WO2017113050A1 - 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 - Google Patents

猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 Download PDF

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WO2017113050A1
WO2017113050A1 PCT/CN2015/099172 CN2015099172W WO2017113050A1 WO 2017113050 A1 WO2017113050 A1 WO 2017113050A1 CN 2015099172 W CN2015099172 W CN 2015099172W WO 2017113050 A1 WO2017113050 A1 WO 2017113050A1
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seq
protein
sumo
dna
porcine
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PCT/CN2015/099172
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English (en)
French (fr)
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林俊宏
陈正文
王志鹏
彭子庭
李蕙宇
黄文正
王仕蓉
杨程尧
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财团法人农业科技研究院
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Priority to CA3009903A priority Critical patent/CA3009903C/en
Application filed by 财团法人农业科技研究院 filed Critical 财团法人农业科技研究院
Priority to DK15911682.1T priority patent/DK3399040T3/da
Priority to JP2018534063A priority patent/JP6629975B2/ja
Priority to KR1020187018139A priority patent/KR102055215B1/ko
Priority to RU2018127469A priority patent/RU2701816C1/ru
Priority to PCT/CN2015/099172 priority patent/WO2017113050A1/zh
Priority to EP15911682.1A priority patent/EP3399040B1/en
Priority to CN201580082991.5A priority patent/CN108026538B/zh
Priority to BR112018013007-9A priority patent/BR112018013007B1/pt
Priority to ES15911682T priority patent/ES2881625T3/es
Priority to US15/778,062 priority patent/US10767185B2/en
Publication of WO2017113050A1 publication Critical patent/WO2017113050A1/zh
Priority to PH12018501183A priority patent/PH12018501183A1/en

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Definitions

  • the invention relates to a method for preparing a sheath protein of porcine circovirus type 2; in particular to a method for preparing a sheath protein of porcine circovirus type 2 using a prokaryotic cell expression system.
  • Porcine circovirus type 2 is a viral pathogen that affects the global pig industry. It mainly causes post-weaning multisystemic wasting syndrome (PMWS). Symptoms include fever, swollen lymph nodes, weight loss or weakness, difficulty breathing, diarrhea, paleness, and occasional jaundice. It may also cause porcine dermatitis and nephropathy syndrome (PDNS), infectious congenital tremor (ICT) and reproductive disorders.
  • PDNS porcine dermatitis and nephropathy syndrome
  • ICT infectious congenital tremor
  • PRDC porcine respiratory disease complex
  • PCV2 In the field, the prevention and control of PCV2 proposes 20 points of feeding management, such as unified management, good health management, obsolescence or isolation and vaccination. Among them, vaccination can effectively reduce the infection rate of PCV2, thereby increasing the breeding rate.
  • the current PCV2 vaccines are divided into three categories, including inactivated PCV2 vaccine, inactivated baculovirus subunit vaccine, and inactivated porcine circovirus type 1 and type 2 (PCV1-PCV2) chimeric virus vaccines (Beach and Meng). , 2012; Chanhee, 2012).
  • the inactivated PCV2 vaccine is prepared by infecting the porcine kidney cell line PK-15 with PCV2, and inoculating and mixing the adjuvant with the virus solution; the inactivated baculovirus subunit seedling will carry the PCV2 outer sheath protein (capsid)
  • the protein ORF2 gene baculovirus is transfected into insect cells, and the expression of the immunogenic ORF2 is performed. If the antigen is expressed in the cell, the vaccine is prepared by ultrasonically disrupting the culture solution containing the cells, followed by inactivation treatment and mixing of the adjuvant.
  • the cell culture supernatant is collected, and then the virus vector is inactivated and mixed with the adjuvant to prepare a vaccine; inactivation of the PCV1-PCV2 chimeric virus vaccine is to replace the ORF2 in PCV1 with
  • the ORF2 of PCV2 was prepared by cell infection, viral fluid collection, virus inactivation, and mixed adjuvant.
  • Another object of the present invention is to provide a composition for preventing and treating porcine circovirus type 2 infection, which comprises an outer sheath protein of porcine circovirus type 2 as an active ingredient, and comprises a suitable adjuvant to provide industrial pig control A tool for circovirus type 2 infection.
  • Still another object of the present invention is to provide a method for preparing porcine interferon to reduce the time and cost required for the production of porcine interferon, and to facilitate the use of porcine interferon for the prevention and treatment of porcine circovirus type 2 infection.
  • the present invention provides a method for expressing a protein, comprising: (a) obtaining an arabinose-inducible expression vector; wherein the aforementioned arabinose-inducible expression vector comprises a nucleotide sequence of an expression element and a protein of interest; wherein the expression The element comprises: a promoter; a T7 phage translation enhancing element having the sequence of SEQ ID NO: 01; and a ribosome binding site having the sequence of SEQ ID NO: 02; (b) the aforementioned arabinose-inducible expression vector Transforming into an E. coli host and performing induced expression of the target protein; wherein the aforementioned target protein is: outer sheath protein of porcine circovirus type 2 or porcine interferon.
  • the -16 portion of the aforementioned promoter has the sequence shown in SEQ ID NO:03.
  • the aforementioned expression element has the sequence shown in SEQ ID NO:04.
  • the aforementioned arabinose-inducible expression vector further comprises a nucleotide sequence of a fusion partner, and/or a nucleotide sequence of a marker molecule.
  • the aforementioned fusion partner is: Escherichia coli MsyB, Escherichia coli YjgD gene, Lambda phage D protein, Baker's yeast SUMO protein, or a combination thereof.
  • the aforementioned labeling molecule is: a His tag, a Strep II tag, a FLAG tag, or a combination thereof.
  • the aforementioned target protein is an outer sheath protein of porcine circovirus type 2, and the nucleotide sequence thereof has the sequence of SEQ ID NO: 09 or SEQ ID NO: 24.
  • the aforementioned arabinose-inducible expression vector has the sequence shown in SEQ ID NO:46.
  • the aforementioned porcine interferon is porcine interferon alpha or porcine interferon gamma.
  • the aforementioned target protein is porcine interferon, and the nucleotide sequence thereof has the sequence of SEQ ID NO: 64 or SEQ ID NO: 76.
  • the aforementioned arabinose-inducible expression vector has the sequence of SEQ ID NO:80, SEQ ID NO:87, or SEQ ID NO:95.
  • the aforementioned method does not comprise the folding step of the aforementioned porcine interferon.
  • the aforementioned step (b) further comprises the step (c) of purifying the aforementioned target protein.
  • the aforementioned steps Step (c) is further included after step (c): treating the aforementioned target protein with SUMO protease.
  • the weight ratio of the target protein to the SUMO protease is 4 to 20.
  • the present invention further provides a composition for preventing and treating porcine circovirus type 2 infection, comprising: outer sheath protein of porcine circovirus type 2 of 2.5 to 250 ⁇ g/mL; porcine interferon alpha of 2.5 to 25 ⁇ g/mL; Swine interferon gamma up to 25 ⁇ g/mL; and a pharmaceutically acceptable carrier.
  • the aforementioned composition further comprises a pharmaceutically acceptable adjuvant.
  • the aforementioned pharmaceutically acceptable adjuvant is: MONTANIDETM ISA 536 VG adjuvant, MONTANIDETM GEL 01 adjuvant, Freund's complete or incomplete adjuvant, aluminum gel, surfactant, anionic polymer, peptide, oil An emulsion, or a combination thereof.
  • the aforementioned composition comprises: 3.5 to 170 ⁇ g/mL of outer coat protein of porcine circovirus type 2; 5 to 20 ⁇ g/mL of porcine interferon alpha; 5 to 20 ⁇ g/mL of porcine interferon ⁇ ; Acceptable carrier.
  • the present invention mainly provides a method of expressing a protein which expresses a protein by using an arabinose-inducing expression vector.
  • the method of the present invention contributes to the synthesis of the outer sheath protein of porcine circovirus type 2 and the porcine interferon used as an adjuvant in vaccines with higher efficiency.
  • the pharmaceutical composition of the present invention combines the outer sheath protein and other advantageous components in an appropriate ratio to obtain an excellent immune-inducing effect. Accordingly, the disclosure of the present invention is of significant benefit to the prevention and treatment of porcine circovirus type 2 in the field.
  • Fig. 1 is a schematic diagram showing the outer sheath protein expression vectors of five porcine circovirus type 2 prepared in Example 1.
  • Figure 2 shows the protein-induced expression of the five expression vectors prepared in Example 1 after transformation into E. coli host by protein electrophoresis and Western Blot.
