NL2025015B1

NL2025015B1 - Method for Efficiently Expressing PCV2 Cap and PCV3 Cap Fusion Protein

Info

Publication number: NL2025015B1
Application number: NL2025015A
Authority: NL
Inventors: Shi Jianli; Li Chen; Xu Shaojian; Li Jun; Wu Xiaoyan; Wang Shuo; Peng Zhe
Original assignee: Inst Animal Science & Veterinary Medicine Shandong Aas
Priority date: 2020-01-17
Filing date: 2020-02-28
Publication date: 2022-03-18
Also published as: NL2025015A; CN111187353A; CN111187353B

Abstract

The present invention provides a method for efficiently expressing a PCV2 Cap and PCV3 Cap fusion protein. First, a recombinant baculovirus that efficiently expresses the PCV2 Cap protein and the PCV3 Cap protein is constructed: the nucleotide sequence of the PCV2 Cap protein with its nuclear localization signal truncated is linked to the nucleotide sequence of the PCV3 Cap protein through a hydrophobic flexible protein linker sequence, and a honeybee melittin signal peptide is added to the N-terminal of the PCV2 Cap protein sequence and the PCV3 Cap protein sequence to promote their secretion and expression. The proteins expressed by sf9 insect cells infected with the recombinant positive baculovirus have unden/vent a variety of post-translational modifications, are close to natural virus-encoded proteins, and have high biological activity. Moreover, the baculovirus is highly species-specific, only infects Insect cells and are non-infectious to vertebrate cells, and their expression products are safe and reliable and can be used in subsequent experiments after simple processing. Therefore, for expression of PCV2 Cap and PCV3 Cap fusion proteins, the method of the present invention is safer and more effective than traditional methods.

Description

Method for Efficiently Expressing PCV2 Cap and PCV3 Cap Fusion Protein Technical Field

[0001] The present invention pertains to the field of molecular biology, and particularly relates to a method for efficiently expressing a PCV2 Cap and PCV3 Cap fusion protein. Background

[0002] Porcine circovirus (PCV) mainly infects weaned piglets and growing pigs, and can cause a variety of porcine circovirus-associated diseases, posing a huge threat to the healthy development of the pig industry. According to the difference in antigenicity, pathogenicity and genetic homology, PCV is divided into three genotypes, including Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), and Porcine circovirus 3 (PCV3). It is known that PCV1 is non-pathogenic, while PCV2 is highly pathogenic and can cause a variety of Porcine circovirus-associated diseases (PCVADs), the most influential of which is Postweaning multisystemic wasting syndrome (PMWS). This disease is widespread in China and even the world, and has become a common and frequently-occurring disease, which is often associated with infections caused by other pathogens, causing huge losses to the pig industry. PCV3 infection can cause Porcine dermatitis and nephropathy syndrome, reproductive disorders, and inflammations in the heart and multiple systems. The diseased sows have reduced conception rate, increased miscarriage rate, and may deliver dead fetuses and mummified fetuses of different gestational ages; and the diseased piglets have dysfunctions in multiple organs and systems, seriously affecting the development of the pig industry worldwide. The amino acid homology of PCV2 and PCV3 Cap proteins is only 30%, and the possibility of cross-immunity protection between the two is low. At present, there are no vaccines and drugs that can prevent and control both PCV2 and PCV3. Therefore, the development of a safe and effective vaccine to prevent both PCV2 and PCV3 is of great significance for the prevention and control of PCV infection.

[0003]At present, it is common to use E. coli to optimize codon to express fusion proteins. Due to its obvious advantages such as simple and economical culture conditions, rapid growth and reproduction, and availability of a variety of genetic modification tools and host species, it has always been the preferred system for foreign protein expression. However, in many practical applications, the production efficiency and product quality of fusion proteins vary greatly depending on the specific system. In some systems, the protein expression is inefficient, the product concentration is low, product collection and purification are difficult, and it is hard to meet the needs of practical production and application. In other systems, although foreign proteins can be expressed at high levels, a large number of inactive inclusion bodies are formed and few soluble proteins with biological activity are available, and the active expression is even completely undetectable, causing waste of material and energy. Subsequent processing of proteins involves in vitro renaturation and purification of inactive inclusion body proteins. However, in vitro renaturation increases the complexity of the actual production process. Also, it has high production cost and low yield, cannot be widely promoted and applied.

