A PCV3 Cap protein and its application

Abstract

The present invention belongs to the technical field of protein recombinant expression, and provides a PCV3 Cap protein and its application. The amino acid sequence of the PCV3 Cap recombinant protein is shown in SEQ ID NO: 1. The PCV3 Cap recombinant protein can be used to prepare PCV3 detection reagents or PCV3 drugs and vaccines. The present invention optimizes the PCV3 ORF2 sequence, retains the complete protein structure, does not delete the nuclear localization signal sequence, and can express a large amount of complete PCV3 Cap protein at 37°C, thereby reducing the production cost of PCV3 vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of protein recombinant expression, and specifically relates to the expression and application of PCV3 Cap protein. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Porcine circovirus (PCV) has long been a threat to the healthy development of my country's pig industry. In particular, the emergence of PCV3 in recent years has caused huge economic losses to my country's pig industry. Current research shows that PCV is mainly divided into two types and four genotypes. PCV1 is generally considered non-pathogenic, PCV2 mainly causes post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis nephrotic syndrome (PDNS), and PCV3 was first detected in pigs with porcine dermatitis nephrotic syndrome and reproductive disorders in 2016. Subsequently, there have been reports of the disease in countries including Europe, Asia, North America and South America. my country first detected and reported the existence of PCV3 in 2016. After testing, it was found that the virus has always had a high positive rate in the affected pigs. In recent years, studies have shown that PCV3 is pathogenic to pigs of all ages. The clinical symptoms mainly include dermatitis and nephropathy syndrome, reproductive disorders in sows, congenital tremor in piglets, heart disease and multi-system inflammation, etc. Moreover, PCV3 infection alone can cause relatively obvious clinical symptoms, indicating that it has a high pathogenicity and has caused certain economic losses to the breeding industry. However, there is still a shortage of products related to the prevention of PCV3.

[0004] PCV3 virus particles are icosahedral in structure, without envelope, with a diameter of 17-20nm, single-stranded circular DNA, a total length of 2000bp, and contain 3 major open reading frames (ORFs), of which ORF2 is 645bp in length and encodes the major antigen capsid protein (Cap protein) of PCV type 3. At present, there are cases of using eukaryotic cell expression systems to prepare PCV3 virus-like particles, but the use of eukaryotic expression systems to prepare virus-like particles has the disadvantages of complex methods and high costs. Related studies have found that the first 36 aa at the N-terminus of the Cap protein of PCV3 is a potential nuclear localization region. Due to the presence of more arginine, the protein is highly hydrophobic, which easily leads to the expression of the full-length Cap gene in the form of inclusion bodies in the prokaryotic expression system. In order to improve soluble expression, a method of expressing a Cap protein truncation with the nuclear localization amino acids removed using a prokaryotic expression system has been studied. Although this method achieves soluble expression of the Cap protein, the prepared Cap protein truncation has the disadvantages of low expression level and poor immunogenicity. Therefore, the preparation of highly expressed and immunogenic PCV3 Cap protein is the key to the development of PCV3 vaccine. Summary of the invention

[0005] In view of the problems in the prior art, the present invention provides a PCV3 Cap recombinant protein that can be highly expressed in Escherichia coli, contains the full length of PCV3 Cap, and has good immunogenicity.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A PCV3 Cap recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 1.

[0008] A gene encoding a PCV3 Cap recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0009] An expression cassette, vector or Escherichia coli comprising the gene encoding the PCV3 Cap recombinant protein.

[0010] A method for preparing the above-mentioned PCV3 Cap recombinant protein comprises the following steps:

[0011] (1) modifying the amino acid sequence as shown in SEQ ID NO: 1 according to the codon preference of Escherichia coli to synthesize the coding gene;

[0012] (2) Connect the coding gene to the plasmid vector and screen to obtain the recombinant plasmid;

[0013] (3) Transfecting the recombinant plasmid into Escherichia coli and screening to obtain engineered bacteria;

[0014] (4) Inducing the expression of the engineered bacteria to obtain bacterial cells;

[0015] (5) Disrupt the bacterial cells to separate and purify the target protein;

[0016] (6) Renaturing the target protein to obtain the recombinant protein.

[0017] Preferably, the above step (1) is: synthesizing the coding gene according to the nucleotide sequence shown in SEQ ID NO: 2.

