Expression plasmid system of phaneric antigen-loaded phaneric nanoparticles and preparation and application thereof

Abstract

The application discloses an expression plasmid system of antigen-loaded PHA nanoparticles and preparation and application thereof. The expression plasmid system comprises pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid, nucleotide sequences of the 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 a PhaA and PhaB protein expression frame inserted into a multiple cloning site of a vector pACYCDuet-1 and driven by two T7 promoters respectively; the pET28a-Phac-Target plasmid is a PhaC-vaccine antigen fusion protein expression frame inserted into a multiple cloning site of a vector pET-28a. The application lays a good foundation for preparation of PHA nanoparticle vaccines and related research.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to an expression plasmid system of antigen-loaded PHA nanoparticles and preparation and application thereof. BACKGROUND

[0002] PHA particles are a general term of renewable polyhydroxyalkanoate, which is widely used in the fields of biotechnology and biological medicine due to excellent biocompatibility and easy modification of microsphere surface.

[0003] Both gram-negative and gram-positive bacteria produce PHA particles, which are used as carbon source and energy storage materials when environmental pressure exists, such as accumulation in bacteria when carbon is excessive and important growth nutrients such as nitrogen or phosphate are limited. Some bacteria can even collect up to 90% of PHA particles of the weight of dry cells. PHA particles are deposited in the form of insoluble spherical inclusions in the cells of bacteria, and the size of the particles is generally about 500 nm.

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

[0005] In recent years, PHA nanoparticles that present antigens on the surface have been gradually confirmed as a reliable new vaccine. Many microorganisms can be used to produce PHA, and the E. coli expression system is considered to be a relatively 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 has higher conversion rate, which can significantly reduce production cost.

[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 fermentation to ensure high assembly efficiency and high protein yield. Secondly, the PHA extracted from E. coli needs to remove the cell wall and other impurities, and the purification process is complex and costly. SUMMARY

[0007] The present application aims at overcoming the deficiencies of the prior art, and provides an expression plasmid system of antigen-loaded PHA nanoparticles and preparation and application thereof. The present application constructs a plasmid capable of expressing PhaA and PhaB proteins and a plasmid expressing a PhaC-vaccine antigen fusion protein, optimizes the expression conditions in a Rosetta strain, and obtains a large amount of antigen-loaded PHA nanoparticles, purifies the antigen-loaded PHA nanoparticles using three different purification buffers, removes the impurities therein, and finally obtains pure antigen-loaded PHA nanoparticles.

[0008] To achieve the above-mentioned object, the technical scheme designed by the present application is as follows:

[0009] The present application provides an expression plasmid system of antigen-loaded PHA nanoparticles, which comprises a pACYC-PhaA-PhaB plasmid and a pET28a-PhaC-Target plasmid; wherein,

[0010] The nucleotide sequences of the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Target plasmid are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively;

[0011] The pACYC-PhaA-PhaB plasmid is a PhaA and PhaB protein expression frame inserted into the multiple cloning site of the vector pACYCDuet-1 and driven by two T7 promoters, respectively;

[0012] The pET28a-PhaC-Target plasmid is a PhaC-vaccine antigen fusion protein expression frame inserted into the multiple cloning site of the vector pET-28a.

[0013] Further, the nucleotide sequences of PhaA, PhaB and PhaC are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0014] Still further, the vaccine antigen fusion protein is Afua protein, and the nucleotide sequence of the Afua protein is shown in SEQ ID NO: 6.

[0015] The present application also provides a PHA purification buffer combination, which consists of a PHA purification buffer A, a PHA purification buffer B and a PHA purification buffer C; wherein,

[0016] The PHA purification buffer A comprises 1.18-1.23 g / L of Tris, 1.43-1.49 g / L of ethylenediaminetetraacetic acid, and 0.39-0.41 g / L of urea, and has a pH of 10.5-11.5.

