which two T7 promoters are respectively inserted into the multiple cloning sites of the vector pACYCDuet-1; the pET28a-Phac-Target plasmid is a PhaC-vaccine antigen fusion protein expression cassette inserted into the multiple cloning sites of the vector pET-28a.

[0009] Furthermore, the nucleotide sequences of PhaA, PhaB and PhaC are respectively shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0010] Still further, the vaccine antigen fusion protein is an Afua protein, and the nucleotide sequence of the Afua protein is shown in SEQ ID NO: 6.

[0011] The present invention also provides a PHA purification buffer combination, and the PHA purification buffer combination is composed of PHA purification buffer A, PHA purification buffer B and PHA purification buffer C; wherein, the PHA purification buffer A includes 1.18 - 1.23 g / L of Tris, 1.43 - 1.49 g / L of ethylenediaminetetraacetic acid, 0.39 - 0.41 g / L of urea, and the pH is 10.5 - 11.5; The PHA purification buffer B includes 1.18 - 1.23 g / L of Tris, 1.43 - 1.49 g / L of ethylenediaminetetraacetic acid (EDTA), 117 - 123 g / L of urea, and has a pH of 7.0 - 8.0; The PHA purification buffer C includes 1.18 - 1.23 g / L of Tris and has a pH of 7.0 - 8.0.

[0012] Further, the PHA purification buffer A includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid, 0.4 g / L of urea, and has a pH of 11; The PHA purification buffer B includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid (EDTA), 120 g / L of urea, and has a pH of 7.5; The PHA purification buffer C includes 1.21 g / L of Tris and has a pH of 7.5.

[0013] The present invention also provides a method for preparing antigen - loaded PHA nanoparticles using the said expression plasmid system, comprising the following steps: (1) Electroporate the pACYC - PhaA - PhaB plasmid and the pET28a - PhaC - Target plasmid into Escherichia coli Rosetta (DE3) competent cells, and obtain single colonies of recombinant strains through antibiotic resistance screening; (2) Inoculate the single colonies into an LB medium containing kanamycin and chloramphenicol, and culture with shaking; (3) Transfer the bacterial liquid cultured in step (2) into an LB medium containing kanamycin, chloramphenicol, and glucose, and culture with shaking until OD 600 = 0.6 - 0.8; add IPTG and then culture with shaking; (4) Centrifuge the bacterial liquid cultured in step (3) to obtain a precipitate, resuspend the precipitate in Tris buffer, ultrasonically disrupt the cells, and then centrifuge to obtain a precipitate; (5) Resuspend and centrifuge the precipitate obtained in step (4) successively with PHA purification buffer A and PHA purification buffer B in the said PHA purification buffer combination, then incubate, centrifuge to obtain a precipitate, and finally resuspend with PHA purification buffer C in the said PHA purification buffer combination, and centrifuge to obtain a precipitate; (6) Resuspend the precipitate finally obtained in step (5) with Tris buffer to obtain antigen - loaded PHA nanoparticles.

[0014] Further, in step (1), the antibiotics for the resistance screening are a combination of chloramphenicol at a final concentration of 0.05 mg / mL and kanamycin at a final concentration of 0.05 mg / mL; In step (2), in the LB medium, the kanamycin concentration is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, and the culture is shaken at 37 °C and 220 rpm for 8 - 10 h; In step (3), in the LB medium, the kanamycin concentration is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, the glucose concentration is 0.01 - 0.1 g / mL, and the shaking culture conditions are 37 °C and 220 rpm; the final concentration of IPTG added is 0.1 - 1.6 mM, and the shaking culture conditions after adding IPTG are 16 - 37 °C, 200 rpm, and 8 - 24 h; In step (5), incubate for 1 - 1.5 h.

[0015] Furthermore, the PHA purification buffer A includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid, 0.4 g / L of urea, and the pH is 11; The PHA purification buffer B includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid (EDTA), 120 g / L of urea, and the pH is 7.5; The PHA purification buffer C includes 1.21 g / L of Tris, and the pH is 7.5; The Tris buffer includes 2.42 g / L of Tris and 8.76 g / L of NaCl, and the pH is 7.4.

[0016] The present invention also provides PHA nanoparticles loaded with antigens prepared by the method described above.