[0004] Production of PCV2 Cap-PCV3 Cap fusion protein by CHO cells features high expression, but there are also many disadvantages. The current technique of using CHO cells to produce the protein of interest cannot meet the development and production requirements of biological drugs, and there are still many problems in its upstream production, such as: (1) Some glycosylated expression products are unstable and difficult to purify; (2) The upstream construction of recombination CHO cells is disconnected from the downstream purification in that the upstream construction focuses on high-efficiency expression and seldom considers the efficient extraction, that is isolation and purification, of the product; (3) Recombinant cell culture is expensive with low level of automation. Currently, CHO cells produce PCV2 Cap-PCV3 Cap fusion proteins with an expression level of 0.8 mg/mL, which is the maximum yield obtained after scale-up culture, optimization of culture conditions, feeding time and feeding amount, and affinity chromatography and normal saline dialysis. It still has the problems of low expression level, and complicated production and purification processes when applied to vaccine production. Although there are techniques using recombinant baculovirus to express PCV2 ORF2 gene with expression levels of about 0.1-0.4 mg/mL in China, the low-level expression of recombinant proteins has become an obstacle to further development and utilization of these techniques. Summary

[0005] In view of the current problems of low PCV2 Cap-PCV3 Cap fusion protein expression and complicated purification process, the present invention provides a new method for expressing PCV2 Cap and PCV3 Cap fusion proteins. In this method, the virus purification and identification steps are simple, and the protein, after multiple post- translational modification, is close to the natural virus-encoded protein and has high biological activity.

[0006] To achieve the above objective, the present invention adopts the following technical solutions.

[0007]A PCV2 Cap and PCV3 Cap fusion protein, which comprises a truncated PCV2 Cap protein segment, a linker sequence, a PCV3 Cap protein segment and a honeybee melittin signal peptide region. The truncated PCV2 Cap protein segment is a PCV2 Cap protein with its nuclear localization signal removed.

[0008] The linker sequence is preferably a hydrophobic flexible peptide chain. The linker sequence is as set forth in SEQ ID NO: 1.

[0009] A nucleotide sequence of the abovementioned fusion protein. Preferably, the nucleotide sequence comprises a sequence as set forth in SEQ ID NO: 2.

[0010] The nucleotide sequence may further include a restriction site for a restriction enzyme.

[0011] A vector, recombinant bacmid and recombinant baculovirus comprising the aforementioned nucleotide sequence.

[0012] Preferably, there is a dual copy of the nucleotide sequence in the vector, recombinant bacmid or recombinant baculovirus.

[0013] Preferably, the vector is a pFastBac™ plasmid; more preferably, a pFastBac™ Dual plasmid.

[0014] Use of the vector, recombinant bacmid, or recombinant baculovirus in expressing a PCV2 Cap and PCV3 Cap fusion protein.

[0015] A method for preparing the fusion protein, which comprises the following steps: transfecting competent cells comprising a bacmid skeleton with a vector comprising the abovementioned nucleotide sequence to obtain recombinant bacmids; transfecting insect host cells with the recombinant bacmids to obtain a supernatant containing a recombinant baculovirus; inoculating insect host cells with the supernatant containing the recombinant baculovirus to obtain a supernatant after culturing; subjecting the supernatant to isolation and purification to obtain a PCV2 Cap and PCV3 Cap fusion 5 protein.

[0016] The insect host cell is selected from Sf9 cells, Sf21 cells, Hi-5 cells or S2 cells.

[0017] The MOI of the inoculation is 0.1-1.0, the density of the insect host cells is 2.5 x 108 cells/mL, and the culture time is 1-5 days after inoculation.

[0018] The isolation and purification can be implemented by conventional protein purification methods, such as centrifugation, dialysis, ultrafiltration, affinity column chromatography, to remove impurities such as cells or cell residues, miscellaneous proteins, and components of the culture medium.

[0019] Specifically, the following steps are included: (1) inserting the sequence as set forth in SEQ ID NO: 2 into the two promoters of the pFastBacTM Dual plasmid to obtain a recombinant plasmid pFD-H-2Cap-3Cap; (2) transforming the recombinant plasmid pFD-H-2Cap-3Cap into DH10Bac E. coli competent cells, and screening and extracting to obtain a recombinant bacmid rBacmid- H-2Cap-3Cap; (3) transfecting the recombinant bacmid rBacmid-H-2Cap-3Cap into insect host cells to culture to obtain a supernatant containing a recombinant baculovirus rpFB-H-2Cap- 3Cap; (4) inoculating insect host cells with the supernatant of step (3) to culture to obtain a supernatant containing a PCV2 Cap and PCV3 Cap fusion protein, and subjecting the supernatant to isolation and purification to obtain the PCV2 Cap and PCV3 Cap fusion protein.

[0020] Use of the above fusion protein in in the preparation of a porcine circovirus vaccine, antibody, antigen, or detection kit.

[0021] A porcine circovirus vaccine containing the abovementioned fusion protein.

[0022] The present invention has the following advantages: The present invention constructs a recombinant baculovirus that efficiently expresses PCV2Cap protein and PCV3Cap protein. Insect baculovirus expression system can speed up the expression of foreign proteins, not only make foreign proteins have high expression efficiency, but also make them exist in active soluble form as far as possible, shortening the time for later purification of proteins, reducing the production cost, and optimizing the expression of the fusion protein. In addition, insect cells grow in suspension, are easy to scale up, and are conducive to large-scale expression of recombinant proteins. They have important economic and social value for developing PCV2 and PCV3 Cap protein vaccines.