[0017] The PHA purification buffer B comprises 1.18-1.23 g / L of Tris, 1.43-1.49 g / L of ethylenediamine tetraacetic acid (EDTA), 117-123 g / L of urea, and has a pH of 7.0-8.0;

[0018] The PHA purification buffer C comprises 1.18-1.23 g / L of Tris and has a pH of 7.0-8.0.

[0019] Further, the PHA purification buffer A comprises 1.21 g / L of Tris, 1.46 g / L of ethylenediamine tetraacetic acid, 0.4 g / L of urea, and has a pH of 11;

[0020] The PHA purification buffer B comprises 1.21 g / L of Tris, 1.46 g / L of ethylenediamine tetraacetic acid (EDTA), 120 g / L of urea, and has a pH of 7.5;

[0021] The PHA purification buffer C comprises 1.21 g / L of Tris and has a pH of 7.5.

[0022] The application further provides a method for preparing the antigen-loaded PHA nanoparticles by using the expression plasmid system, comprising the following steps:

[0023] (1) electrotransforming the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Target plasmid into Escherichia coli Rosetta (DE3) competent cells, and obtaining single colonies of the recombinant strain through resistance screening;

[0024] (2) inoculating the single colonies into LB medium containing kanamycin and chloramphenicol, and performing shock culture;

[0025] (3) transferring the bacterial liquid cultured in the step (2) into LB medium containing kanamycin, chloramphenicol and glucose, and performing shock culture until the OD 600 =0.6-0.8; after adding IPTG, performing shock culture;

[0026] (4) centrifuging the bacterial liquid cultured in the step (3) to obtain a precipitate, resuspending the precipitate in Tris buffer, performing ultrasonic cell disruption, and centrifuging to obtain a precipitate;

[0027] (5) resuspending and centrifuging the precipitate obtained in the step (4) with the PHA purification buffer A and the PHA purification buffer B in the PHA purification buffer combination in sequence, then performing incubation, centrifuging to obtain a precipitate, and finally resuspending the precipitate in the PHA purification buffer C in the PHA purification buffer combination and centrifuging to obtain a precipitate;

[0028] (6) resuspend the precipitate obtained in step (5) with Tris buffer to obtain the antigen-loaded PHA nanoparticles.

[0029] Further, in the step (1), the antibiotic for the resistance screening is a combination of chloramphenicol with a final concentration of 0.05 mg / mL and kanamycin with a final concentration of 0.05 mg / mL;

[0030] In the step (2), the kanamycin concentration in the LB medium is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, and the culture is carried out at 37℃ with 220 rpm shaking for 8-10 h;

[0031] In the step (3), the kanamycin concentration in the LB medium is 0.05 mg / mL, the chloramphenicol concentration is 0.05 mg / mL, and the glucose concentration is 0.01-0.1 g / mL, and the shaking culture condition is 37℃, 220 rpm; the final concentration of IPTG added is 0.1-1.6 mM, and the shaking culture condition after adding IPTG is 16-37℃, 200 rpm, 8-24 h;

[0032] In the step (5), the incubation is carried out for 1-1.5 h.

[0033] Further, the PHA purification buffer A comprises 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid, and 0.4 g / L of urea, and the pH is 11;

[0034] The PHA purification buffer B comprises 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid (EDTA), and 120 g / L of urea, and the pH is 7.5;

[0035] The PHA purification buffer C comprises 1.21 g / L of Tris, and the pH is 7.5;

[0036] The Tris buffer comprises 2.42 g / L of Tris and 8.76 g / L of NaCl, and the pH is 7.4.

[0037] The application further provides the antigen-loaded PHA nanoparticles prepared by the method.

[0038] The application further provides the use of the antigen-loaded PHA nanoparticles in the preparation of vaccines.