[0017] The present invention also provides the application of the PHA nanoparticles loaded with antigens in the preparation of vaccines.

[0018] The beneficial effects of the present invention: The present invention transfers the prokaryotic expression vectors pACYC - PhaA - PhaB and pET28a - PhaC - Target into the Rosetta strain, induces the expression of PHA nanoparticles loaded with antigens with IPTG, and obtains the optimal conditions for IPTG induction, including the induction dose, induction time, induction temperature, and the optimal concentration of glucose in the medium. Under the optimal induction conditions, the expression level of PHA nanoparticles loaded with antigens in prokaryotic cells is greatly improved, and the PHA nanoparticles loaded with antigens are purified by using the purification buffer. The present invention lays a good foundation for the preparation of PHA nanoparticle vaccines and related research. Description of the Drawings

[0019] Figure 1SDS-PAGE diagrams of PHA nanoparticles loaded with antigen at different times; Figure 2 SDS-PAGE diagrams of PHA nanoparticles loaded with antigen at different final concentrations of inducer; Figure 3 SDS-PAGE diagrams of PHA nanoparticles loaded with antigen at different induction temperatures; Figure 4 SDS-PAGE diagrams of PHA nanoparticles loaded with antigen under induction with different final concentrations of glucose; Figure 5 SDS-PAGE diagrams of purified PHA nanoparticles loaded with antigen; Figure 6 Transmission electron microscopy images of PHA nanoparticles loaded with antigen; Figure 7 Plasmid map of the basic vector pACYCDuet-1; Figure 8 Plasmid map of the basic vector pET-28a; Figure 9 Results diagram of antibody dilution of mice in each group after immunization; Figure 10 Results diagram of whole blood killing rate of mice in each group after immunization. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.

[0021] Example 1 Expression plasmid system of PHA nanoparticles loaded with antigen The expression plasmid system in this example includes pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid. The amino acid sequences of PhaA, PhaB and PhaC were obtained through query. After codon optimization according to the codon preference of Escherichia coli, the synthesis of nucleotide sequences and plasmid construction were all completed by Beijing Tsingke Biotechnology Co., Ltd. The nucleotide sequences of PhaA, PhaB and PhaC after codon optimization are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the plasmid sequences of pACYC-PhaA-PhaB and pET28a-PhaC-Target are shown as SEQ ID NO: 4 and SEQ ID NO: 5 respectively.

[0022] The pACYC-PhaA-PhaB plasmid inserts two expression cassettes of PhaA and PhaB proteins each driven by the T7 promoter into the multiple cloning sites of the basic vector pACYCDuet-1; the plasmid map of the basic vector pACYCDuet-1 is as shown in Figure 7 shown; The pET28a-Phac-Target plasmid inserts the expression cassette of the PhaC-vaccine antigen fusion protein driven by the T7 promoter into the multiple cloning sites of the basic vector pET-28a; the plasmid map of the basic vector pET-28a is as shown in Figure 8 shown.

[0023] Example 2 Construction of pET28a-PhaC-Afua plasmid 1. In this example, the selected vaccine antigen Target is the Afua protein of Glasser's disease caused by Haemophilus parasuis, and its nucleotide sequence was synthesized by Beijing Tsingke Biotechnology Co., Ltd. The nucleotide sequence of the Afua protein is as shown in SEQ ID NO: 6.

[0024] 2. Design primers for linearizing the pET28a-PhaC-Target plasmid: pET-28a-F and pET-28a-R, and their nucleotide sequences are as shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively. Using the pET28a-PhaC-Target plasmid as a template, the plasmid was linearized by inverse PCR amplification and the linearized plasmid fragment was recovered.

[0025] 3. Design primers for amplifying the full length of the Afua gene with vector adapters: Afua-F and Afua-R, and their nucleotide sequences are as shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively. Using the synthesized Afua gene as a template for PCR amplification, the Afua gene fragment with vector adapters was recovered.

[0026] 4. Use a fusion enzyme to fuse the linearized plasmid fragment with the Afua gene fragment. The fusion product was transformed into Escherichia coli Rosetta (DE3) competent cells by heat shock method. Single colonies were screened by kanamycin resistance with a final concentration of 0.05 mg / mL, identified by PCR amplification and sequenced. Finally, the correct pET28a-PhaC-Afua recombinant plasmid was obtained, and its nucleotide sequence is as shown in SEQ ID NO: 11.