[0023] The recombinant baculovirus of the present invention connects the nucleotide sequence of the PCV2Cap protein with the nuclear localization signal truncated to the nucleotide sequence of the PCV3Cap protein through a hydrophobic flexible protein linker sequence. A honeybee melittin signal peptide sequence is added to the N- terminal of the protein sequence of the PCV2Cap protein and the PCV3Cap protein to promote their secretion and expression. The Bac-to-Bac baculovirus expression system uses baculovirus shuttle vector technology, which greatly shortens the time for virus purification and identification. The proteins expressed by sf9 insect cells infected with recombinant positive baculovirus have underwent a variety of post-translational modifications, are close to the natural virus-encoded protein, and have high biological activity. Moreover, baculovirus is highly species-specific, only infects insect cells, and is non-infectious to vertebrate cells. Its expression products are safe and reliable, and can be used in subsequent experiments after simple processing. Therefore, using this system to simultaneously express the PCV2 Cap protein and the PCV3 Cap protein, and to carry out subsequent purification and vaccine development, is safer and more effective than traditional methods. Compared with other methods for producing fusion proteins using the baculovirus system, this method is simple to operate and needs only simple centrifugation for purification, which saves the cost and time of later scale-up culture. The expression level in this method and is 2-8 times that in other methods, which greatly increases the expression efficiency and is beneficial to the subsequent product development. Brief Description of Figures

[0024] Fig. 1 is a gel electropherogram of the gene of interest; Fig. 2 is a gel electropherogram of the recombinant plasmid after digestion; Fig. 3 is a schematic structural diagram of the recombinant plasmid pF D-H-2Cap-3Cap; Fig. 4 shows the blue-white selection of DH10Bac containing the recombinant plasmid pFD-H-2Cap-3Cap; Fig. 5 shows the microscopic images of normal sf9 cells and those transfected with recombinant baculovirus;

Fig. 6 shows indirect immunofluorescence identification of sf9 cells transfected with recombinant baculovirus; Fig. 7 shows the SDS-PAGE electrophoretograms of the supernatants of different samples; Fig. 8 shows the Western-Blot pictures of the supernatants of different samples; Fig. 9 shows the SDS-PAGE electrophoretograms of the supernatants of cells inoculated at different MOIS. Detailed Description

[0025] The present invention is further described below with reference to examples and accompanying drawings, but is not limited by the following examples.

[0026] Example 1 Construction of baculovirus

1. Acquisition of the gene of interest With reference to "The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province" (Zheng S, Wu X, Zhang L, et al. The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province [J]. Transboundary and Emerging Diseases, 2017.), the plasmids pEASY-Blunt-PCV2 and pEASY-Blunt- PCV3 of porcine circovirus type 2 and 3 were constructed, and stored at -80°C for future use.

[0027] Table 1 PCR primer sequences of the gene of interest Primer name Primer sequence Restriction

I Ph-HBM 5-TCCCACCATCGGGCGCGGATCCATGAAA | BamH | | remeromeemsscoremmies |

Co] CTATGCGATGACGTATCCAAGGAGG-3 | Linker1-PCV2Cap R 5-GCTTCCTCCTCCTCCGCTTCCTCCTCCT CCTTAGGGTTAAGTGGGGGGT-3 Linker2-PCV3Cap-phF | 5-TCCTCCTCCTCCGCTTCCTCCTCCTCCAT GAGACACAGAGCTA-3 PCV3CapR Ph 5-ACAAGCTTGTCGAGACTGCAGGAGAACG | Pst | GACTTGTAG-3 p10-HBM 5-ATCCCAACTCCATAAGCATGCATGAAATT | Sphl CTTAGTCAACGTTGCCCTTGTTTTTATG-3 PCV3CapR-p10 5-GCCTCCCCCATCTCCCGGTACCGAGAAC | Kpn | GGACTTGTAG-3

[0028] The primers were synthesized according to Table 1. The PCV2 Cap base sequence was amplified using pEASY-Blunt-PCV2 as a template, HBM-PCV2Cap F and Linker1-PCV2Cap R as primers, and the amplified product was recovered using a gel recovery kit and named 2Cap. The PCV3 Cap base sequence was amplified using pEASY-Blunt-PCV3 as a template and Linker2-PCV3Cap-phF and PCV3Cap-phR as primers, and the amplified product was recovered using a gel recovery kit and named 3Cap. Then, the recombinant sequence was amplified using the recovered products as templates, and Ph-HBM and PCV3CapR PH; p10-HBM and PCV3CapR-p10 as primers, respectively. The resultant sequences was named as Ph-H-2Cap-3Cap and p10-H- 2Cap-3Cap, with their gene sequence structure being as follows: vector homologous arm sequence-restriction site-PCV2 Cap-Linker-PCV3 Cap-honeybee melittin signal peptide- restriction site e-vector homologous arm sequence, with the electropherogram as shown in Fig. 1, where 1 represents Marker, 2 and 3 both represent Ph-H-2Cap- 3Cap, and 4 and 5 both represent p10-H-2Cap-3Cap. According to the electropherogram, a fragment of 1495bp in size was obtained, which conformed to the size of the recombination sequence. The PCR product was recovered using a gel recovery kit.