[0039] The present application has the following beneficial effects: the present application introduces the prokaryotic expression vectors pACYC-PhaA-PhaB and pET28a-PhaC-Target into the Rosetta strain, and induces the expression of the antigen-loaded PHA nanoparticles by using IPTG, and obtains the optimal conditions for the IPTG induction, including the induction dose, the induction time, the induction temperature and the optimal concentration of glucose in the culture medium. Under the optimal induction conditions, the expression amount of the antigen-loaded PHA nanoparticles in the prokaryotic cells is greatly improved, and the antigen-loaded PHA nanoparticles are purified by using the purification buffer, and the present application lays a good foundation for the preparation of the PHA nanoparticle vaccine and related research. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 SDS-PAGE diagram of the antigen-loaded PHA nanoparticles under different induction times;

[0041] Figure 2 SDS-PAGE diagram of the antigen-loaded PHA nanoparticles under different final concentrations of the induction agent;

[0042] Figure 3 SDS-PAGE diagram of the antigen-loaded PHA nanoparticles under different induction temperatures;

[0043] Figure 4 SDS-PAGE diagram of the antigen-loaded PHA nanoparticles under different final concentrations of glucose;

[0044] Figure 5 SDS-PAGE diagram of the purified antigen-loaded PHA nanoparticles;

[0045] Figure 6 Transmission electron microscope diagram of the antigen-loaded PHA nanoparticles;

[0046] Figure 7 Plasmid map diagram of the basic vector pACYCDuet-1;

[0047] Figure 8 Plasmid map diagram of the basic vector pET-28a;

[0048] Figure 9 Antibody dilution result diagram of the mice in each group after immunization;

[0049] Figure 10 Whole blood killing rate result diagram of the mice in each group after immunization. DETAILED DESCRIPTION

[0050] The present application will be further described in detail below in combination with specific embodiments, so as to be understood by those skilled in the art.

[0051] Example 1

[0052] Expression plasmid system of antigen-loaded PHA nanoparticle

[0053] The expression plasmid system of the present example comprises pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid. The amino acid sequences of PhaA, PhaB and PhaC were obtained by inquiry, and after codon optimization according to the codon bias of Escherichia coli, the synthesis of nucleotide sequences and the construction of plasmids were completed by Beijing Qikexing Biotechnology Co., Ltd. The codon-optimized nucleotide sequences of PhaA, PhaB and PhaC are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the sequences of pACYC-PhaA-PhaB and pET28a-PhaC-Target plasmids are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

[0054] The pACYC-PhaA-PhaB plasmid is a plasmid in which two PhaA and PhaB protein expression frames initiated by T7 promoter are inserted into the multiple cloning site of the basic vector pACYCDuet-1; the plasmid map of the basic vector pACYCDuet-1 is shown in Figure 7 .

[0055] The pET28a-PhaC-Target plasmid is a plasmid in which a PhaC-vaccine antigen fusion protein expression frame initiated by T7 promoter is inserted into the multiple cloning site of the basic vector pET-28a; the plasmid map of the basic vector pET-28a is shown in Figure 8 .

[0056] Example 2

[0057] Construction of pET28a-PhaC-Afua plasmid

[0058] 1. The vaccine antigen Target selected in the present example is Afua protein of Paraglaser porcinum, and the nucleotide sequence thereof is synthesized by Beijing Qikexing Biotechnology Co., Ltd. The nucleotide sequence of Afua protein is shown in SEQ ID NO: 6.

[0059] 2. Primers for linearizing the pET28a-PhaC-Target plasmid, pET-28a-F and pET-28a-R, are designed, and their nucleotide sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The plasmid is linearized by reverse PCR amplification using the pET28a-PhaC-Target plasmid as a template, and the linearized plasmid fragment is recovered.

[0060] 3、Design the primers of Afua gene full-length with vector adapter: Afua-F and Afua-R, the nucleotide sequences are shown as SEQ ID NO: 9 and SEQ ID NO: 10. The synthesized Afua gene is used as a template for PCR amplification, and the Afua gene fragment with vector adapter is recovered.