[0027] Example 3 Optimization of induction expression conditions of PHA nanoparticles loaded with antigens I. Construction of recombinant strains The correct pACYC-PhaA-PhaB and pET28a-PhaC-Afua plasmids were co-electroporated into Escherichia coli Rosetta (DE3) competent cells. Single colonies containing both pACYC-PhaA-PhaB and pET28a-PhaC-Afua plasmids were screened using double resistance to chloramphenicol at a final concentration of 0.05 mg / mL and kanamycin at a final concentration of 0.05 mg / mL. The colonies were inoculated into 2 mL of LB medium containing kanamycin at a final concentration of 0.05 mg / mL and chloramphenicol at a final concentration of 0.05 mg / mL, and cultured in a shaker at 37 °C and 220 rpm for 8 h. Then, colony PCR was performed to identify the correct single colonies as the strains for expression, namely the PhaA-PhaB-PhaC-Afua recombinant strain.

[0028] II. Optimization of the induction time of antigen-loaded PHA nanoparticles 1. Preparation method of Tris buffer: Take 2.42 g of Tris and 8.76 g of NaCl, add them to 800 mL of ddH2O, adjust the pH value to 7.4 using concentrated hydrochloric acid, and then add ddH2O to make the volume of the solution up to 1 L.

[0029] 2. The PhaA-PhaB-PhaC-Afua recombinant strain was transferred to a 20 mL conical flask containing LB medium with kanamycin at a final concentration of 0.05 mg / mL and chloramphenicol at a final concentration of 0.05 mg / mL at a ratio of 1:100, and cultured in a shaker at 37 °C and 220 rpm until OD600 = 0.6.

[0030] 3. IPTG with a final concentration of 0.8 mM was added, and the culture was continued in a shaker at 16 °C and 200 rpm for 8, 16, and 24 h respectively.

[0031] 4. The bacterial solution was centrifuged at 8000 rpm for 5 min at 4 °C, the supernatant was discarded, the precipitate was resuspended in 4 mL of Tris buffer, the bacteria were ultrasonically disrupted, centrifuged at 8000 rpm for 5 min at 4 °C again, the supernatant was discarded, the precipitate was resuspended in 4 mL of Tris buffer again, 5×loading buffer was added to each centrifuge tube and mixed well, immediately placed in an ice bath after boiling water bath for 10 min, 20 μL of sample was taken from each tube, and the content of antigen-loaded PHA nanoparticles was detected by SDS-PAGE.

[0032] The results of SDS-PAGE are as Figure 1As shown in the figure, the protein size of the antigen-loaded PHA nanoparticles is 103 kDa. It can be seen from the figure that at the final concentration of IPTG of 0.8 mM and the same induction temperature, when inducing for different times, the expression level of the antigen-loaded PHA nanoparticles shows a gradually increasing trend, and the expression level of the antigen-loaded PHA nanoparticles at the induction time of 24 h is the highest. Therefore, 24 h is the optimal induction time for the antigen-loaded PHA nanoparticles.

[0033] III. Optimization of the final concentration of the inducer for the antigen-loaded PHA nanoparticles 1. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain into a conical flask containing 20 mL of LB medium with a final concentration of 0.05 mg / mL kanamycin and a final concentration of 0.05 mg / mL chloramphenicol at a ratio of 1:100, and place it in a shaker at 37°C and 220 rpm for cultivation until OD600 = 0.6.

[0034] 2. Add IPTG to the bacterial solution at final concentrations of 0.1, 0.8, and 1.6 mM respectively, and cultivate in a shaker at 16°C and 200 rpm for 24 h.

[0035] 3. Centrifuge the bacterial solution at 4°C and 8000 rpm for 5 min, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer, ultrasonically disrupt the bacteria, centrifuge at 4°C and 8000 rpm for 5 min again, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer again, add 5×loading buffer to each centrifuge tube and mix well, immediately place it in an ice bath after boiling water bath for 10 min, take 20 μL of sample from each tube, and detect the content of the antigen-loaded PHA nanoparticles by SDS-PAGE.