[0029] The PCR system and reaction conditions are as follows: 50 x PCR system: Ingredient Volume Template plasmid 1 uL Primer Star mix 25ulL HBM-PCV2Cap F/Linker2-PCV3Cap-phF 1.5 ul Linker1-PCV2Cap R/PCV3Cap-phR 1.5 ul ddH,0 21 pL Total volume 50 uL PCR conditions: Temperature Time 98h 5 min 98h 30s 550 30s 35 cycles 720 30s 72 7 min

[0030] 2. Construction of recombinant plasmid (1) The sequence downstream of the Ph promoter of pFastBacTM Dual vector was digested with BamH | and Pst |, and the digested linear vector was recovered using a gel recovery Kit. The following system was configured using a seamless ligation kit and the linear vector was connected to the recombinant sequence Ph-H-2Cap-3Cap: Component Amount Linearized vector 80ng Recombination fragment 100ng 5xCE Il Buffer 4ul

Exnase lI 2uL ddH,0 made up to 20uL

(2) DH5a competent cells were placed on ice, to which the ligation product was added at an amount of one-tenth by volume of the cells.

The mixture was mixed gently, allowed to stand still on ice for 30 min, placed in a thermostatic water bath at 42°C for 90 s, and then in an ice bath for 2-3 min.

Next, 900 pL of non-resistant LB liquid medium pre-heated to 37°C was added thereto on a clean bench.

The mixture was incubated on a constant temperature shaker at 37°C for 1 h, and centrifuged at 12000 rpm for 1 min. 700 pL of the resultant supernatant was discarded, and the pellet was resuspended. 50 UL of the resuspended bacterial solution was taken and coated onto Amp + resistant LB solid medium to culture in an inverted manner at 37°C for 12 h.

Monoclones were picked to culture in Amp + LB medium at 37°C with shaking for 12 h, and plasmids were extracted using a plasmid mini-kit.

The extracted plasmid was identified by double- enzyme digestion and sent to Shanghai Sangon Biotech Company for sequencing and identification, and the correct recombinant plasmid was stored for future use (3) The gene sequence downstream of p10 promoter of the recombinant plasmid obtained in step (2) was double-digested with Sph | and Kpn I.

With the same method, the linear plasmid and p10-H-2Cap-3Cap recombinant plasmid were ligated with DNA ligase, transformed, and plasmid was extracted and identified.

The obtained plasmid was verified by double enzyme digestion, and the results of nucleic acid electrophoresis was shown in Fig. 2, where 1 represents Marker, both 2 and 3 represent those after double digestion with BamH | and Pst |, and both 4 and 5 represents those after double digestion with Sph | and Kpn I.

It can be seen from the pictures that the size of both the vector fragment and the fragment of interest is consistent with what's expected,

indicating that the recombinant fragment has been cloned into the pFastBac Dual vector correctly. Thus, a donor plasmid containing double recombinant sequences was constructed and named pFD-H-2Cap-3Cap. The structure of the recombinant plasmid is shown in Fig. 3.

[0031] 3. Preparation of recombinant bacmids The correctly identified recombinant plasmid pFD-H-2Cap-3Cap was transformed into DH10Bac competent cells, and then coated onto the three-antibiotic blue-white LB solid medium to culture at 37°C for 48 h in an inverted manner (see Fig. 4). The white colonies were picked and streaked onto the blue-white screening LB solid medium for purification and screening. Monoclonal white colonies were again picked and inoculated into the three-antibiotic LB liquid medium to culture with shaking at 37°C for 16 h. The recombinant bacmid rBacmid-H-2Cap-3Cap was extracted from the bacterial solution. PCR amplification was performed according to the primer sequences in Table 2. The electrophoresis of the product showed that a fragment with a size corresponding to that of the fragment of interest was successfully amplified. Correct sequencing proved that the recombinant plasmid was successfully constructed.

[0032] Table 2 PCR primers for recombinant plasmid identification

[0033]4. Rescue of recombinant baculovirus sf9 cells were cultured in sf900 II medium containing 10% fetal bovine serum (FBS) and 1% double-antibiotics at 27°C, and the cells were passaged at 48-72 h. Before transfection, the cells were plated in a 6-well plate to ensure that the cells were in good growth and in the logarithmic growth phase, and the cell culture density (1 x 10° - 2 x 10° cells/mL) was controlled by a cell counter. The rBacmid-H-2Cap-3Cap recombinant bacmid DNA transfection was performed by using the Cellfectin II Reagent as the transfection reagent, and culture was carried out at 27°C for 72-96 h. The cell culture supernatant was harvested, and centrifuged at 4°C and 3000 rpm for 30 min to remove cell debris, thereby obtaining the P1 generation, and the recombinant baculovirus was named rpFB-H-2Cap-3Cap. Fig. 5 shows a photomicrograph of sf9 cells transfected with the recombinant plasmid and normal sf9 cells after 96 h of culture. The results show that compared with the normal adherent sf9 cells, the transfected sf9 cells were observed under the microscope to become expanded and round, with the nucleus filling the whole cell, and finally become dissociated or suffer other cytopathies.