[0061] 4、The plasmid linearization fragment is fused with the Afua gene fragment by using the fusion enzyme, and the fusion product is transformed into the E. coli Rosetta (DE3) competent cells by heat shock method. The single colony is screened by the resistance of 0.05 mg / mL kanamycin, identified by PCR amplification and sequenced, and finally the correct pET28a-PhaC-Afua recombinant plasmid is obtained, and the nucleotide sequence is shown as SEQ ID NO: 11.

[0062] Example 3

[0063] Optimization of induced expression conditions of antigen-loaded PHA nanoparticles

[0064] I. Construction of recombinant strain

[0065] The correct pACYC-PhaA-PhaB and pET28a-PhaC-Afua plasmids are simultaneously electrotransformed into E. coli Rosetta (DE3) competent cells, and the single colony with pACYC-PhaA-PhaB and pET28a-PhaC-Afua plasmids is screened by using the final concentration of 0.05 mg / mL chloramphenicol and the final concentration of 0.05 mg / mL kanamycin. The colony is inoculated into 2 mL LB medium containing the final concentration of 0.05 mg / mL kanamycin and the final concentration of 0.05 mg / mL chloramphenicol, and cultured at 37°C, 220 rpm for 8 h. Then, the bacterial liquid PCR is performed, and the correct single colony is identified as the expression strain, i.e. PhaA-PhaB-PhaC-Afua recombinant strain.

[0066] II. Optimization of induction time of antigen-loaded PHA nanoparticles

[0067] 1. The configuration method of Tris buffer: take 2.42 g Tris and 8.76 g NaCl into 800 mL ddH2O, adjust the pH value to 7.4 by using concentrated hydrochloric acid, and then add ddH2O to make the solution constant volume to 1 L.

[0068] 2. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain to a 20 mL Erlenmeyer flask containing 0.05 mg / mL kanamycin and 0.05 mg / mL chloramphenicol at a ratio of 1:100. Incubate at 37°C and 220 rpm on a shaker until OD600 = 0.6.

[0069] 3. Add IPTG to a final concentration of 0.8 mM, and incubate at 16℃ and 200 rpm for 8, 16 and 24 h respectively.

[0070] 4. Centrifuge the bacterial culture at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer, sonicate to disrupt the bacteria, centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer again, add 5× loading buffer to each centrifuge tube and mix well, incubate in a boiling water bath for 10 min and immediately place on ice to cool, take 20 μL of sample from each tube, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE.

[0071] SDS-PAGE results are as follows Figure 1 As shown in the figure, the protein size of the antigen-loaded PHA nanoparticles is 103 kDa. The figure also shows that, at a final IPTG concentration of 0.8 mM and the same induction temperature, the expression level of the antigen-loaded PHA nanoparticles gradually increases with different induction times, reaching its highest level after 24 h of induction. Therefore, 24 h is the optimal induction time for antigen-loaded PHA nanoparticles.

[0072] III. Optimization of the final concentration of inducer for antigen-loaded PHA nanoparticles

[0073] 1. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain to a 20 mL Erlenmeyer flask containing 0.05 mg / mL kanamycin and 0.05 mg / mL chloramphenicol at a ratio of 1:100. Incubate at 37°C and 220 rpm on a shaker until OD600 = 0.6.

[0074] 2. IPTG was added to the bacterial culture at final concentrations of 0.1, 0.8 and 1.6 mM, respectively, and the cultures were incubated at 16°C and 200 rpm for 24 h.

[0075] 3. Centrifuge the bacterial culture at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer, sonicate to disrupt the bacteria, centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer again, add 5× loading buffer to each centrifuge tube and mix well, incubate in boiling water for 10 min and immediately place on ice to cool, take 20 μL of sample from each tube, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE.

[0076] SDS-PAGE test results are as follows: Figure 2 As shown. From Figure 2 It can be observed that after the addition of the inducer IPTG, the expression level of PHA nanoparticles loaded with antigen showed a trend of first increasing and then decreasing. The optimal induction dose of IPTG was selected with a final concentration of 0.8 mM.