  • A Protein electrophoresis results.
  • B Western blotting results; Lane 1: BL21(DE3)/pET29a; Lane 2: BL21(DE3)/pET-SUMO-ORF2; Lane 3: BL21(DE3)/pET-OPTSUMO-ORF2; Lane 4: Rosetta2 /pET-SUMO-ORF2; Lane 5: BL21(DE3)/pET-SUMO-OPTORF2; Lane 6: BL21(DE3)/pET-OPTSUMO-OPTORF2; Lane 7: BL21/pBA-OPTSUMO-OPTORF2.
  • Figure 3 shows the soluble expression of the fusion protein after transformation of the four expression vectors prepared in Example 1 into E. coli host by protein electrophoresis.
  • T total cell disruption
  • S soluble protein
  • IS insoluble protein.
  • the arrow indicates the target protein.
  • Figure 4 is a protein electropherogram showing the results of purification of the fusion protein expressed by pBA-OPTSUMO-OPTORF2 in host cells (E. coli BL21) by immobilized metal ion affinity chromatography. Lane 1: Total cell disruption of E. coli BL21 (pBA-OPTSUMO-OPTORF2); Lane 2: purified fusion protein.
  • Figure 5 shows the expression of recombinant SUMO protease (SUMOPH) and recombinant D-SUMO protease (DSUMOPH) in host cells [E. coli BL21 (DE3)] by protein electrophoresis and Western blotting.
  • A Protein electrophoresis results.
  • B Results of Western blotting. T: total cell disruption; S: soluble protein; IS: insoluble protein. The arrow indicates the target protein.
  • Figure 6 is a protein electropherogram showing the results of purification of recombinant proteases expressed by pET-SUMOPH and pET-D-SUMOPH in host cells [E. coli BL21 (DE3)] by immobilized metal ion affinity chromatography.
  • Lane 1 Purified SUMO protease (SUMOPH);
  • Lane 2 purified D-SUMO protease (DSUMOPH).
  • Figure 7 is a protein electropherogram showing purification, cleavage and filtration of recombinant SUMO-ORF2 fusion protein.
  • Lane 1 Purified SUMO-ORF2 fusion protein.
  • Lane 2 Sheared SUMO-ORF2 fusion protein.
  • Lane 3 ORF2 fusion protein obtained after shearing and filtration (100 kDa filter membrane).
  • Figure 8 is a transmission electron microscope image showing the SUMO-ORF2 fusion protein (A), the protease-cleaved recombinant SUMO-ORF2 fusion protein (B), and the ORF2 fusion protein obtained by protease cleavage and filtration.
  • Figure 9 is a protein electropherogram showing the expression of recombinant porcine interferon of Example 3; T: total cell disruption; S: soluble protein.
  • A pET-OPTPIFNAH/E.coli Shuffle;
  • B pBA-OPTPIFNAH/E.coli Shuffle;
  • C pET-SUMO-OPTPIFNAH/E.coli Shuffle;
  • D pET-OPTSUMO-OPTPIFNAH/E.coli Shuffle;
  • E pBA-OPTSUMO-OPTPIFNAH/E.coli Shuffle;
  • F pET-OPTPIFNRH/E.coli BL21(DE3);
  • G pET-SUMO-OPTPIFNRH/E.coli BL21(DE3);
  • I pBA-OPTSUMO-OPTPIFNRH/E.coli BL21 (DE3).
  • Figure 10 is a protein electropherogram showing the results of purification of recombinant porcine interferon of Example 3.
  • Lane 1 Purification of the fusion protein obtained by E. coli Shuffle (pET-OPTPIFNAH) expression
  • Lane 2 E. coli Shuffle (pBA-OPTSUMO-OPTPIFNAH) expression of the fusion protein by D-SUMO protease [pET-D- SUMOP/E.coli BL21 (DE3) cell disrupted product] the result of purification after cleavage
  • Lane 3 E.
  • coli BL21 (DE3) (pET-OPTSUMO-OPTPIFNRH) expression of the fusion protein by D-SUMO protease [ pET-D-SUMOP/E.coli BL21 (DE3) cell disrupted product] the result of purification after shearing.
  • Figure 11 is a ELISA test result showing the anti-PCV2 antibody titer produced by each sample in the experiment of Example 4 in the pig.
  • Figure 12 shows the extent to which each sample in Example 4 Experiment 3 reduced viremia in pigs.
  • Figure 13 is a ELISA test result showing anti-PCV2 produced in pigs of each sample in Example 4, Experiment 4. Antibody titer.
  • Figure 14 shows the extent to which each sample in Example 4 Experiment 4 reduced viremia in pigs.
  • Figure 15 is a ELISA test result showing the anti-PCV2 antibody titer produced by each sample in the experiment of Example 4 in Experiment 5.
  • the method of the present invention is to prepare an porcine circovirus type 2 by using the arabinose-inducible expression element disclosed in the applicant's invention patent application No. 103146225 (application date: December 30, 2014) of the present invention. Outer sheath protein.
  • Taiwan Patent No. 103146225 The entire contents of the aforementioned Taiwan Patent No. 103146225 are incorporated herein by reference.
  • target protein refers to a protein to be expressed by a prokaryotic expression system.
  • the aforementioned target protein is an outer sheath protein of porcine circovirus type 2, porcine interferon alpha, or porcine interferon gamma.
  • nucleotide sequence of a protein of interest refers to a nucleotide sequence which, upon in vivo or in vitro transcription/translation mechanisms, forms the aforementioned protein of interest. Accordingly, the "nucleotide sequence of the outer sheath protein of porcine circovirus type 2" or “the nucleotide sequence of porcine interferon” of the present invention is also defined as before. Similarly, the “nucleotide sequence of the fusion partner" or “nucleotide sequence of the labeled molecule” of the present invention is also defined as before.
  • the "fusion partner" as used in the present invention refers to a molecule which is used to enhance the water solubility of the aforementioned target protein for synthesis.
  • the nucleotide sequence of the fusion partner and the nucleotide sequence of the aforementioned target protein are genetically engineered on the same expression vector, thereby synthesizing the aforementioned target protein and the aforementioned fusion partner into a fusion protein.
  • the aforementioned fusion partners are, for example but not limited to, Escherichia coli MsyB, E. coli YjgD, lambda phage D protein, baker's yeast SUMO protein, or a combination thereof.
  • the "marker molecule” as used in the present invention refers to a molecule which is useful for observing the synthesis of the aforementioned target protein or for facilitating purification of the aforementioned target protein.
  • the nucleotide sequence of the marker molecule and the nucleotide sequence of the aforementioned target protein are genetically engineered on the same expression vector, thereby synthesizing the target protein and the aforementioned marker molecule into a fusion protein.
  • the aforementioned labeling molecules are for example but not limited to: His tags, Strep II tags, FLAG tags, or a combination thereof.
  • a first aspect of the invention is a method for preparing an outer sheath protein of porcine circovirus type 2, porcine interferon alpha, or porcine interferon gamma.
  • the aforementioned method comprises (a) obtaining an arabinose-inducible expression vector; wherein the aforementioned arabinose induction table The vector comprises a nucleotide sequence of the expression element and the protein of interest; and (b) transforming the aforementioned arabinose-inducible expression vector into an E. coli host for expression of the protein of interest.
  • the target protein is an outer sheath protein of porcine circovirus type 2.
  • the aforementioned target protein is porcine interferon alpha or porcine interferon gamma.
  • the aforementioned expression element is as described in the applicant's invention patent application No. 103146225 (application date: December 30, 2014). Specifically, the aforementioned expression element comprises: a promoter; a T7 phage translation enhancing element; and a ribosome binding site.
  • the aforementioned expression element is the araB-M11 expression element described in the Chinese Patent Application No. 103146225.
  • the aforementioned T7 phage translation enhancing element has the sequence set forth in SEQ ID NO:01.
  • the ribosome binding site has the sequence set forth in SEQ ID NO:02.
  • the -16 portion of the aforementioned promoter has the sequence set forth in SEQ ID NO:03.
  • the aforementioned expression element has the sequence shown in SEQ ID NO:04.
  • the foregoing step (b) further comprises the step (c): purifying the target protein.
  • the aforementioned target protein can be purified by immobilized-metal ion affinity chromatography.
  • the step (c) is further included after the step (c): treating the target protein with SUMO protease.
  • treatment means that the aforementioned SUMO protease cleaves the aforementioned SUMO fusion partner to separate the aforementioned target protein from the aforementioned SUMO protein.