[0034] Then, the P1 generation of recombinant baculovirus was passaged in sf9 cells again. The specific method is as follows: sf cells were cultured at 27°C for 12 h, inoculated with the baculovirus at a concentration of 1%, and cultured at 27°C, and the supernatant was harvested at 72-96 h. Recombinant baculovirus was blindly passaged to the P4 generation, and stored at 4°C in the dark, and at -80°C for long-term storage.

[0035] 5. Identification of recombinant baculovirus

5.1 Recombinant baculovirus titer determination The P2 generation virus was subjected to culture expansion and the titer of the recombinant baculovirus was measured by the TCIDso method, the cells were observed for cytopathies under microscope and the virus titer was calculated according to the formulas below:

Proportional distance = (% next above 50% cytopathy - 50%) / (% next above 50% cytopathy -% next below 50% cytopathy) Log (TCIDso) = proportional distance x difference between logarithm of dilution + Log (dilution next above 50% cytopathy).

[0036] Table 3 P2 virus titer determination Viral Number of Number of Number of Number of Percentage of dilution wells with cytopathy- . wells with cytopathy | free wells | Wells with | cytopathy- | cytopathy cytopathy free wells fo” 8 jo 4 0 | 10 8 jo 0 0 |] 10 102 18 jo fs jo p10 104 18 jo {24 JO 0000 100 | 10 5 139 0000/4 0 6 072 fe a wae fof 2 8 2% Ar 109 Jo 18 jo [24 | 0 The results shown in Table 3 indicate that the recombinant baculovirus was successfully rescued, and the titer of the P2 generation virus was determined to be 1 x 1085 IFU/mL.

[0037]5.2 Indirect immunofluorescence identification The P1 generation recombinant baculovirus rBac-H-2Cap-3Cap was passaged for 3 10 generations blindly, and the genome was extracted for PCR identification. The P4 generation was inoculated into adherent sf9 cells to further culture for 48 h. Indirect immunofluorescence identification was performed as follows: The fourth-generation virus solution was inoculated into a 6-well plate according to the virus culture method, 100 pL per well; after 72 h, the nutrient solution was discarded and the plate was washed 3 times with PBS; 4% pre-chilled paraformaldehyde stationary liquid was added to each well to fix for 30min, and the wells were washed 3 times with PBS, and allowed to stand still for 2 min each time; 200 pL of 0.2% TritonX-100 was added to each well, and the wells were allowed to stand still for 15min to permeate the cells; 1% BSA solution was added in each well to block for 30 min; and the wells were washed 3 times with PBS and allowed to stand still for 2 min each time; a mouse monoclonal antibody against PCV2 Cap protein and a mouse monoclonal antibody against PCV3 Cap protein diluted at 1: 200 were added, and the wells were incubated at 37°C for 1 h, washed with PBS 3 times, and allowed to stand still for 2 min each time; Alexa Fluor® 488-labeled goat anti-mouse IgG (H + L) secondary antibody diluted at 1: 5000 was added in each well, and the wells were incubated for 1 h at room temperature, washed 3 times with PBS, and allowed to stand still for 2 min each time; observation was conducted using an inverted fluorescence microscope.

[0038] The results are shown in Fig. 6, where A is an image showing virus-exposed cells with a PCV2 Cap monoclonal antibody, B is an image showing virus-exposed cells with a PCV3 Cap monoclonal antibody, and C and D are images of uninfected cells. The results show that compared with the normal cell control, the cells inoculated with baculovirus rBac-H-2Cap-3Cap were observed to have clear green fluorescence under a fluorescence microscope, indicating that rBac-H-2Cap-3Cap can specifically bind to the corresponding anti-PCV2 Cap mouse serum and anti-PCV3 Cap mouse serum, and recombinant baculovirus rBac-H-2Cap-3Cap was successfully constructed.

[0039] 5.3 SDS-PAGE and Western-Blot identification Supernatants from 72 h and 96 h of culture of cells inoculated with the P2 generation virus and cell lysates of uninfected sf9 cells were identified using anti-mouse monoclonal antibodies by SDS-PAGE and Western-Blot.