[0077] IV. Optimization of Induction Temperature for Antigen-Loaded PHA Nanoparticles

[0078] 1. Transfer the PhaA-PhaB-PhaC-Afua recombinant strain to a 20 mL Erlenmeyer flask containing 0.05 mg / mL kanamycin and 0.05 mg / mL chloramphenicol at a ratio of 1:100. Incubate at 37°C and 220 rpm on a shaker until OD600 = 0.6.

[0079] 2. Add IPTG to a final concentration of 0.8 mM and incubate at 16, 28 and 37 °C for 24 h at 200 rpm.

[0080] 3. Centrifuge the bacterial culture at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer, sonicate to disrupt the bacteria, centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant, resuspend the precipitate in 4 mL of Tris buffer again, add 5× loading buffer to each centrifuge tube and mix well, incubate in boiling water for 10 min and immediately place on ice to cool, take 20 μL of sample from each tube, and detect the content of PHA nanoparticles loaded with antigen by SDS-PAGE.

[0081] SDS-PAGE test results are as follows: Figure 3 As shown. From Figure 3 As can be seen, the expression level of antigen-loaded PHA nanoparticles was highest when the final IPTG concentration was 0.8 mM and the induction temperature was 28℃ for 24 h. Therefore, the optimal induction temperature for antigen-loaded PHA nanoparticles is 28℃.

[0082] In summary, the optimal induction conditions for IPTG-induced expression of antigen-loaded PHA nanoparticles are: an IPTG final concentration of 0.8 mM, an induction temperature of 28°C, and an induction time of 24 h.

[0083] V. Optimization of the final glucose concentration of antigen-loaded PHA nanoparticles

[0084] Under the optimal IPTG induction dose, induction time, and induction temperature conditions, the glucose concentration in the culture medium was changed, and antigen-loaded PHA nanoparticle expression was induced.

[0085] 1. The PhaA-PhaB-PhaC-Afua recombinant strain was transferred to a 20 mL conical flask containing 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 at a ratio of 1:100, and was cultured at 37°C and 220 rpm until the OD 600 = 0.6.

[0086] 2. IPTG was added at a final concentration of 0.8 mM, and the culture was incubated at 28°C and 200 rpm for 24 h.

[0087] 3. The bacterial solution was centrifuged at 8000 rpm and 4°C for 5 min, the supernatant was discarded, the precipitate was resuspended in 4 mL Tris buffer, the bacteria were broken by ultrasonic, the mixture was centrifuged at 8000 rpm and 4°C for 5 min, the supernatant was discarded, the precipitate was resuspended in 4 mL Tris buffer, 5x loading buffer was added to each centrifuge tube, the mixture was boiled in a water bath for 10 min and then immediately placed on ice, 20 μL of sample was taken from each tube, and the content of antigen-loaded PHA nanoparticles was detected by SDS-PAGE.

[0088] The SDS-PAGE detection results are shown in Figure 4 From Figure 4 it can be seen that, under the optimal IPTG induction conditions, the expression amount of antigen-loaded PHA nanoparticles is the highest when the culture medium contains glucose at a final concentration of 0.05 g / mL.

[0089] Example 4

[0090] A PHA purification buffer combination

[0091] The present example provides a PHA purification buffer combination, which comprises PHA purification buffer A, PHA purification buffer B, and PHA purification buffer C.

[0092] PHA purification buffer A configuration method: take 1.21 g Tris, 1.46 g EDTA, 0.4 g urea (urea) into 800 mL ddH2O, use NaOH solution to adjust the pH value to 11, then add ddH2O to constant volume to 1 L.

[0093] PHA purification buffer B configuration method: take 1.21 g Tris, 1.46 g EDTA, 120 g Urea, 20 mL Triton X-100, add 800 mL ddH2O, use concentrated hydrochloric acid to adjust the pH value to 7.5, then add ddH2O to constant volume to 1 L.