  • the aforementioned SUMO protease is produced by a T7 expression vector.
  • the weight ratio of the target protein to the SUMO protease is 4 to 20.
  • the foregoing method does not comprise the folding step of the aforementioned porcine interferon.
  • the "folding step" in the prokaryotic cell expression system means that the produced peptide is folded into a tertiary structure by using urea or guanidine hydrochloride to dissolve the inclusion body, and then dialysis or the like. Or the process of a four-level structure.
  • the "folding step without the aforementioned porcine interferon" as described in the present invention means that the peptide prepared in the method of the present invention can be self-folded into a desired protein without using the aforementioned urea or guanidine hydrochloride. And human steps such as dialysis.
  • the aforementioned host is Escherichia coli.
  • the aforementioned Escherichia coli is BL21, BL21 (DE3), Rosetta 2, or Shuffle.
  • a second aspect of the invention is a composition for preventing and treating porcine circovirus type 2 infection, comprising: pig ring disease Toxic protein of toxic type 2, porcine interferon alpha, porcine interferon gamma, and a pharmaceutically acceptable carrier.
  • the composition for preventing porcine circovirus type 2 infection comprises: 2.5 to 250 ⁇ g/mL of outer coat protein of porcine circovirus type 2; and 2.5 to 25 ⁇ g/mL of porcine interferon alpha 2.5 to 25 ⁇ g/mL of porcine interferon gamma; and a pharmaceutically acceptable carrier.
  • the aforementioned composition for controlling porcine circovirus type 2 infection comprises: outer sheath protein of porcine circovirus type 2 of 3.5 to 170 ⁇ g/mL; porcine interference of 5 to 20 ⁇ g/mL ⁇ ; 5 to 20 ⁇ g/mL of porcine interferon gamma; and a pharmaceutically acceptable carrier.
  • the outer sheath protein of the porcine circovirus type 2 is prepared according to the method of the present invention.
  • the aforementioned porcine interferon alpha, and/or the aforementioned porcine interferon gamma is produced according to the method of the present invention.
  • the "pharmaceutically acceptable carrier” as used in the present invention means that the outer sheath protein of the aforementioned porcine circovirus type 2, the aforementioned porcine interferon alpha, and/or the aforementioned porcine interferon gamma are not considered from the viewpoint of medicine/pharmacy.
  • the aforementioned pharmaceutically acceptable carrier is, for example, but not limited to, water, phosphate buffered saline, alcohol, glycerin, chitin, alginate, chondroitin, vitamin E, minerals, or combination.
  • the aforementioned composition further comprises a pharmaceutically acceptable adjuvant.
  • pharmaceutically acceptable adjuvant means that the outer sheath protein of the aforementioned porcine circovirus type 2, the aforementioned porcine interferon alpha, and/or the aforementioned porcine interference is contributed from a medical/pharmaceutical point of view.
  • the pharmaceutically acceptable carrier such as, but not limited to: MONTANIDE TM ISA 536 VG adjuvant, MONTANIDE TM GEL 01 adjuvant, Freund's complete or incomplete adjuvant, aluminum gel, a surfactant Agent, anionic polymer, peptide, oil emulsion, or a combination thereof.
  • the pharmaceutically acceptable adjuvant is MONTANIDE TM ISA 536 VG adjuvant, MONTANIDE TM GEL 01 adjuvant, or combinations thereof.
  • Example 1 Construction of a porcine circovirus type 2 outer sheath protein (PCV2 ORF2) expression vector.
  • the spleen and lymph nodes of the weak pigs were obtained from the pig farm infected with PCV2 (Yunlin, Taiwan, China), and then cut with sterile scissors, and then the lymphatic organs were ground with a sterilized grinding pest and a grinding rod. Add an appropriate amount of sterilized phosphate buffer solution and mix well to prepare an emulsion. The supernatant was collected by centrifugation (6,000 x g, 20 minutes) and filtered through a sieve to remove tissue debris.
  • Use DNA purification kit (DNeasy Blood&Tissue kit; Qiagen, USA) The extraction of viral DNA is performed.
  • the centrifuge tube was placed in a new collection tube, and 500 ⁇ L of AW2Buffer was added to the centrifuge tube and centrifuged at 20,630 ⁇ g for 5 minutes. Place the centrifuge tube into a sterile microcentrifuge tube and drain the DNA by adding appropriate amount of sterile deionized water.
  • the primers PCVF (5'-ACCAGCGCACTTCGGCAGC-3'; SEQ ID NO: 05) and PCVR (5'-AATACTTACAGCGCACTTCTTTCGT TTTC-3'; SEQ ID NO: 06) were designed and polymerase chain reaction (PCR) was used. Amplification of PCV2 gene DNA.
  • the volume of the PCR reaction mixture was 100 ⁇ L, including 10 ⁇ L of the aforementioned DNA extracted from lymphoid organs, 10 ⁇ L of 10X Taq buffer, 200 ⁇ M of dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer and 2.5 U DreamTaq DNA polymerase (Thermo, USA). ).
  • the PCR reaction conditions were 94 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 59 ° C for 30 seconds, 72 ° C for 1 minute and 30 seconds (35 cycles); 72 ° C for 7 minutes (1 step) . DNA fragments were confirmed by DNA electrophoresis for the estimated size.
  • TA clone was performed using the probiotic yT&A cloning vector reagent set (Yeastern, Taiwan). The experimental procedure was carried out by referring to the manufacturer's yT&A cloning vector reagent set operation manual. 5 ⁇ L of the purified PCR product was mixed with 2 ⁇ L of yT&A vector, 1 ⁇ L of ligation buffer A, 1 ⁇ L of ligation buffer B, and 1 ⁇ L of T4 DNA ligase (2 unit/ ⁇ L), and then allowed to act at 22 ° C for 30 minutes. 1 ⁇ L of the ligation mixture was transformed into E.
  • the transformed cells were added to 1 mL of SOC regeneration medium and shaken at 37 ° C for 60 minutes at 250 rpm. Thereafter, an appropriate amount of the bacterial solution was applied to a solid medium containing ampicillin (final concentration: 100 ⁇ g/mL), and cultured at 37 ° C for 16 hours.
  • the colony polymerase chain reaction experimental procedure is as follows. First, prepare a microcentrifuge tube to add 50 ⁇ L of 2 times Premix reaction buffer (GMbiolab, Taiwan), 0.5 ⁇ L of 100 mM PCVF primer, 0.5 ⁇ L of 100 mM PCVR primer and 49 ⁇ L of sterilized water, mix well, and then carry out PCR reaction. The fraction was placed in a PCR vial (10 ⁇ L/tube). After the colony is spotted into the PCR vial with a toothpick, the PCR reaction can be performed.
  • Premix reaction buffer GMbiolab, Taiwan
  • 0.5 ⁇ L of 100 mM PCVF primer 0.5 ⁇ L of 100 mM PCVR primer and 49 ⁇ L of sterilized water
  • the fraction was placed in a PCR vial (10 ⁇ L/tube). After the colony is spotted into the PCR vial with a toothpick, the PCR reaction can be performed.
  • the PCR reaction conditions were 95 ° C for 5 minutes (1 step); 95 ° C reaction for 30 seconds, 59 ° C reaction for 30 seconds, 72 ° C, reaction for 1 minute and 30 seconds (25 cycles); 72 ° C reaction for 7 minutes (1 step) ).
  • Use of DNA Electrophoresis confirmed the presence or absence of a DNA fragment of an estimated size. After confirming that the recombinant plasmid in the transformant carries the insert DNA, the plasmid in the transformant is extracted and subjected to DNA sequencing (Source International Biotech Co., Ltd.), and the plasmid containing PCV2 DNA is named pTA- PCV2.
  • ORF2F/ORF2R primer combination (ORF2F; 5'-CAATATGGATCCATGA CGTATCCAAGGAGGCGT TTC-3'; SEQ ID NO: 07 and ORF2R; 5'-GATATAGTCGACTTAGGGT TTAAGTGGGGGGTCTTTAAGATTAA-3'; SEQ ID NO: 08) Amplification of the ORF2 gene was performed.
  • One-fold GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pTA-PCV2 and 1 U GDP-HiFi DNA polymerase were included in the 50 ⁇ L PCR reaction mixture.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step). Agarose gel electrophoresis was used to confirm whether the PCR product contained a DNA fragment of an estimated size.
  • recovery of the PCR product was carried out using a PCR- MTM Clean Up kit. According to the sequencing result, the sequence of the aforementioned ORF2 gene is shown as SEQ ID NO:09.