[0040] 5.3.1 SDS-PAGE analysis The sample supernatant was taken, 20 uL of sample per well. The voltage was adjusted to 90 V to start electrophoresis. After the sample entered the separation gel, the voltage was increased to 120 V to continue electrophoresis. When the bromophenol blue indicator reached the bottom, electrophoresis was stopped. A part of the separation gel was cut off, to which an appropriate amount of a Coomassie Brilliant Blue staining solution was added to stain with slow shaking for 1-2 h. Then, the stained gel was taken out, to which an appropriate amount of destaining solution was added to destain with slow shaking until the destaining solution became colorless. Photographs were taken and the experimental results were analyzed. The results are shown in Fig. 7, where 1 represents the protein marker, 2 represents the supernatant cultured for 72 h, 3 represents the supernatant cultured for 96 h, and 4 represents the cell lysate. This shows that the recombinant baculovirus rBac-H-2Cap-3Cap was successfully constructed and it is capable of expressing a foreign protein with a size of about 60 kDa.

[0041]5.3.2 Western-Blot analysis The collected recombinant baculovirus rBac-H-2Cap-3Cap stock solution was mixed with 4 x SDS loading buffer at a ratio of 4: 1, and the mixture was placed in a water bath to boil for 10 min; 1 x protein electrophoresis buffer was added to the electrophoresis tank, 20 yL of protein sample and 7 pL of pre-stained Marker were added to the gel well, and an electrophoresis apparatus was connected; the initial voltage was set to 90 V to start electrophoresis. After the bromophenol blue band was concentrated and entered the separation gel (over about 30 min), the voltage was adjusted to 120 V. When the bromophenol blue band reached the bottom of the separation gel (over about 1.5 h-2 h),

the electrophoresis was stopped; thereafter, based on the size of the gel, two pieces of thick filter paper and one piece of NC membrane were clipped out, and were both fully immersed in the transfer buffer to become moist.

They were placed in the of order of thick filter paper - NC membrane - protein gel - thick filter paper from bottom to top.

After the stacking was completed, the air bubbles between the filter paper, the NC membrane and the gel were removed using a glass rod.

The stacking was placed in a semi-dry type electric transfer instrument, the power was turned on (15 V) for 30 min; the NC membrane was then taken out, put into a blocking buffer and then placed onto a shaker to shake gently at room temperature for 2 h for blocking; then, the blocking solution was discarded, and the NC membrane was washed quickly with TBST 5 times, 5 min each time.

After the TBST was discarded, the monoclonal antibody was diluted at 1000-fold with Western primary antibody diluent, shaken gently on the shaker for 2 h, and then incubated in a chromatography refrigerator at 4°C overnight; the NC membrane was quickly washed with TBST for 5 times, 5 min each time; the HRP- labeled goat anti-mouse IgG antibody was diluted at 5000-fold with the blocking buffer, shaken gently on the shaker for 1 h; after the secondary antibody was discarded, the NC membrane was washed quickly with TBST 5 times, 5 min each time.

The NC membrane was developed with ECL luminescent liquid and exposed through the ChemiDoc MP gel imaging system.

The results are shown in Fig. 8, where Fig. 8-(a) is the picture of anti-PCV2 Cap mouse serum; Fig. 8-(b) is the picture of anti-PCV3 Cap mouse serum.

In Fig. 8, M represents the protein Marker, both 1 and 2 represent the samples, and 3 represents the blank control.

The results show that the foreign protein expressed by baculovirus is a fusion protein of PCV2 Cap and PCV3 Cap and is immunogenic.

[0042] Example 2 Expression of fusion proteins Recombinant P2 baculovirus was inoculated into sf9 cells with a density of 2.5 x 10° cells/mL at MOI = 1, 0.5, and 0.1 respectively. 200 pL of cell supernatant was collected every 24 h for 5 consecutive days. The protein samples were centrifuged and purified as follows: 1) Centrifuging the collected supernatant at 4°C and 5000 x g for 30 min to remove cell debris and impurities, and collecting the culture supernatant; 2) Centrifuging the cell supernatant at 30000 rpm for 1 h, harvesting the pellet, and then resuspending the pellet in sterile PBS; 3) Adding the pellet resuspended in sterile PBS to the upper surface of a sucrose solution with a density gradient of 10% - 30% - 50%, centrifuging them at 35000 rpm for

1.5 h for purification; carefully collecting the white flocculent strip between the 30% -50% sucrose layers; resuspending the strip in PBS, centrifuging it at 30000 rpm for 1.5 h to remove sucrose, and collecting the pellet; resuspending the pellet in 2 mL of sterile PBS to store at -80°C until use. The SDS-PAGE electrophoresis analysis was performed according to the method described in Example 5.3 in Example 1. The total protein concentration was determined by the BCA method: 1) Preparation of working solution: On the basis of the standard and the number of samples, the BCA working solution was prepared from 50 volumes of BCA reagent plus 1 volume of Cu reagent (50: 1) with thorough mixing (the solution may become turbid during mixing, and this phenomena will disappear after thorough mixing). The BCA working solution is stable within 24 h at room temperature; 2) Dilution of the standard: 10 pL of the BSA standard was measured and diluted to 100 UL with PBS to reach a final concentration of 0.5 mg/mL. The standard was added at 0, 2,4,6, 8, 12, 16, 20 pL respectively into the protein standard wells of a 96-well plate, and PBS was added to make up to 20 pL; 3) The sample was diluted at 2-fold, 4-fold and 8-fold, 20 pL of each was added to the sample well of the 96-well plate, and the sample point was made to fall behind 1/2 of the standard line as far as possible; 4) 200 pL of the BCA working solution was added to each well to leave at 37°C for 15- 30 min. Ass2nm Was measured using a microplate reader, and the protein concentration was calculated according to the standard curve.