[0018] Preferably, the vector in step (2) is pET-30a, pET-28a or pET-32a.

[0019] Preferably, the Escherichia coli in step (3) is BL21.

[0020] Preferably, in step (4), the conditions for inducing expression are: 600 When the absorbance is 0.4-1.4, add 0.4mM-1.0 mM IPTG and induce at 18-40℃ for more than 2 hours. The more preferred induction expression conditions are: when the bacterial solution OD 600 When the absorbance is 0.4-1.0, add 0.4 mM-1.0 mM IPTG and induce at 25℃-40℃ for 4 h-8 h.

[0021] Preferably, in step (5), the separation and purification step is: Ni-NTA resin column purification. Specifically, the separation and purification step is: the cells are broken and then centrifuged, the precipitate is resuspended and denatured with a denaturing solution, then centrifuged with the denaturing solution, the supernatant is mixed with the Ni-NTA resin and eluted with a washing solution and an elution solution in sequence to obtain the target protein.

[0022] Preferably, the denaturing solution is composed of: 25 mM Tris-HCl, 500 mM NaCl, 8 M urea, 0.1 mM PMSF, 1 mM DTT / β-mercaptoethanol;

[0023] The washing solution is composed of: 25 mM Tris-HCl, 500 mM NaCl, 8 M urea, 0.1 mM PMSF, 10-50 mM imidazole;

[0024] The composition of the elution solution is: 25 mM Tris-HCl, 500 mM NaCl, 8 M urea, 0.1 mM PMSF, 100-500 mM imidazole.

[0025] Preferably, in step (6), the renaturation step is: dialyzing the target protein solution in a renaturation solution with a urea content of 6M-0M.

[0026] The composition of the renaturation solution with 6M-0M urea content is: 10 mM Tris pH 8.0, 5 mM EDTA, 6 M-0 M urea, 500 mM NaCl, 50 mM glycine, 5% glycerol, and 0.5 mM PMSF.

[0027] A use of the PCV3 Cap recombinant protein in the preparation of a PCV3 detection reagent or a PCV3 drug.

[0028] A PCV3 detection reagent or PCV3 vaccine prepared with the above PCV3 Cap recombinant protein.

[0029] The present invention has the following advantages:

[0030] The present invention optimizes the PCV3 ORF2 sequence, retains the complete protein structure, does not delete the nuclear localization signal sequence, can express a large amount of complete PCV3 Cap protein at 37°C, and reduces the production cost of PCV3 vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is an amino acid alignment diagram of different designed sequences of PCV3 Cap protein;

[0032] Figure 2 The SDS-PAGE and Western Blot images of different transformants; wherein, lanes 5 and 10 are the supernatant and precipitate of empty vector, respectively; lanes 4 and 9 are the supernatant and precipitate of PCV3 Cap NLS modification, respectively; lanes 2 and 7 are the supernatant and precipitate of PCV3 Cap ΔNLS, respectively; lanes 3 and 8 are the supernatant and precipitate of PCV3 Cap NLS+, respectively; lanes 1 and 6 are the supernatant and precipitate of PCV3 Cap NLS replacement, respectively;

[0033] Figure 3 It is the SDS-PAGE picture of the purified PCV3 Cap protein. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0035] Example 1 Preparation of the gene encoding PCV3 Cap recombinant protein and recombinant vector

[0036] Based on the PCV3 Cap coding sequence of PP506117.1, codon optimization was performed according to the design sequence of PCV3 Cap in Table 1 according to the preference of Escherichia coli, as shown in SEQ ID NO: 2, 4, 6 and 8, respectively. After adding restriction sites, each target gene was synthesized and seamlessly cloned into the XhoI site of the pET-30a vector. After successful verification, the recombinant vector was obtained.

[0037] Table 1 Sequences of different purposes

[0038]

[0039] The Genbank ID of the amino acid sequence of PCV3 Cap protein before modification is WYC12411.1. The amino acid alignment of the proteins expressed by each sequence in Table 1 is as follows: Figure 1 As shown: the modifications were mainly made to the presence or absence and specific sequence of the NLS region. The modified amino acid sequences are shown in SEQ ID NOs: 9, 10, 1 and 11, respectively. Through 3D structure simulation, the amino acid modification in the non-NLS region did not change its secondary structure.