  • Codon optimization ORF2 (OPTORF2) gene synthesis
  • ORF2 The amino acid sequence of ORF2 was reverse deduced into a nucleotide sequence according to the preferred codons of E. coli. Design primers based on the aforementioned nucleotide sequence: OPTORF2-T1, OPTORF2-T2, OPTORF2-T3, OPTORF2-T4, OPTORF2-T5, OPTORF2-T6, OPTORF2-T7, OPTORF2-T8, OPTORF2-T9, OPTORF2-T10, OPTORF2 -T11, OPTORF2-T12, OPTORF2F and OPTORF2R, the sequences of which are shown in Table 1 below.
  • Table 1 Codon-optimized primers for ORF2 (OPTORF2) gene synthesis.
  • OPTORF2-T1 ⁇ OPTORF2-T12 was used as a template primer, and OPTORF2 and OPTORF2R were used as amplification primers.
  • the codon-optimized ORF2 gene was amplified in a large amount by an overlay-extension polymerase chain reaction (OEPCR).
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M of each primer and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit. According to the sequencing result, the sequence of the aforementioned codon-optimized ORF2 gene is shown in SEQ ID NO: 24.
  • SUMOF 5'-GATATAGGTACCATGTCGGACTCAGAAGTCAATCAAG-3'; SEQ ID NO: 25
  • SUMOR 5'-CAATATGGATCCACCACCAATCTG TTCTCTGTGAGC-3; SEQ ID NO: 26
  • primer combination Amplification of the SUMO gene.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 200 ng baker's yeast genome and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the amino acid sequence of SUMO is reversely deduced into a nucleotide sequence according to the preferred codon of E. coli.
  • Primers were designed based on the above nucleotide sequences: OPTSUMO-T1, OPTSUMO-T2, OPTSUMO-T3, OPTSUMO-T4, OPTSUMO-T5, OPTSUMO-T6, OPTSUMO-T7, OPTSUMO-T8, OPTSUMOF and OPTSUMOR, the sequences of which are shown in Table 2 below. Shown.
  • OPTSUMO-T1 to OPTSUMO-T8 were used as template primers, and OPTSUMOF and OPTSUMOR were used as amplification primers.
  • a large number of codon-optimized SUMO genes were amplified by overlap extension polymerase chain reaction.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M of each primer and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • the PCR reaction was carried out using pEF29a as a template using a combination of primers of DRAF (5'-GATATACATATGAAAAAAAAATTCGTATCGCATCACCATCACCATCACAGCGGTGGTGGTACCCCAGATCTGGGTACCCTGG-3'; SEQ ID NO: 38) / T7terminator (GCTAGTTATTGCTCAGCGG; SEQ ID NO: 39).
  • DRAF 5'-GATATACATATGAAAAAAAAATTCGTATCGCATCACCATCACCATCACAGCGGTGGTGGTACCCCAGATCTGGGTACCCTGG-3'; SEQ ID NO: 38
  • T7terminator GCTAGTTATTGCTCAGCGG; SEQ ID NO: 39
  • 1 comprising double Ex Taq TM buffer 50 ⁇ L PCR reaction mixture, 200 ⁇ M of dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100ng pET29a and 1.25U TakaRa Ex Taq
  • the reaction conditions of the PCR were 94 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 50 seconds (35 cycles); 72 ° C for 7 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli XL1-blue (Protech, Taiwan). Transformants were randomly selected for DNA sequencing confirmation.
  • the plasmid with the correct DNA sequence was named pET-DRAHIS.
  • the start code of this plasmid is followed by a downstream sequence (DS) AAAAAAAAATTCGTATCG (SEQ ID NO: 40) and a His-tagged DNA sequence CATCACCATCACCATCAC (SEQ ID NO: 41).
  • the SUMO gene amplified from the baker's yeast genome is cleaved with KpnI and BamHI, and then ligated with T4 DNA.
  • the enzyme ligates the DNA fragment into pET-DRAHIS which is cleaved by the same restriction enzyme.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMO.
  • the ORF2 gene amplified from the PCV2 Yunlin virus genome was cleaved with BamHI and SalI, and the DNA fragment was inserted into pET-SUMO which was cleaved by the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMO-ORF2, which has the sequence shown as SEQ ID NO:42.
  • the DNA fragment was ligated into pET-DRAHIS cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTSUMO.
  • the ORF2 gene amplified from the PCV2 Yunlin virus genome was cleaved with BamHI and SalI, and the DNA fragment was ligated into pET-OPTSUMO which was cleaved by the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTSUMO-ORF2, which has the sequence shown as SEQ ID NO:43.
  • the DNA fragment was ligated into pET-SUMO cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMO-OPTORF2, which has the sequence shown as SEQ ID NO:44.
  • the DNA fragment was ligated into pET-OPTSUMO cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. Confirmation of recombinant plasmids in transformants by DNA electrophoresis After inserting the DNA, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTSUMO-OPTORF2, which has the sequence shown as SEQ ID NO:45.
  • the pBA-OPTSUMO-OPTORF2 constructed in this experiment was obtained by embedding the DNA fragment of OPTSUMO-OPTORF2 into a novel arabinose-inducible expression vector pBCM-araM11.
  • pBCM-araM11 is an arabinose-inducible expression element disclosed in the Chinese Patent Application No. 103146225 (Application Date: December 30, 2014) of the applicant of the present invention and the Taiwan Patent Application No. 103142753
  • the vector pBRCMMCS SEQ ID NO: 100 disclosed in the case (application date: December 9, 2014) was constructed. The construction process of the expression vector is described below.
  • pARABM11-GFPT was cleaved with EcoRI and NdeI
  • a DNA fragment containing araC and araB-M11 expression elements was recovered using a Gel- MTM gel extraction system kit (GMbiolab, Taiwan).
  • the araC and araB-M11 expression elements were ligated into the same restriction enzyme cleavage pBRCMMCS using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • the transformants were selected by colony polymerase chain reaction and plasmids were extracted for DNA sequencing confirmation.
  • the plasmid with the correct sequence was named pBCM-araM11, which has the sequence shown in SEQ ID NO:98.
  • pET-OPTSUMO-OPTORF2 was cleaved with NdeI and SalI
  • a DNA fragment containing OPTSUMO-OPTORF2 was recovered using a Gel- MTM gel extraction system kit.
  • OPTSUMO-OPTORF2 was ligated into pBCM-araM11 cut with the same restriction enzymes using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • the transformants were selected by colony polymerase chain reaction and plasmids were extracted for DNA sequencing confirmation.
  • the plasmid with the correct sequence was named pBA-OPTSUMO-OPTORF2, which has the sequence shown as SEQ ID NO:46.
  • the aforementioned DNA fragment containing the araB-M11 expression element is the arabinose-inducible expression element of the present invention, which comprises a promoter (the 16th portion thereof is represented by SEQ ID NO: 03), and a T7 phage translation enhancing element (SEQ ID NO: 01). ), and the ribosome binding site (SEQ ID NO: 02).
  • the aforementioned arabinose-inducible expression element is shown in the case of Chinese Patent Application No. 103146225 (Application Date: December 30, 2014), which has the sequence shown in SEQ ID NO:04.
  • porcine circovirus type 2 outer sheath protein expression vectors were prepared in this example: pET-SUMO-ORF2 (SEQ ID NO: 42) and pET-OPTSUMO-ORF2 (SEQ ID) NO: 43), pET-SUMO-OPTORF2 (SEQ ID NO: 44), pET-OPTSUMO-OPTORF2 (SEQ ID NO: 45), and pBA-OPTSUMO-OPTORF2 (SEQ ID NO: 46), and see Figure 1 Shown.
  • Example 2 Preparation of the outer sheath protein of the porcine circovirus type 2 of the present invention.
  • each of the vectors (SEQ ID NOS: 42 to 46) prepared in Example 1 contained the DNA of the outer sheath protein ORF2, and was applicable to the production of outer sheath proteins.
  • the target protein is synthesized as a fusion protein with SUMO protein and His tag, and the fusion protein is referred to herein as a SUMO-ORF2 fusion protein, and the fusion protein is not described here. His tag.
  • This example will be used to prepare the SUMO-ORF2 fusion protein of the present invention using the expression vector described in Example 1.
  • Expression vectors such as pET-SUMO-ORF2, pET-OPTSUMO-ORF2, pET-SUMO-OPTORF2, and pET-OPTSUMO-OPTORF2 were transformed into E. coli BL21 (DE3) (Yeastern, Taiwan), respectively.