[0036] The SDS-PAGE detection results are as Figure 2 shown. It can be found from Figure 2 that after adding the inducer IPTG, the expression level of the antigen-loaded PHA nanoparticles shows a trend of increasing first and then decreasing, and the optimal induction dose of IPTG is selected as the final concentration of 0.8 mM.

[0037] IV. Optimization of the induction temperature for the antigen-loaded PHA nanoparticles 1. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain into a conical flask containing 20 mL of LB medium with a final concentration of 0.05 mg / mL kanamycin and a final concentration of 0.05 mg / mL chloramphenicol at a ratio of 1:100, and place it in a shaker at 37°C and 220 rpm for cultivation until OD600 = 0.6.

[0038] 2. Add IPTG in an amount corresponding to a final concentration of 0.8 mM, and culture at 200 rpm for 24 h at 16, 28, and 37 °C respectively.

[0039] 3. Centrifuge the bacterial solution at 8000 rpm for 5 min at 4 °C, discard the supernatant, resuspend the pellet in 4 mL of Tris buffer, sonicate to lyse the bacteria, centrifuge at 8000 rpm for 5 min at 4 °C again, discard the supernatant, resuspend the pellet in 4 mL of Tris buffer again, add 5×loading buffer to each centrifuge tube and mix well, immediately cool on ice after boiling water bath for 10 min, take 20 μL of sample from each tube, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE.

[0040] The results of SDS-PAGE detection are as Figure 3 shown. It can be seen from Figure 3 that when the final concentration of IPTG is 0.8 mM and induced at 28 °C for 24 h, the expression level of PHA nanoparticles loaded with antigen is the highest. Therefore, the optimal induction temperature for PHA nanoparticles loaded with antigen is 28 °C.

[0041] In summary, the optimal induction conditions for IPTG-induced expression of PHA nanoparticles loaded with antigen are: final concentration of IPTG 0.8 mM, induction temperature 28 °C, and induction time 24 h.

[0042] V. Optimization of the final glucose concentration of PHA nanoparticles loaded with antigen Under the optimal IPTG induction dose, induction time, and induction temperature conditions, change the glucose concentration in the medium to induce the expression of PHA nanoparticles loaded with antigen.

[0043] 1. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain at a ratio of 1:100 to a conical flask containing 20 mL of LB medium with a final concentration of 0.05 mg / mL kanamycin, a final concentration of 0.05 mg / mL chloramphenicol, and a final concentration of 0.01, 0.05, or 0.1 g / mL glucose, place it on a shaker at 37 °C and 220 rpm for culture until OD 600 = 0.6.

[0044] 2. Add IPTG in an amount corresponding to a final concentration of 0.8 mM, and culture at 28 °C and 200 rpm for 24 h.

[0045] 3. Centrifuge the bacterial solution at 4°C and 8000 rpm for 5 min, discard the supernatant, resuspend the pellet in 4 mL of Tris buffer, sonicate the bacteria, centrifuge at 4°C and 8000 rpm for 5 min, discard the supernatant, resuspend the pellet in 4 mL of Tris buffer again, add 5×loading buffer to each centrifuge tube and mix well, immediately cool on ice after boiling water bath for 10 min, take 20 μL of sample from each tube, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE.

[0046] The SDS-PAGE detection results are as Figure 4 shown. It can be seen from Figure 4 that under the optimal IPTG induction conditions, when the medium contains glucose with a final concentration of 0.05 g / mL, the expression level of PHA nanoparticles loaded with antigen is the highest.

[0047] Example 4 A combination of PHA purification buffers This example provides a combination of PHA purification buffers, including PHA purification buffer A, PHA purification buffer B, and PHA purification buffer C; Method for preparing PHA purification buffer A: Take 1.21 g of Tris, 1.46 g of EDTA, 0.4 g of urea, add to 800 mL of ddH2O, adjust the pH value to 11 with NaOH solution, and then add ddH2O to make the volume of the solution up to 1 L.

[0048] Method for preparing PHA purification buffer B: Take 1.21 g of Tris, 1.46 g of EDTA, 120 g of urea, 20 mL of Triton X-100, add to 800 mL of ddH2O, adjust the pH value to 7.5 with concentrated hydrochloric acid, and then add ddH2O to make the volume of the solution up to 1 L.