[0040] Example 2 Construction of an engineered bacterium expressing PCV3 Cap recombinant protein

[0041] The recombinant plasmids obtained in Example 1 were heat-shock transformed into BL21 competent cells, coated with LB solid culture medium containing kanamycin, and inverted cultured at 37°C overnight. Positive single colonies were picked and inoculated into LB liquid culture medium containing kanamycin to obtain recombinant bacterial seed liquid.

[0042] The seed liquid of each recombinant strain was inoculated into LB liquid medium containing kanamycin at a ratio of 3% to induce expression at 37°C. 600 When the absorbance value was greater than 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to induce protein expression. The induction conditions were 37°C, 7 h, and IPTG concentration was 0.5 mmol / L. After the induction, the cells were separated by centrifugation. The cell pellet (precipitate) and supernatant (supernatant) were subjected to SDS-PAGE and protein imprint analysis (Western Blot), respectively.

[0043] SDS-PAGE and Western Blot (His monoclonal antibody) of each recombinant strain were as follows Figure 2 As shown: each recombinant bacteria can express the target protein, however, the transformant of the recombinant plasmid No. 4 can significantly increase the expression level of the target protein, and its expression form is mainly endosomal expression.

[0044] The positive transformants of the recombinant plasmid No. 4 were screened for induction conditions. The results showed that: 600When the absorbance is 0.4-1.4, adding IPTG can induce protein expression; when the concentration of IPTG added is 0.4-1.0 mM, induction can be achieved; when the induction time is 2 h-8 h at 18-40℃, the target band will appear.

[0045] Example 3 Purification and preparation of PCV3 Cap recombinant protein

[0046] According to the initial induction method of Example 2, the transformants of the recombinant plasmid No. 4 were induced and cultured, and the cells were collected and washed. The cells were resuspended in a cell disruption solution (25 mM Tris-HCl, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM PMSF, pH 7.5) and ultrasonically disrupted, and centrifuged at 10,000 rpm for 30 min. The precipitate after centrifugation was collected, washed with PBS, and resuspended in an inclusion body denaturation solution (25 mM Tris-HCl, 500 mM NaCl; 8 M urea; 0.1 mM PMSF (phenylmethylsulfonyl fluoride); 1 mM DTT / β-mercaptoethanol, pH 7.5) at a ratio of 1:10 (g / mL), and denatured overnight at 4°C with magnetic stirring.

[0047] Prepare the solution required for purification according to the following formula:

[0048] Inclusion body Ni column purification column equilibrium solution: 25 mM Tris-HCl, 500 mM NaCl; 8 M urea; 0.1 mM PMSF (added before use), pH 7.5;

[0049] Inclusion body Ni column purification wash solution: 25 mM Tris-HCl, 500 mM NaCl; 8 M urea; 0.1 mM PMSF (added before use); 10-50 mM imidazole, pH 7.5;

[0050] Inclusion body Ni column purification eluent: 25 mM Tris-HCl, 500 mM NaCl; 8 M urea; 0.1 mM PMSF (added before use); 100-500 mM imidazole;

[0051] Inclusion body refolding solution 1: 10 mM Tris pH 8.0, 5 mM EDTA, 6 M urea, 500 mM NaCl, 50 mM glycine, 5% glycerol, 0.5 mM PMSF;

[0052] Inclusion body refolding solution 2: 10 mM Tris pH 8.0, 5 mM EDTA, 4 M urea, 500 mM NaCl, 50 mM glycine, 5% glycerol, 0.5 mM PMSF;

[0053] Inclusion body refolding solution 3: 10 mM Tris pH 8.0, 5 mM EDTA, 2 M urea, 500 mM NaCl, 50 mM glycine, 5% glycerol, 0.5 mM PMSF;

[0054] Inclusion body refolding solution 4: 10 mM Tris pH 8.0, 5 mM EDTA, 1 M urea, 500 mM NaCl, 50 mM glycine, 5% glycerol, 0.5 mM PMSF;

[0055] Inclusion body refolding solution 5: 10 mM Tris pH 8.0, 5 mM EDTA, 500 mM NaCl, 50 mM glycine, 5% glycerol, 0.5 mM PMSF.