  • pET-SUMO-ORF2 was transformed into E. coli Rosetta 2 (EMD Millipore, USA).
  • pBA-OPTSUMO-OPTORF2 was transformed into E. coli BL21 (New England Biolabs, USA). The method of conversion is carried out by referring to the steps provided by the manufacturer.
  • the E. coli BL21 (DE3) transformant was inoculated into LB medium containing kanamycin (final concentration: 30 ⁇ g/mL), and shake culture was carried out at 37 ° C and 180 rpm. After overnight incubation, the bacterial solution was inoculated at a ratio of 1:100 to LB medium containing kanamycin (final concentration 30 ⁇ g/mL). The shaking culture was carried out at 37 ° C and 180 rpm. The bacteria were cultured to measure the cell concentration by spectrophotometry to an OD 600 of about 0.4 to 0.6, and 0.1 mM isopropyl- ⁇ -D-thiogalactoside (IPTG) was added for protein induction. expression.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the primary and secondary antibodies used in Western blotting are rabbit anti-His polyclonal antibody (rabbit anti-6 ⁇ His polyclonal antibody; Protech, Taiwan) and alkaline phosphatase conjugated goat anti-rabbit antibody [alkaline] Phosphatase-conjugated goat anti-rabbit IgG (H+L)]; the coloring agent used was NBT/BCIP (Thermo, USA).
  • the division of soluble and insoluble proteins was also performed on the cells, and the soluble expression of the SUMO-ORF2 fusion protein was observed by protein electrophoresis.
  • the E. coli Rosetta 2 transformant was inoculated into LB medium containing chloramphenicol (final concentration 34 ⁇ g/mL) and connamycin (final concentration 30 ⁇ g/mL) at 37 ° C and 180 rpm.
  • the shaking culture was carried out. After overnight incubation, the bacterial solution was inoculated at a ratio of 1:100 to LB medium containing chloramphenicol (final concentration 34 ⁇ g/mL) and connamycin (final concentration 30 ⁇ g/mL).
  • the shaking culture was carried out at 37 ° C and 180 rpm.
  • the bacteria were cultured to measure the cell concentration by spectrophotometry to an OD 600 of about 0.4 to 0.6, and 0.1 mM IPTG was added for protein-induced expression. After 4 hours of induction, the bacterial fraction was collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C) and the expression of the SUMO-ORF2 fusion protein was observed by protein electrophoresis and Western blotting. The soluble protein and insoluble protein were also divided into the cells, and the soluble expression of the SUMO-ORF2 fusion protein was observed by protein electrophoresis.
  • the E. coli BL21 transformant was inoculated into LB medium containing chloramphenicol (25 ⁇ g/mL), and shake culture was carried out at 37 ° C and 180 rpm. After overnight incubation, the bacterial solution was inoculated to a LB medium containing chloramphenicol (25 ⁇ g/mL) at a ratio of 1:100. The shaking culture was carried out at 37 ° C and 180 rpm. The bacteria were cultured to measure the cell concentration by spectrophotometry to an OD 600 of about 0.4 to 0.6, and 0.2% arabinose was added for protein-induced expression.
  • the bacterial fraction was collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C) and the expression of the SUMO-ORF2 fusion protein was observed by protein electrophoresis and Western blotting.
  • the soluble protein and insoluble protein were also divided into the cells, and the soluble expression of the SUMO-ORF2 fusion protein was observed by protein electrophoresis.
  • the expression percentage of the recombinant SUMO-ORF2 fusion protein was estimated using Image Quant TL 7.0 (GE Healthcare Life Sciences, USA) software, and the yield of the fusion protein was further calculated.
  • the pET-OPTSUMO-OPTORF2 expression vector with the codon-optimized ORF2 full-length gene and the codon-optimized SUMO gene was transformed into E. coli BL21 (DE3) and induced.
  • the results showed that the recombinant SUMO-ORF2 fusion protein was successfully expressed (Fig. 2) and was mainly soluble protein (Fig. 3); the yield of the soluble recombinant SUMO-ORF2 fusion protein was 81.66 mg/L.
  • This result indicates that the codons of the fusion partner gene can be further optimized to further enhance the expression of the ORF2 fusion protein in E. coli.
  • Past studies have not exemplified the optimization of the SUMO gene codon to increase the amount of fusion protein expression.
  • the present invention confirms the SUMO gene The optimized codons increase the yield of the SUMO-ORF2 fusion protein.
  • Table 3 Yield of soluble SUMO-ORF2 fusion protein.
  • the N-terminus of the recombinant SUMO-ORF2 fusion protein with His-tag can form a coordinating bond with nickel or cobalt ions, and the protein is purified by immobilized metal ion affinity chromatography. Protein liquid chromatography system prime plus (GE Healthcare, Sweden) with 5mL HiTrap TM Ni excel column (GE Healthcare, Sweden) performed.
  • the cells were suspended in a Lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8.0), and the cells were disrupted by a sonicator, and the supernatant fraction was collected by centrifugation (8,000 ⁇ g, 15 minutes).
  • a Lysis buffer 50 mM Tris-HCl, 500 mM NaCl, pH 8.0
  • the supernatant fraction was collected by centrifugation (8,000 ⁇ g, 15 minutes).
  • the supernatant of disrupted bacteria injection HiTrap TM Ni excel column.
  • the specifically bound protein was washed with 100 mL of washing buffer (50 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 8.0).
  • the recombinant protein on the resin was eluted with 150 mL of Elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0), which competes with the recombinant protein for the resin binding site by high concentration of imidazole, resulting in recombinant SUMO-ORF2 fusion protein. It is eluted from the resin. The purification of recombinant SUMO-ORF2 fusion protein was observed by protein electrophoresis. The experimental results are shown in Figure 4.
  • the SUMO-ORF2 fusion protein of the present invention is cleaved by SUMO protease
  • This experiment used SUMO protease to cleave the ORF2 fusion protein produced by the E. coli expression system. Cut After excision, a His-tagged SUMO fusion partner fragment and an outer sheath protein fragment will be obtained.
  • SUMO protease will be produced by the E. coli expression system and applied to the aforementioned uses.
  • One skilled in the art can also carry out this step using SUMO protease obtained in other ways.
  • Amplification of SUMO protease gene using SUMPPF (5'-CAATATGGATCCCTTGTTCCTGAATTAAATGAAAAAGACG-3'; SEQ ID NO: 47) / SUMOPENZHISR (5'-GATATACTCGAGTTAGTGATGGTGATGGTGATGACCACTGCCGCTACCTTTTAAAGCGTCGGTTAAAATCAAATG-3; SEQ ID NO: 48) primer combination using the baker's yeast genome as a template .
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 200 ng baker's yeast genome and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with BamHI and SalI using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMOPH, which has the sequence shown in SEQ ID NO:49.
  • ⁇ phage DNA (Promega, USA) as a template, using DF (5'-GATATAGGTACCATGACGAGCAAAGAAACCTTTACC-3'; SEQ ID NO: 50) and DR (5'-CAATATGGATCCAACGATGCTGATTGCCGTTC-3'; SEQ ID NO: 51) primer combination Amplification of protein genes.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng lambda phage DNA and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET-D cut with BamHI and SalI using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-D-SUMOPH, which has the sequence shown in SEQ ID NO:52.
  • Expression vectors such as pET-SUMOPH and pET-D-SUMOPH were transformed into E. coli BL21 (DE3), respectively.
  • the E. coli BL21 (DE3) transformant was inoculated into LB medium containing kanamycin (final concentration: 30 ⁇ g/mL), and shake culture was carried out at 37 ° C and 180 rpm. After overnight incubation, the bacterial solution was inoculated at a ratio of 1:100 to LB medium containing connamycin (final concentration of 30 ⁇ g/mL). The shaking culture was carried out at 37 ° C and 180 rpm.
  • the bacteria were cultured to measure the cell concentration by spectrophotometry to an OD 600 of about 0.4 to 0.6, and 0.1 mM IPTG was added for protein-induced expression.
  • the bacterial fraction was collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C) to divide the soluble protein and the insoluble protein, and the soluble expression of the recombinant protease was observed by protein electrophoresis and Western blotting.
  • the primary antibody and the secondary antibody used in the Western blotting method were a rabbit anti-His tag polyclonal antibody and an alkaline phosphatase conjugate goat anti-rabbit antibody, respectively; the coloring agent used was NBT/BCIP.
  • the purification method of the recombinant protease is the same as the purification method of the recombinant ORF2 fusion protein.