[0094] PHA purification buffer C configuration method: take 1.21 g Tris, add 800 mL ddH2O, use concentrated hydrochloric acid to adjust the pH value to 7.5, then add ddH2O to constant volume to 1 L.

[0095] Example 5

[0096] Expression and purification of antigen-loaded PHA nanoparticles

[0097] This example uses the combination of PHA purification buffer of Example 4 to express and purify antigen-loaded PHA nanoparticles.

[0098] 1. The PhaA-PhaB-PhaC-Afua recombinant strain single colony was inoculated into 2 mL LB medium containing a final concentration of 0.05 mg / mL kanamycin and a final concentration of 0.05 mg / mL chloramphenicol, and cultured at 37°C, 220 rpm for 8 h.

[0099] 2. The above bacterial solution was transferred to a 20 mL conical flask containing 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 0.05 g / mL glucose at a ratio of 1:100, and cultured at 37°C, 220 rpm until the OD 600 =0.6.

[0100] 3. IPTG was added at a final concentration of 0.8 mM, and cultured at 28°C, 200 rpm for 24 h.

[0101] 4. The bacterial solution was centrifuged at 4°C, 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with 4 mL Tris buffer. The cells were broken by ultrasonic, and centrifuged at 4°C, 8000 rpm for 5 min. The supernatant was discarded, and the precipitate was obtained.

[0102] 5, the precipitate was resuspended with 4 mL PHA purification buffer A, centrifuged at 8000 rpm for 5 min at 4°C; the supernatant was discarded, the precipitate was resuspended with 4 mL PHA purification buffer B, incubated at 4°C for 1 h, centrifuged at 8000 rpm for 5 min at 4°C; the supernatant was discarded, the precipitate was resuspended with 4 mL PHA purification buffer C, centrifuged at 8000 rpm for 5 min at 4°C;

[0103] 6, the supernatant was discarded, the precipitate was resuspended with 4 mL Tris buffer; 5x loading buffer was added to the tube and mixed, then placed in boiling water for 10 min and immediately cooled on ice, 20 μL sample was taken, and the content of the PHA nanoparticle loaded with antigen was detected by SDS-PAGE, and the SDS-PAGE diagram is shown in Figure 5 As can be seen from Figure 5 , after purification, the impure proteins were removed, and the pure PHA nanoparticle loaded with antigen was obtained.

[0104] The purified PHA nanoparticle loaded with antigen sample was negatively stained, and observed under a transmission electron microscope, as shown in Figure 6 , in the field of view, it can be seen that it is successfully aggregated into spherical particles with a diameter of 500 nm.

[0105] The prokaryotic expression plasmids pACYC-PhaA-PhaB and pET28a-PhaC-Afua are co-transformed into E. coli Rosetta (DE3) competent cells, and expression is induced by IPTG, the optimal conditions for IPTG induction and the optimal concentration of glucose in the culture medium are obtained, the final concentration of IPTG is 0.8 mM, the induction temperature is 28°C, the induction time is 24 h, and the final concentration of glucose is 0.05 g / mL. The results show that under the optimal induction conditions, the expression of the PHA nanoparticle loaded with antigen in the E. coli Rosetta (DE3) strain can be greatly improved, after obtaining the PHA nanoparticle loaded with antigen, the purification buffer is used for purification, and finally the pure PHA nanoparticle loaded with antigen is obtained, and the present application lays a good foundation for preparing a PHA nanoparticle vaccine and related research.