[0049] Method for preparing PHA purification buffer C: Take 1.21 g of Tris, add to 800 mL of ddH2O, adjust the pH value to 7.5 with concentrated hydrochloric acid, and then add ddH2O to make the volume of the solution up to 1 L.

[0050] Example 5 Expression and purification of PHA nanoparticles loaded with antigen In this example, the combination of PHA purification buffers in Example 4 was used for the expression and purification of PHA nanoparticles loaded with antigen.

[0051] 1. Inoculate a single colony of the PhaA-PhaB-PhaC-Afua recombinant strain into 2 mL of LB medium containing kanamycin at a final concentration of 0.05 mg / mL and chloramphenicol at a final concentration of 0.05 mg / mL, and culture it in a shaker at 37 °C and 220 rpm for 8 h.

[0052] 2. Transfer the above bacterial solution to a conical flask containing 20 mL of LB medium with kanamycin at a final concentration of 0.05 mg / mL, chloramphenicol at a final concentration of 0.05 mg / mL, and glucose at a final concentration of 0.05 g / mL according to a ratio of 1:100, and place it in a shaker at 37 °C and 220 rpm for culture until OD 600 = 0.6.

[0053] 3. Add IPTG at a final concentration of 0.8 mM and culture it at 28 °C and 200 rpm for 24 h.

[0054] 4. Centrifuge the bacterial solution at 4 °C and 8000 rpm for 5 min, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer, ultrasonically disrupt the cells, centrifuge at 4 °C and 8000 rpm for 5 min, and discard the supernatant to obtain the precipitate.

[0055] 5. Resuspend the precipitate in 4 mL of PHA purification buffer A, centrifuge at 4 °C and 8000 rpm for 5 min; discard the supernatant, resuspend the precipitate in 4 mL of PHA purification buffer B, incubate at 4 °C for 1 h, centrifuge at 4 °C and 8000 rpm for 5 min; discard the supernatant, resuspend the precipitate in 4 mL of PHA purification buffer C, centrifuge at 4 °C and 8000 rpm for 5 min; 6. Discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer; add 5×loading buffer to the tube, mix well, immediately place it in an ice bath for 10 min after boiling water bath, take 20 μL of the sample, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE. The SDS-PAGE diagram is as Figure 5 shown. It can be seen from Figure 5 that after purification, the miscellaneous proteins are removed, and pure PHA nanoparticles loaded with antigen are obtained.

[0056] After negative staining of the purified PHA nanoparticle sample loaded with antigen, observe it under a transmission electron microscope. As Figure 6 shown, spherical particles with a diameter of 500 nm can be seen successfully polymerized in the field of view.

[0057] The prokaryotic expression plasmids pACYC-PhaA-PhaB and pET28a-PhaC-Afua were co-transformed into Escherichia coli Rosetta (DE3) competent cells, and their expression was induced with IPTG to obtain the optimal conditions for IPTG induction and the optimal concentration of glucose in the medium. The final concentration of IPTG was 0.8 mM, the induction temperature was 28 °C, the induction time was 24 h, and the final concentration of glucose was 0.05 g / mL. The results showed that the expression of antigen-loaded PHA nanoparticles in Escherichia coli Rosetta (DE3) strain could be greatly improved under the optimal induction conditions. After obtaining the antigen-loaded PHA nanoparticles, they were purified using a purification buffer, and finally pure antigen-loaded PHA nanoparticles were obtained. This invention laid a good foundation for the preparation of PHA nanoparticle vaccines and related research.

[0058] Example 6 Immune response of antigen-loaded PHA nanoparticles Three groups, namely 20 μg of antigen-loaded PHA nanoparticles (Pha-Afua), 20 μg of Afua single antigen, and 100 μL of PBS, were respectively mixed with the adjuvant MontanideTM ISA206 VG (ISA206) at a ratio of 1:1 and used to immunize 10 mice. The second immunization was carried out 14 days after the first immunization. The mice were bled 14 days after the second immunization to obtain the sera of the Pha-Afua group, the Afua group, and the PBS group. The obtained sera were diluted from 100-fold to 25,600-fold, and the titers of Afua-specific IgG antibodies in the sera were measured by indirect ELISA. The measurement steps were as follows: (1) The purified protein Afua was diluted in carbonate coating buffer to 800 μL per well, and 100 μL was coated onto the ELISA plate and incubated overnight at 4 °C.