  • the protein was purified by immobilized metal ion affinity chromatography using the His tagging property of the C-terminus of the recombinant protease.
  • the results showed that the intracellular soluble recombinant SUMO protease and D-SUMO protease could be purified by immobilized metal ion affinity column (Fig. 6).
  • the purified yield of D-SUMO protease was higher, which could be purified from 1L culture solution. 21.50 mg of protein, about 1.4 times the purified yield of SUMO protease (15.33 mg).
  • the purified recombinant SUMO-ORF2 fusion protein is mixed with a recombinant protease (SUMO protease or D-SUMO protease) at a weight ratio of 1:0.05 (for example, 1 mg of recombinant ORF2 fusion protein and 0.05 mg of recombinant protease), and the mixture is allowed to be 4 Act at °C for 16 hours.
  • a recombinant protease SUMO protease or D-SUMO protease
  • the mixture is allowed to be 4 Act at °C for 16 hours.
  • the filtered protein was then filtered using a 100 kDa regeberated cellulose filter membrane. The results show that the 100kDa filter membrane can effectively remove the fusion partner, and it
  • the SUMO-ORF2 fusion protein, the protease-cleaved SUMO-ORF2 fusion protein, and the ORF2 fusion protein obtained by protease cleavage and filtration were placed on a copper grid and placed at room temperature. minute. Then, the excess water is blotted dry with a filter paper, and a uranyl acetate dye is added for negative staining for about 40 seconds to 1 minute. Thereafter, the excess dye was blotted dry using a filter paper, and the virus-like particles were observed by a field emission transmission electron microscope JEM-2100F (JEOL, Japan).
  • the average particle size of the viroid-like particles was calculated from a transmission electron microscope pattern to be about 19 nm.
  • porcine interferon disclosed in the present invention is an adjuvant particularly suitable as a subunit vaccine of porcine circovirus type 2. Therefore, porcine interferon alpha and porcine interferon gamma required for producing the subunit vaccine of the present invention in E. coli host cells in this example are used.
  • the amino acid sequence of mature porcine interferon alpha-6 was reversely deduced into a nucleotide sequence according to the preferred codon of E. coli.
  • Primers were designed based on the aforementioned nucleotide sequences: OPTIFNA-T1, OPTIFNA-T2, OPTIFNA-T3, OPTIFNA-T4, OPTIFNA-T5, OPTIFNA-T6, OPTIFNA-T7, OPTIFNA-T8, OPTIFNAF and OPTIFNAR, the sequences of which are shown in Table 4 below. Shown.
  • Table 4 Codons used to optimize the synthesis of porcine interferon alpha-6 gene.
  • OPTIFNA-T1 to OPTIFNA-T8 were used as template primers, and OPTIFNAF and OPTIFNAR were used as amplification primers.
  • the codon-optimized IFN- ⁇ gene was amplified in large numbers by overlap extension polymerase chain reaction.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M of each primer and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 58 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the amino acid sequence of mature porcine interferon gamma was reversely deduced into a nucleotide sequence according to the preferred codon of E. coli.
  • Primers were designed based on the aforementioned nucleotide sequences: OPTIFNR-T1, OPTIFNR-T2, OPTIFNR-T3, OPTIFNR-T4, OPTIFNR-T5, OPTIFNR-T6, OPTIFNR-T7, OPTIFNR-T8, OPTIFNRF and OPTIFNRR, the sequences of which are shown in Table 5 below. Shown.
  • Table 5 Codons used to optimize the synthesis of the porcine interferon gamma gene.
  • OPTIFNR-T1 to OPTIFNR-T8 were used as template primers, and OPTIFNRF and OPTIFNRR were used as amplification primers.
  • the codon-optimized IFN- ⁇ gene was amplified in large numbers by overlap extension polymerase chain reaction.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M of each primer and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 57 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • IFN- ⁇ was performed using PIFNANDEIF (5'-CAATATCATATGTGCGATCTGCCGCAAACC-3'; SEQ ID NO: 77)/PIFNAHISSALIR (5'-GATATAGTCGATTATTAGTGATGGTGATGGTGATGTTCCTTTTTACGCAGGCGGTC-3'; SEQ ID NO: 78) primer combination Amplification of genes.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pJET-IFNA-6 and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTPIFNAH, which has the sequence shown in SEQ ID NO:79.
  • the DNA fragment was separately introduced into pBCM-araM11 which was cleaved by the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction.
  • the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pBA-OPTPIFNAH, which has the sequence shown in SEQ ID NO:80.
  • Amplification of the SUMO gene was carried out using the baker's yeast genome as a template and using SUMOF (SEQ ID NO: 25) / SUMOR2 (5'-ACCACCAATCTGTTCTCTGTGAGC-3'; SEQ ID NO: 81) primer combinations.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 200 ng baker's yeast genome and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • Amplification of the IFN- ⁇ gene was carried out using the pJET-IFNA-6 plasmid as a template using SUMOIFNAF (5'-GCTCACAGAGAACAGATTGGTGGTTGCGATCTGCCGCAAACC-3'; SEQ ID NO: 82) / PIFNAHISSALIR (SEQ ID NO 78) primer combinations.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pJET-IFNA-6 and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • the SUMO-IFN- ⁇ fusion gene can be obtained by polymerase chain reaction using the SUMOF (SEQ ID NO: 25) / PIFNAHISSALIR (SEQ ID NO: 78) primer combination using the above two PCR products as a template.
  • SUMOF SEQ ID NO: 25
  • PIFNAHISSALIR SEQ ID NO: 78
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMO-OPTPIFNAH, which has the sequence shown in SEQ ID NO:83.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pET-OPTSUMO-ORF2 and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • Amplification of the IFN- ⁇ gene was carried out using the pJET-IFNA-6 plasmid (SEQ ID NO: 63) as a template using the primer combination of OPTSUMOIFNAF (CCGTGAACAAATCGGCGGCTGCGATCTGCCGCAAACC; SEQ ID NO: 85) / PIFNAHISSALIR (SEQ ID NO: 78).
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pJET-IFNA-6 and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • the OPTSUMO-IFN- ⁇ fusion gene can be obtained by polymerase chain reaction using the above two PCR products as a template and using the OPTSUMOF (SEQ ID NO: 35) / PIFNAHISSALIR (SEQ ID NO: 78) primer combination.
  • OPTSUMOF SEQ ID NO: 35
  • PIFNAHISSALIR SEQ ID NO: 78
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5. Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTSUMO-OPTPIFNAH, which has the sequence shown in SEQ ID NO:86.
  • the DNA fragment containing the OPTSUMO-IFN- ⁇ fusion gene was recovered using the Gel-M TM gel extraction system kit.
  • the DNA fragment was ligated into pBCM-araM11 cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5. Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pBA-OPTSUMO-OPTPIFNAH, which has the sequence shown in SEQ ID NO:87.
  • the IFN- ⁇ gene was carried out using the primer combination of PIFNRNDEIF (5'-CAATATCATATGCAAGCCCCGTTTTTCAAAGAA-3'; SEQ ID NO: 88)/PIFNRHISSALIR (5'-GATATAGTCGACTTATTAGTGATG GTGATGGTGATGTTTGCTGGCACGCTGACC-3'; SEQ ID NO: 89) Amplification.
  • One-fold GDP-HiFi PCR buffer B 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pJET-IFNR and 1 U GDP-HiFi DNA polymerase were included in the 50 ⁇ L PCR reaction mixture.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTPIFNRH, which has the sequence shown in SEQ ID NO:90.
  • Amplification of the SUMO gene was carried out using the baker's yeast genome as a template and the SUMOF (SEQ ID NO: 25) / SURMS2 (SEQ ID NO: 81) primer combination.
  • the amplification conditions and PCR recovery methods are as described above.
  • the IFN- ⁇ gene was carried out using SUMOIFNRF (5'-GCTCACAG AGAACAGATTGGTGGTCAAGCCCCGTTTTTCAAAGAA-3'; SEQ ID NO: 91) / PIFNRHISSALIR (SEQ ID NO: 89) primer combinations.
  • SUMOIFNRF 5'-GCTCACAG AGAACAGATTGGTGGTCAAGCCCCGTTTTTCAAAGAA-3'; SEQ ID NO: 91
  • PIFNRHISSALIR SEQ ID NO: 89
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • the SUMO-IFN- ⁇ fusion gene can be obtained by polymerase chain reaction using the SUMOF (SEQ ID NO: 25) / PIFNRHISSALIR (SEQ ID NO: 89) primer combination using the above two PCR products as a template. Includes 1x GDP-HiFi PCR Buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100ng SUMO PCR product, 100ng IFN- ⁇ PCR product and 1U GDP-HiFi DNA polymerase in 50 ⁇ L PCR reaction mixture .