[0106] Example 6

[0107] Immune response of the PHA nanoparticle loaded with antigen

[0108] Ten mice were immunized with three groups respectively, each group mixed with 20 μg Afua single antigen, 20 μg antigen-loaded PHA nanoparticles (Pha-Afua) and 100 μL PBS 1:1 with adjuvant Montanide™ ISA206 VG (ISA206), the second immunization was performed 14 days after the first immunization, and the mice were bled 14 days after the second immunization, to obtain serum of Pha-Afua group, serum of Afua group and serum of PBS group, the obtained serum was diluted from 100 times to 25600 times, and the Afua specific IgG antibody titer in the serum was determined by indirect ELISA, and the determination steps were as follows:

[0109] (1) The purified protein Afua was added to the carbonate coating buffer and diluted to 800 μL per well, 100 μL was coated in the ELISA plate, and the coating was performed at 4°C overnight.

[0110] (2) The coating solution in the ELISA plate was discarded, and the plate was washed with PBST once.

[0111] (3) 120 μL of blocking solution was added to each well, and the plate was blocked at 37°C for 1 h.

[0112] (4) The blocking solution was discarded, and the plate was washed once with PBST.

[0113] (5) The obtained serum was incubated at dilution ratios 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 was added to each well of the ELISA plate, and the plate was incubated at 37°C for 1 h.

[0114] (6) The liquid in the ELISA plate was discarded, and the plate was washed with PBST for 5 times.

[0115] (7) HRP-labeled goat anti-mouse IgG diluted 1:10000 was added, and the plate was incubated at 37°C for 45 min.

[0116] (8) The liquid in the ELSIA plate was discarded, and the plate was washed with PBST for 5 times.

[0117] (9) 100 μL TMB was added to each well, and the plate was protected from light for 15 min.

[0118] (10) The reaction was terminated by adding the termination solution, and the OD450 was read by the enzyme label instrument.

[0119] (11) The detection results were determined: the OD450 value of the PBS group serum at 1:100 dilution ratio was used as the positive determination standard.

[0120] The results are as follows:Figure 9 As shown, the immunogenicity of PHA nanoparticles loaded with antigen is higher than that of Afua single antigen, and they have a higher antibody dilution, with a serum dilution of up to 12,800 times, which is higher than the serum dilution of 6,400 times after immunization with Afua single antigen, and can significantly improve the antibody response level.

[0121] Example 7

[0122] bactericidal properties of antigen-loaded PHA nanoparticles

[0123] (1) Take the serum from the Pha-Afua group, Afua group and PBS group of Example 6 for later use (immune group serum).

[0124] (2) Gerasimov serotype 5 (strain 0165) was cultured to the logarithmic growth phase, and then its concentration was precisely adjusted to 1×10⁻⁶ using sterile PBS (phosphate buffered saline). 4 CFU / ml (colony forming units per milliliter).

[0125] (3) Take 50 μL of serum from step (1) and 50 μL of *Glassie suis* culture from step (2), and place them in 1.5 mL sterile EP tubes respectively, and incubate them at 37°C for 20 minutes. At the same time, set up serum from healthy mice that have not undergone immunization treatment as a blank control group for subsequent data comparison and analysis.

[0126] (4) Add 100 μL of heparin sodium anticoagulated blood sample from unimmunized healthy mice to each of the above EP tubes, and then incubate at 37°C for 1 hour. During the incubation, gently shake the EP tube once every 10 minutes to ensure that the reaction is sufficient and uniform.

[0127] (5) After diluting each group of samples 10-fold with sterile PBS, 100 μL of the diluted sample was spread onto TSA plates and incubated at 37°C for 24 to 48 hours. Once colony formation stabilized, the colony count on each plate was recorded. Based on the recorded colony count, the bactericidal rate was calculated using the following formula: Bactericidal rate × 100% = (Number of CFUs in blank control serum after 1 hour of incubation - Number of CFUs in immune serum after 1 hour of incubation) / Number of CFUs in blank control serum after 1 hour of incubation. This bactericidal rate was used to assess the difference in bactericidal efficacy between the immune group serum and the blank group serum against the corresponding bacterial strains.

[0128] The results are as follows Figure 10 As shown, compared with the single antigen and PBS groups, the whole blood killing effect of the antigen-loaded PHA nanoparticle group was significantly improved, reaching up to 60%, and it had a certain immune enhancement effect.