[0043] The total protein content of each sample was determined by the BCA method, and a standard curve was plotted according to the standard to obtain a linear regression equation of c = 0.511A - 0.046, with a correlation coefficient of r = 0.9001. The total protein concentration of the sample was calculated from the absorbance value, and the total protein contents at different time for different MOI values are shown in Table 4: Table 4 Total protein content expressed at different time for different MOI values Content 24 48 72 120

[0044] The ratio of the protein of interest to the total protein was analyzed with reference to the image analysis system to obtain the specific expression of the protein of interest.

The electrophoresis results of the SDS-PAGE sample are shown in Fig. 9. The results show that when the virus was inoculated at different MOls, sf9 cells began to express the recombinant protein 24 h after infection; and with the increase of the culture time, the expression of the protein of interest increased, peaked at 72-96 h, and then stabilized. Through analysis of the content of the protein of interest by calculation of the ratio of the target band to the total band in conjunction with the analysis software, it can be known that when MOI = 0.1, the cell density of sf9 cells in the later stage increased compared to the initial density, the infected cells showed significant advantages in the expression level of recombinant protein, the band of the protein of interest accounted for 38% of the total protein on average, with an average expression level of about 0.7 mg/mL, and the highest expression level can reach 0.85 mg/mL. When MOI = 0.5, the band of the protein of interest accounted for 38% of the total protein on average, and the average expression level of protein was about 0.6 mg/mL. When MOI = 1, the band of the protein of interest accounted for 29% of the total protein on average, and the average expression of the protein of interest was about 0.4 mg/mL. On the fifth day of infection, a large number of sf9 cells were broken, and the cell survival rate was less than 50%. At that time, the cell density was lower than the previous cell density. It was obvious that the cells cast off and died due to cell lysis, and the total protein content increased, but the protein of interest was significantly degraded and had a reduced content. Therefore, the optimal conditions for obtaining the fusion protein are: MOI = 0.1, sf9 cells at 2.5 x 10° cells/mL are inoculated, and the fusion protein is harvested 4 days after inoculation, with an average expression of 0.7 mg/mL and a maximum expression of 0.85 mg/mL.

SEQLTXT

SEQUENCE LISTING <110> Institute of Animal Science & Veterinary Medicine Shandong Academy of Agricultural Sciences <120> Method for Efficiently Expressing PCV2 Cap and PCV3 Cap Fusion Protein Technical Field <130> P6092179NL <150> CN 202010052203.0 <151> 20200117 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 1 Gly Gly Gly Gly Ser Ser Ser 1 5 <210> 2 <211> 1452 <212> DNA <213> Artificial Sequence <220> <223> H-2Cap-3Cap <400> 2 atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tgtacatttc ttacatctat 60 gcgatgacgt atccaaggag gcgtttccgc agacgaagac accgcccccg cagccatctt 120 ggccagatcc tccgccgcecg cccctggctc gtccaccccc gcctccgtta ccgctggaga 180 aggaaaaatg gcatcttcaa cacccgcctc tcccgcacca tcggttatac tgtcaagaaa 240 accacagtca gaacgccctc ctggaatgtg gacatgatga gatttaatat taatgatttt 300 cttcccccag gagggggctc aaaccccctc actgtgccct ttgaatacta cagaataagg 360 Pagina 1