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5. Colony polymerase chain reaction Transformants should be selected.
  • the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-SUMO-OPTPIFNRH, which has the sequence shown in SEQ ID NO:92.
  • Amplification of the OPTSUMO gene was carried out using pET-OPTSUMO-ORF2 (SEQ ID NO: 43) as a template using the primer combination of OPTSUMOF (SEQ ID NO: 35) / OPTSUMOR2 (SEQ ID NO: 84).
  • the amplification conditions and PCR recovery methods are as described above.
  • the porcine interferon gamma gene was subjected to the primer combination of OPTSUMOIFNRF (5'-CCGTGAACAAATCGGCGGCCAAGCCCCGTTTTTCAAAGAAATC-3'; SEQ ID NO: 93)/PIFNRHISSALIR (SEQ ID NO: 89).
  • OPTSUMOIFNRF 5'-CCGTGAACAAATCGGCGGCCAAGCCCCGTTTTTCAAAGAAATC-3'; SEQ ID NO: 93
  • PEFNRHISSALIR SEQ ID NO: 89.
  • Amplification One-fold GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100 ng pJET-IFNR and 1 U GDP-HiFi DNA polymerase were included in the 50 ⁇ L PCR reaction mixture.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using a Gel- MTM gel extraction system kit.
  • the OPTSUMO-IFN- ⁇ fusion gene can be obtained by polymerase chain reaction using the above two PCR products as a template and using the OPTSUMOF (SEQ ID NO: 35) / PIFNRHISSALIR (SEQ ID NO: 89) primer combination.
  • 1x GDP-HiFi PCR buffer B 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 100ng OPTSUMO PCR product, 100ng porcine interferon gamma PCR product and 1U GDP-HiFi DNA polymerization in 50 ⁇ L PCR reaction mixture Enzyme.
  • the PCR reaction conditions were 96 ° C for 2 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 1 minute (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET29a cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-OPTSUMO-OPTPIFNRH, which has the sequence shown in SEQ ID NO:94.
  • pET-OPTSUMO-OPTPIFNRH was cleaved with NdeI and SalI
  • a DNA fragment containing the OPTSUMO-IFR- ⁇ fusion gene was recovered using a Gel- MTM gel extraction system kit.
  • the DNA fragment was ligated into pBCM-araM11 cleaved with the same restriction enzyme using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5. Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pBA-OPTSUMO-OPTPIFNRH, which has the sequence shown in SEQ ID NO:95.
  • pET-OPTPIFNAH (SEQ ID NO: 79), pBA-OPTPIFNAH (SEQ ID NO: 80), pET-SUMO-OPTPIFNAH (SEQ ID NO: 83), pET-OPTSUMO-OPTPIFNAH (SEQ ID NO: 86), and pBA -OPTSUMO-OPTPIFNAH (SEQ ID NO: 87) was transformed into E.
  • the bacterial solution was inoculated at a ratio of 1:100 to LB medium containing connamycin (final concentration of 30 ⁇ g/mL).
  • the shaking culture was carried out at 37 ° C and 180 rpm.
  • the bacteria were cultured to measure the cell concentration by spectrophotometry to an OD600 of about 0.4 to 0.6, and induced expression of the protein was carried out by adding 0.1 mM IPTG at 25 ° C and 180 rpm.
  • the cells were collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C) and the expression of recombinant porcine interferon was observed by protein electrophoresis.
  • the soluble protein and insoluble protein were also divided into the cells, and the soluble expression of recombinant porcine interferon was observed by protein electrophoresis.
  • Amplification of the SUMO protease gene was carried out using the baker's yeast gene as a template and using SUMOPF (SEQ ID NO: 47) / SUMOPENZR (5'-GATATACTCGAGTTATTTTAAAGCGTCGGT TAAAATCAAATG-3; SEQ ID NO: 96) primer combinations.
  • the 50 ⁇ L PCR reaction mixture contained 1 ⁇ GDP-HiFi PCR buffer B, 200 ⁇ M dATP, dTTP, dGTP and dCTP, 1 ⁇ M amplification primer, 200 ng baker's yeast genome and 1 U GDP-HiFi DNA polymerase.
  • the PCR reaction conditions were 96 ° C for 5 minutes (1 step); 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds (35 cycles); 68 ° C for 5 minutes (1 step).
  • agarose gel electrophoresis was used to confirm the presence or absence of a DNA fragment of an estimated size.
  • the PCR product was then recovered using the PCR- MTM Clean Up system kit.
  • the DNA fragment was ligated into pET-D cut with BamHI and SalI using T4 DNA ligase.
  • the bound product was transformed into E. coli ECOS 9-5.
  • Transformants were selected by colony polymerase chain reaction. After confirming the recombinant plasmid in the transformant with extrapolated DNA by DNA electrophoresis, the plasmid in the transformant was extracted and subjected to DNA sequencing.
  • the plasmid with the correct DNA sequence was named pET-D-SUMOP, which has the sequence shown in SEQ ID NO:97.
  • E. coli BL21 (DE3) was transformed into E. coli BL21 (DE3).
  • the E. coli BL21 (DE3) transformant was inoculated into LB medium containing connamycin (final concentration: 30 ⁇ g/mL), and shake culture was carried out at 37 ° C and 180 rpm. After overnight incubation, the bacterial solution was inoculated at a ratio of 1:100 to LB medium containing connamycin (final concentration of 30 ⁇ g/mL). The shaking culture was carried out at 37 ° C and 180 rpm.
  • the bacteria were cultured to measure the cell concentration by spectrophotometry to an OD 600 of about 0.4 to 0.6, and the induced expression of the protein was carried out by adding 0.1 mM IPTG at 28 ° C and 180 rpm. After 4 hours of induction, the bacterial fraction was collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C).
  • the transformants carrying the SUMO-porcine interferon fusion protein expression vector and the SUMO protease expression vector were induced to express, and the bacterial fraction was collected by centrifugation (8,000 ⁇ g, 30 minutes, 4 ° C).
  • the collected cells were suspended in an appropriate amount of Lysis buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) to have an absorbance of 50 at 600 nm. After the cells were disrupted by a sonicator, the supernatant fraction was collected by centrifugation (8,000 ⁇ g, 15 minutes, 4 ° C).
  • SUMO protease Purified recombinant SUMO-porcine interferon fusion protein and recombinant protease
  • the mixture was mixed at a weight ratio of 4 and allowed to stand at 4 ° C for 16 hours; in this stage, the SUMO-porcine interferon fusion protein was cleaved by SUMO protease into SUMO protein and His-tagged porcine interferon at the C-terminus.
  • Protein purification is then carried out using immobilized metal ion affinity chromatography.
  • Protein liquid chromatography system With prime plus 5mL HiTrap TM Ni excel column for.
  • fusion protein solution was injected into the shear column HiTrap TM Ni excel.
  • the specifically bound protein was washed with 100 mL of washing buffer (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4).
  • Example 4 Preparation and application of the composition for preventing and treating porcine circovirus type 2 infection of the present invention.
  • This example is a composition for preventing and treating porcine circovirus type 2 infection using the ORF2, SUMO-ORF2 fusion protein prepared in the foregoing Example 2 and Example 3, and porcine interferon.
  • the composition further comprises MONTANIDETM ISA 563 VG adjuvant (SEPPIC, France) and/or MONTANIDETM GEL 01 adjuvant (SEPPIC, France).
  • the ingredients were mixed according to the following experimental designs, and then inoculated into piglets to observe the induced immune response or whether adverse reactions (such as vomiting, trembling, depression, shortness of breath, and swelling of the affected area were observed; The above symptoms and the presence of symptoms are higher than 50%, and it is judged that the composition is less safe).
  • Seventy-three-year-old field piglets were selected and grouped in a random manner, which were divided into two groups: A and B; the number of pigs in group A was 38, and that in group B was 35. Each group was intramuscularly immunized once, and the immunization dose was 2 mL.
  • the relevant vaccine components are listed in Table VIII below. On the day of the vaccine, the next day was observed and the proportion of adverse clinical reactions was recorded.
  • GMOs animally certified organisms
  • 11 pigs with no specific pathogens at 4 weeks of age were randomly divided into five groups: A to E; A to D were experimental groups, and the number of pigs in each group was 2, and the E group was the control group. ), the number of pigs is 3 heads.