[0059] (2) The coating solution in the ELISA plate was discarded, and the plate was washed once with PBST.

[0060] (3) 120 μL of blocking solution was added to each well and incubated at 37 °C for 1 h.

[0061] (4) The blocking solution was discarded, and the plate was washed once with PBST.

[0062] (5) The sera obtained above were incubated at dilution multiples of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600, and serum-free and blank serum control groups were set. 100 μL of each was added to the ELISA plate and incubated at 37 °C for 1 h.

[0063] Discard the liquid in the ELISA plate and wash it 5 times with PBST.

[0064] Add HRP-labeled goat anti-mouse IgG diluted 1:10,000 and incubate at 37 °C for 45 min.

[0065] Discard the liquid in the ELSIA plate and wash it 5 times with PBST.

[0066] Add 100 μL of TMB to each well and react in the dark for 15 min.

[0067] Add the stop solution to terminate the reaction and read OD450 with an enzyme-linked immunosorbent assay reader.

[0068] Judgment of the test results: The OD450 value of the serum diluted 1:100 in the PBS group was used as the positive judgment standard.

[0069] The results were as Figure 9 shown. The immune effect of the PHA nanoparticles loaded with antigen was higher than that of the single antigen of Afua, and had a higher antibody dilution factor. The serum dilution factor was as high as 12,800 times, which was higher than the serum dilution factor of 6,400 times after immunization with the single antigen of Afua, and could significantly improve the antibody response level.

[0070] Example 7 Bactericidal performance of the PHA nanoparticles loaded with antigen Take the sera of the Pha-Afua group, Afua group and PBS group in Example 6 for standby (immune sera).

[0071] Culture Glässeria parasuis serotype 5 (strain 0165) until the logarithmic growth phase, and then accurately adjust its concentration to 1×10 4 CFU / ml (colony-forming units per milliliter) with sterile PBS (phosphate buffer solution).

[0072] Take 50 μL (microliters) of the sera in step (1) and 50 μL of the Glässeria parasuis solution in step (2), respectively, place them in 1.5 mL sterile EP tubes, and incubate at 37 °C for 20 minutes. At the same time, set the serum of healthy mice without immune treatment as the blank control group of the experiment for subsequent data comparison and analysis.

[0073] Add 100 μL of heparinized anticoagulated blood samples of non-immune healthy mice to the above EP tubes respectively, and then culture them at 37 °C for 1 hour. During the culture process, gently shake the EP tubes every 10 minutes to ensure sufficient and uniform reaction.

[0074] (5)After subjecting the samples of each group to 10-fold dilution treatment with sterile PBS, 100 μL of the diluted samples were respectively taken and spread on TSA plates. The plates were placed in an environment of 37°C and statically cultured for 24 to 48 hours. After the colonies were formed stably, the number of colonies on each plate was recorded. According to the recorded number of colonies, the bactericidal rate was calculated according to the following formula: Bactericidal rate × 100% = (CFU number after 1-hour culture in the blank control serum - CFU number after 1-hour culture in the immune serum) / CFU number after 1-hour culture in the blank control serum. This bactericidal rate was used to evaluate the difference in bactericidal efficacy of the immune group serum compared with the blank group serum against the corresponding strain.

[0075] The results are as Figure 10 shown. It can be seen that compared with the single antigen and PBS groups, the whole blood killing effect of the PHA nanoparticles loaded with antigen was significantly improved, up to 60%, showing a certain immune enhancement effect.

[0076] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An expression plasmid system of antigen-loaded PHA nanoparticles, characterized in that: The expression plasmid system includes pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid; among them, the nucleotide sequences of the pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid are shown as SEQ ID NO: 4 and SEQ ID NO: 5 respectively; the pACYC-PhaA-PhaB plasmid is a plasmid in which the PhaA and PhaB protein expression frames respectively driven by two T7 promoters are inserted into the multiple cloning sites of the vector pACYCDuet-1; the pET28a-Phac-Target plasmid is a plasmid in which the PhaC-vaccine antigen fusion protein expression frame is inserted into the multiple cloning sites of the vector pET-28a; the vaccine antigen fusion protein is the Afua protein, and the nucleotide sequence of the Afua protein is shown as SEQ ID NO:

6.