SEQLTXT aaggttaagg ttgaattctg gccctgctcc ccaatcaccc agggtgacag gggagtgggc 420 tccactgctg ttattctaga tgataacttt gtaacaaagg ccaatgccct aacctatgac 480 ccctatgtaa actactcctc ccgccatacc ataacccagc ccttctccta ccactcccgg 540 tactttaccc caaaacctgt cattgatagg acaatcgatt acttccaacc caataacaaa 600 agaaatcaac tctggctgag actacaaact actggaaatg tagaccatgt aggcctcggc 660 actgcgttcg aaaacagtat atacgaccag gactacaata tccgtataac catgtatgta 720 caattcagag aatttaatct taaagacccc ccacttaacc ctaaggcttc ctcctcctcc 780 gcttcetect cctcegcttc ctcctcctcc atgagacaca gagctatatt cagaagaaga 840 CCCCgCCCaa ggagacgacg acgccacaga aggcgctatg ccagaagaag actattcatt 900 aggaggccca cagctggcac atactacaca aagaaatact ccaccatgaa cgtcatttcc 960 gttggaaccc ctcagaataa taagccctgg cacgccaacc acttcattac ccgcctaaac 1020 gaatgggaaa ctgcaattac ctttgaatat tataagatac taaagatgaa agttacactc 1080 agccctgtaa tttctccagc tcagcaaaca aaaactatgt tcgggcacac agccatagat 1140 ctagacggcg cctggaccac aaacacttgg ctccaagacg acccttatgc ggaaagttcc 1200 actcgtaaag ttatgacttc taaaaaaaaa cacagccgtt acttcacccc caaaccactt 1260 ctggcgggaa ctaccagcgc tcacccagga caaagcctct tctttttctc cagacccacc 1320 ccatggctca acacatatga ccccaccgtt caatggggag cactgctttg gagcatttat 1380 gtcccggaaa aaactggaat gacagacttc tacggcacca aagaagtttg gattcgctac 14409 aagtccgttc tc 1452 <210> 3 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Ph-HBM <400> 3 tcccaccatc gggcgcggat ccatgaaatt cttagtcaac gttgcccttg tttttatg 58 Pagina 2

SEQLTXT <210> 4 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> HBM-PCV2Cap F <400> 4 tgtttttatg gtcgtgtaca tttcttacat ctatgcgatg acgtatccaa ggagg 55 <210> 5 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Linker1-PCV2Cap R <400> 5 gcttcetect cctcegcttc ctcctcctcc ttagggttaa gtggggggt 49 <210> 6 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Linker2-PCV3Cap-phF <400> 6 tcctecctect ccgettecte ctcctccatg agacacagag cta 43 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> PCV3CapR Ph <400> 7 acaagcttgt cgagactgca ggagaacgga cttgtag 37 <210> 8 <211> 57 <212> DNA <213> Artificial Sequence Pagina 3

SEQLTXT <220> <223> p19-HBM <400> 8 atcccaactc cataagcatg catgaaattc ttagtcaacg ttgcccttgt ttttatg 57 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PCV3CapR-plo <400> 9 gcctccccca tctcccggta ccgagaacgg acttgtag 38 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> phF <400> 10 cgcggatcca tgaaattctt agtc 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> phR <400> 11 aactgcagga gaacggactt g 21 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> plo F Pagina 4

SEQLTXT

<400> 12 catgcatgca tgaaattctt agtc 24

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> ple R

<400> 13 ggggtaccga gaacggactt gt 22 Pagina 5

Claims

CONCLUSIONS

A PCV2 Cap and PCV3 Cap fusion protein, characterized in that the fusion protein comprises a truncated PCV2 Cap protein segment, a linker sequence, a PCV3 Cap protein segment and a honeybee melittin signal peptide region; and the truncated PCV2 Cap protein segment is a PCV2 Cap protein from which the nuclear localization signal has been deleted.

A fusion protein according to claim 1, characterized in that the linker sequence is a hydrophobic, flexible peptide chain; and preferably the linker sequence is as set forth in SEQ ID NO: I.

The nucleotide sequence of the fusion protein according to claim 1 or 2.

Nucleotide sequence according to claim 3, characterized in that it comprises a sequence as set forth in SEQ ID NO:2.

Vector, recombinant bacmid or recombinant baculovirus for the nucleotide sequence according to claim 3 or 4, characterized in that there is a double copy of the nucleotide sequence in the vector, recombinant bacmid or recombinant baculovirus; preferably the vector is a pFastBac ™ plasmid and more preferably a pFastBac ™ Dual plasmid.

Use of the vector, recombinant bacmid or recombinant baculovirus of claim 5 in expressing a PCV2 Cap and PCV3 Cap fusion protein.

Use according to claim 6, characterized in that the method for expressing a fusion protein by a vector, recombinant bacmid or recombinant baculovirus comprises the following steps: (1) transfecting competent cells comprising a bacmid backbone with a vector to obtain recombinant bacmids; transfecting insect host cells with the recombinant bacmids to obtain a supernatant containing a recombinant baculovirus; (2) inoculating insect host cells with the supernatant containing the recombinant baculovirus to obtain a supernatant after culturing;

(3) subjecting the supernatant to isolation and purification to obtain a PCV2 Cap and PCV3 Cap fusion protein.

Use according to claim 7, characterized in that the insect host cell is selected from Sf9 cells, Sf21 cells, Hi-5 cells or S2 cells.

Use according to claim 7, characterized in that the MOI of the inoculation is 0.1-1.0, the density of the insect host cells is 2.5 x 10° cells/ml and the culture time is 1-5 days after vaccination.

Use of the fusion protein of claim 1 or 2 in the preparation of a porcine circovirus vaccine, antibody, antigen and detection kit; and a porcine circovirus vaccine comprising the fusion protein of claim 1 or 2.