[0056] Purify the target protein as follows:

[0057] The denatured precipitate was centrifuged at 10,000 rpm for 30 min, and the supernatant was retained for later use. The Ni-NTA resin was loaded into the chromatography column. After the 20% ethanol solution in the column flowed out, 5 times the column volume of the equilibration buffer was slowly added to the column to equilibrate the chromatography column. After the equilibration was completed, the supernatant was filtered into a clean centrifuge tube using a 0.22 μm bacterial filter, mixed with the Ni-NTA resin and shaken on a shaker at 4°C for 2 h. After the mixing was completed, the mixed solution was added to the chromatography column. After the mixed solution flowed out, the nickel chromatography column was equilibrated again with 5 times the column volume of the equilibration buffer, and then the impurity protein was washed away with the washing solution, and the target protein was eluted with the elution solution. After the target protein was eluted, it was dialyzed in the inclusion body refolding solution 1-5 in sequence using only a 10 kDa dialysis bag, and each refolding solution was dialyzed for 3 h.

[0058] After dialysis, the solution was subjected to SDS-PAGE detection:

[0059] (1) Prepare protein gel using the PAGE gel rapid preparation kit and follow the instructions;

[0060] (2) Wash the precipitate twice with PBS. After the second wash, centrifuge and discard the supernatant. Add PBS equal to the volume of the bacterial solution before the disruption to resuspend the precipitate. Pipette 80 μL of the precipitate resuspension into 1.5 mL centrifuge tubes and add 20 μL of 5× Loading Buffer to each tube. Mix well and heat in a boiling water bath for 10 min to ensure that the protein in the sample is fully denatured. Centrifuge immediately after heating.

[0061] (3) PageRuler TMPrestained protein molecular weight standards (ThermoFisher, Cat. 26616) and samples were added to the gel wells, 5 μL of standard and 20 μL of sample per well, and the power supply was turned on and the voltage was adjusted to 150 V. Constant voltage electrophoresis was performed for 80 min.

[0062] (4) After the electrophoresis is completed, the gel is cut and stained with a rapid gel staining solution on a shaker for 60 min; after staining, the gel is developed using a gel imager.

[0063] SDS-PAGE results are as follows Figure 3 As shown, PCV3 Cap protein has been purified to a high degree of purity.

[0064] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A PCV3 Cap recombinant protein, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding a PCV3 Cap recombinant protein, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

2.

3. An expression cassette, vector or Escherichia coli comprising the gene encoding the PCV3 Cap recombinant protein according to claim 2.

4. A method for preparing the PCV3 Cap recombinant protein according to claim 1, characterized in that: The following steps are involved: (1) modifying the amino acid sequence as shown in SEQ ID NO: 1 according to the codon preference of Escherichia coli to synthesize the coding gene; (2) Connect the coding gene to the plasmid vector and screen to obtain the recombinant plasmid; (3) Transfecting the recombinant plasmid into Escherichia coli and screening to obtain engineered bacteria; (4) Inducing the expression of the engineered bacteria to obtain bacterial cells; (5) Disrupt the bacterial cells, separate and purify the target protein; (6) Renaturing the target protein to obtain the recombinant protein.

5. The preparation method according to claim 4, characterized in that: Step (1) is: synthesizing the coding gene according to the nucleotide sequence shown in SEQ ID NO:

2.

6. The preparation method according to claim 4, characterized in that: The vector in step (2) is pET-30a, pET-28a or pET-32a; The Escherichia coli in step (3) is BL21.

7. The preparation method according to claim 4, characterized in that: In step (4), the conditions for inducing expression are: 600 When the absorbance is 0.4-1.4, add 0.4 mM-1.0 mM IPTG and induce at 18-40°C for more than 2 h; In step (5), the separation and purification steps are: Ni-NTA resin column purification; In step (6), the renaturation step is: dialyzing the target protein solution in a renaturation solution with a 6M-0M urea content.

8. The preparation method according to claim 4, characterized in that: In step (4), the induction expression conditions are: 600 When the absorbance is 0.4-1.0, add 0.4 mM-1.0 mM IPTG and induce at 25℃-40℃ for 4 h-8 h.

9. Use of the PCV3 Cap recombinant protein according to claim 1 in the preparation of a PCV3 detection reagent or a PCV3 drug.

10. A PCV3 detection reagent or PCV3 vaccine prepared using the PCV3 Cap recombinant protein according to claim 1.

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