2. The expression plasmid system according to claim 1, wherein: the nucleotide sequences of PhaA, PhaB and PhaC are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively.

3. A PHA purification buffer combination, characterized in that: The PHA purification buffer combination consists of PHA purification buffer A, PHA purification buffer B and PHA purification buffer C; among them, the PHA purification buffer A includes 1.18 - 1.23 g / L of Tris, 1.43 - 1.49 g / L of ethylenediaminetetraacetic acid, 0.39 - 0.41 g / L of urea, and the pH is 10.5 - 11.5; the PHA purification buffer B includes 1.18 - 1.23 g / L of Tris, 1.43 - 1.49 g / L of ethylenediaminetetraacetic acid (EDTA), 117 - 123 g / L of urea, and the pH is 7.0 - 8.0; the PHA purification buffer C includes 1.18 - 1.23 g / L of Tris, and the pH is 7.0 - 8.

0.

4. The PHA purification buffer combination according to claim 3, wherein: the PHA purification buffer A includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid, 0.4 g / L of urea, and the pH is 11; the PHA purification buffer B includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid (EDTA), 120 g / L of urea, and the pH is 7.5; the PHA purification buffer C includes 1.21 g / L of Tris, and the pH is 7.

5.

5. A method for preparing antigen-loaded PHA nanoparticles using the expression plasmid system according to any one of claims 1 to 2, characterized in that: It includes the following steps: (1) Electrotransfer the pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid into Escherichia coli Rosetta (DE3) competent cells, and obtain single colonies of recombinant strains through resistance screening; (2) Inoculate the single colonies into the LB medium containing kanamycin and chloramphenicol, and culture with shaking; (3) Transfer the bacterial liquid cultured in step (2) into an LB medium containing kanamycin, chloramphenicol, and glucose, and shake-culture until OD 600 = 0.6 - 0.8; add IPTG and then shake-culture; (4) Centrifuge the bacterial liquid cultured in step (3) to obtain a precipitate, resuspend the precipitate with Tris buffer, ultrasonically disrupt the cells, and centrifuge to obtain a precipitate; (5) Resuspend and centrifuge the precipitate obtained in step (4) successively with PHA purification buffer A and PHA purification buffer B in the PHA purification buffer combination described in claim 3, then incubate, centrifuge to obtain a precipitate, and finally resuspend with PHA purification buffer C in the PHA purification buffer combination described in claim 3, and centrifuge to obtain a precipitate; (6) Resuspend the precipitate finally obtained in step (5) with Tris buffer to obtain antigen-loaded PHA nanoparticles.

6. The method according to claim 5, wherein: In the said step (1), the antibiotics for resistance screening are a combination of chloramphenicol at a final concentration of 0.05 mg / mL and at a final concentration of 0.05 mg / mL; In the said step (2), in the LB medium, the kanamycin concentration is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, at 37 °C, shake culture at 220 rpm for 8 - 10 h; In the said step (3), in the LB medium, the kanamycin concentration is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, the glucose concentration is 0.01 - 0.1 g / mL, the conditions for shake culture are 37 °C, 220 rpm; the final concentration of IPTG added is 0.1 - 1.6 mM, and the shake culture conditions after adding IPTG are 16 - 37 °C, 200 rpm, 8 - 24 h; In the said step (5), incubate for 1 - 1.5 h.

7. The method according to claim 5, characterized in that: The said PHA purification buffer A includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid, 0.4 g / L of urea, and the pH is 11; The said PHA purification buffer B includes 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid (EDTA), 120 g / L of urea, and the pH is 7.5; The said PHA purification buffer C includes 1.21 g / L of Tris, and the pH is 7.5; The said Tris buffer includes 2.42 g / L of Tris and 8.76 g / L of NaCl, and the pH is 7.

4.

8. Antigen-loaded PHA nanoparticles prepared by the method according to any one of claims 5 - 7.

9. Use of the antigen-loaded PHA nanoparticles according to claim 8 in the preparation of a vaccine.

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