[0129] Other parts not described in detail are prior art. Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and other embodiments can be obtained without creativity on the basis of the present embodiment, which are within the protection scope of the present application.

Claims

1. A method for preparing antigen-loaded PHA nanoparticles using an expression plasmid system of antigen-loaded PHA nanoparticles, characterized by: The expression plasmid system comprises a pACYC-PhaA-PhaB plasmid and a pET28a-PhaC-Afua plasmid; wherein, The nucleotide sequences of the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Afua plasmid are shown in SEQ ID NO: 4 and SEQ ID NO: 11, respectively; The pACYC-PhaA-PhaB plasmid is a PhaA and PhaB protein expression frame inserted into the multiple cloning site of the vector pACYCDuet-1, respectively, and driven by two T7 promoters; The pET28a-PhaC-Afua plasmid is a PhaC-vaccine antigen fusion protein expression frame inserted into the multiple cloning site of the vector pET-28a; The vaccine antigen fusion protein is an Afua protein, and the nucleotide sequence of the Afua protein is shown in SEQ ID NO: 6; The nucleotide sequences of the PhaA, PhaB and PhaC are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; The method comprises the following steps: (1) electrotransform the pACYC-PhaA-PhaB plasmid and the pET28a-PhaC-Afua plasmid into E. coli Rosetta (DE3) competent cells, and obtain single colonies of the recombinant strain through resistance screening; (2) inoculate the single colonies into LB medium containing kanamycin and chloramphenicol, and shake culture; (3) The bacteria liquid cultured in step (2) is transferred into LB medium containing kanamycin, chloramphenicol and glucose, and is shaken and cultured until OD 600 = 0.6~0.8; after adding IPTG, it is shaken and cultured; (4) centrifuge the bacterial liquid cultured in step (3) to obtain a precipitate, resuspend the precipitate in Tris buffer, perform ultrasonic cell disruption, and centrifuge to obtain a precipitate; (5) resuspend and centrifuge the precipitate obtained in step (4) with PHA purification buffer A and PHA purification buffer B in the PHA purification buffer combination, then perform incubation, centrifugation to obtain a precipitate, and finally resuspend the precipitate with PHA purification buffer C in the PHA purification buffer combination and centrifuge to obtain a precipitate; (6) resuspend the final precipitate obtained in step (5) in Tris buffer to obtain antigen-loaded PHA nanoparticles; In step (1), the antibiotics for resistance screening are a combination of chloramphenicol with a final concentration of 0.05 mg / mL and kanamycin with a final concentration of 0.05 mg / mL; In step (2), the concentration of kanamycin in the LB medium is 0.05 mg / mL, the concentration of chloramphenicol is 0.05 mg / mL, the culture is performed at 37°C with 220 rpm shaking for 8-10 h; In step (3), the concentration of kanamycin in the LB medium is 0.05 mg / mL, the concentration of chloramphenicol is 0.05 mg / mL, the concentration of glucose is 0.01-0.1 g / mL, 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), the incubation time is 1-1.5 h; The PHA purification buffer A comprises 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 comprises 1.21 g / L of Tris, 1.46 g / L of ethylenediaminetetraacetic acid EDTA, 120 g / L of urea, 20 mL / L Triton X-100, and has a pH of 7.5; The PHA purification buffer C comprises 1.21 g / L of Tris, and has a pH of 7.5; The Tris buffer comprises 2.42 g / L of Tris and 8.76 g / L of NaCl, and has a pH of 7.

4.

2. The antigen-loaded PHA nanoparticle prepared by the method of claim 1.

3. Use of the antigen-loaded PHA nanoparticle of claim 2 in the preparation of a vaccine.

4. Use of the antigen-loaded PHA nanoparticle of claim 2 in the preparation of a product for inhibiting the growth of Lawsonia intracellularis.

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