  • Pigs in groups A to D were intramuscularly immunized once at 4 and 6 weeks of age, and the immunization dose was 2 mL; Group E was not subjected to immunotherapy.
  • the relevant vaccine components are listed in Table 10 below.
  • This experiment was conducted in pastures with low levels of pathogen contamination and no PCV2 infection.
  • Twenty-two-year-old SPF pigs of 4 weeks old without infection with PCV2 were selected. They were grouped in a random manner and divided into five groups of A to E. The number of pigs in each group was 4; A to D were experimental groups, and group E was control group. The pigs in groups A to D were intramuscularly immunized once at 4 and 7 weeks of age, and the immunization dose was 2 mL; the E group was not subjected to immunotherapy. The relevant vaccine components are shown in Table 12 below. Collection of serum samples was performed at specific time points. The titer of anti-PCV2 antibodies in serum was determined using a commercially available ELISA kit.

Abstract

本发明提供了一种猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物。所述制备方法通过使用阿拉伯糖诱导表达载体进行猪圆环病毒2型的外鞘蛋白质的表达。

Description

猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 技术领域
本发明关于一种猪圆环病毒2型的外鞘蛋白质的制备方法；尤指一种使用原核细胞表达系统的猪圆环病毒2型的外鞘蛋白质的制备方法。
背景技术
猪圆环病毒2型(porcine circovirus type 2；PCV2)为影响全球养猪产业甚巨的病毒性病原，其主要造成猪离乳后多系统消耗症候群(post-weaning multisystemic wasting syndrome,PMWS)，临床症状为发烧、淋巴结肿大、体重减轻或消弱、呼吸困难、下痢、苍白及偶发黄疸等症状。另可能引发猪皮肤炎肾病症候群(porcine dermatitis and nephropathy syndrome,PDNS)、先天性震颤(infectious congenital tremor,ICT)及繁殖障碍。此外，PCV2与其他病毒性或细菌性病原混合感染猪只则会引起呼吸道疾病综合症(porcine respiratory disease complex,PRDC)。猪只感染PCV2所引发的疾病会造成育成率与饲料换肉率降低，进而造成养猪业者的严重经济损失。
领域中针对PCV2的预防与控制提出20点饲养管理要点，如统进统出、良好卫生管理、病情严重者淘汰或隔离及疫苗接种等。其中疫苗接种可有效降低PCV2感染率，进而提升育成率。目前领域中的PCV2疫苗分为三类，包括灭活PCV2疫苗、灭活杆状病毒亚单位疫苗及灭活猪圆环病毒1型与2型(PCV1-PCV2)嵌合病毒疫苗(Beach and Meng,2012；Chanhee,2012)。
灭活PCV2疫苗是将PCV2感染猪肾细胞株PK-15后，收取病毒液经灭活处理与混合佐剂所制成；灭活杆状病毒亚单位苗是将带有PCV2外鞘蛋白质(capsid protein)ORF2基因的杆状病毒转染昆虫细胞后，进行免疫原ORF2的表达。若抗原表达于细胞内，疫苗制备的方式是将含有细胞的培养液进行超声波破碎处理，再经灭活处理与混合佐剂制成。若抗原分泌至胞外，则进行细胞培养上清液的收集，再经病毒载体的灭活处理与混合佐剂制成疫苗；灭活PCV1-PCV2嵌合病毒疫苗是将PCV1中的ORF2置换为PCV2的ORF2并进行细胞感染、病毒液收取、病毒灭活及混合佐剂所制成。
综观目前PCV2疫苗的生产方式皆是以培养病毒的方式进行，因而具有制备时间长且生产成本高的缺点。为降低疫苗成本，领域中研究人员尝试利用培养成本较低的重组大肠杆菌进行疫苗抗原ORF2的生产，但仍遭遇ORF2产量低落、重组ORF2无法形成类病毒 颗粒(virus-like particle)、制备过程复杂或免疫效果不佳的瓶颈。
发明内容
爰是，本发明的一个目的为提供一种制备猪圆环病毒2型的外鞘蛋白质的方法，以降低PCV2疫苗的生产时间及成本。
本发明的另一个目的为提供一种防治猪圆环病毒2型感染的组合物，其以猪圆环病毒2型的外鞘蛋白质作为活性成分，并包含合适的佐剂，从而提供产业防治猪圆环病毒2型感染的工具。
本发明的又一个目的为提供一种制备猪干扰素的方法，以降低生产猪干扰素所需的时间及成本，而有利于应用猪干扰素于防治猪圆环病毒2型感染的组合物。
为了达到上述目的，本发明提供一种表达蛋白质的方法，其包含：(a)取得阿拉伯糖诱导表达载体；其中前述阿拉伯糖诱导表达载体包含表达元件及目标蛋白质的核苷酸序列；其中前述表达元件包含：启动子；T7噬菌体转译增强元件，其具有SEQ ID NO：01所示序列；及核糖体结合部位，其具有SEQ ID NO：02所示序列；(b)将前述阿拉伯糖诱导表达载体转化至大肠杆菌宿主中，并进行目标蛋白质的诱导表达；其中前述目标蛋白质为：猪圆环病毒2型的外鞘蛋白质或猪干扰素。
较佳地，前述启动子的-16部位具有SEQ ID NO：03所示序列。
较佳地，前述表达元件具有SEQ ID NO：04所示序列。
较佳地，前述阿拉伯糖诱导表达载体进一步包含融合伴侣的核苷酸序列、及/或标记分子的核苷酸序列。较佳地，前述融合伴侣为：大肠杆菌MsyB、大肠杆菌YjgD基因、λ(Lambda)噬菌体D蛋白质、面包酵母菌SUMO蛋白质、或其组合。较佳地，前述标记分子为：His标签、Strep II标签、FLAG标签、或其组合。
较佳地，前述目标蛋白质为猪圆环病毒2型的外鞘蛋白质，且其核苷酸序列具有SEQ ID NO：09或SEQ ID NO：24所示序列。较佳地，前述阿拉伯糖诱导表达载体具有SEQ ID NO：46所示序列。
较佳地，前述猪干扰素为猪干扰素α或猪干扰素γ。较佳地，前述目标蛋白质为猪干扰素，且其核苷酸序列具有SEQ ID NO：64或SEQ ID NO：76所示序列。较佳地，前述阿拉伯糖诱导表达载体具有SEQ ID NO：80、SEQ ID NO：87、或SEQ ID NO：95所示序列。较佳地，前述方法不包含前述猪干扰素的折叠步骤。
较佳地，前述步骤(b)之后进一步包含步骤(c)：纯化前述目标蛋白质。较佳地，前述步 骤(c)之后进一步包含步骤(d)：以SUMO蛋白酶处理前述目标蛋白质。较佳地，步骤(d)的处理中，前述目标蛋白质与前述SUMO蛋白酶的重量比值为4至20。
本发明又提供一种防治猪圆环病毒2型感染的组合物，其包含：2.5至250μg/mL的猪圆环病毒2型的外鞘蛋白质；2.5至25μg/mL的猪干扰素α；2.5至25μg/mL的猪干扰素γ；及医药可接受的载剂。
较佳地，前述组合物进一步包含医药可接受的佐剂。较佳地，前述医药可接受的佐剂为：MONTANIDETM ISA 536 VG佐剂、MONTANIDETM GEL 01佐剂、弗氏完全或不完全佐剂、铝胶、表面活性剂、阴离子型聚合物、肽、油乳液、或其组合。
较佳地，前述组合物包含：3.5至170μg/mL的猪圆环病毒2型的外鞘蛋白质；5至20μg/mL的猪干扰素α；5至20μg/mL的猪干扰素γ；及医药可接受的载剂。
综上所述，本发明主要提供一种表达蛋白质的方法，其通过使用阿拉伯糖诱导表达载体表达蛋白质。本发明方法有助于以更高的效率合成猪圆环病毒2型的外鞘蛋白质及疫苗中作为佐剂使用的猪干扰素。另一方面，本发明医药组合物以合适比例组合前述外鞘蛋白质及其他有利成分而取得优异的免疫诱发效果。据此，本发明揭露内容对于领域中猪圆环病毒2型的防治工作有显著助益。
附图说明
图1为实施例一所制得的5个猪圆环病毒2型的外鞘蛋白质表达载体的示意图。
图2显示以蛋白质电泳及蛋白质印迹法(Western Blot)观察实施例一所制得的5个表达载体转化至大肠杆菌宿主后的蛋白质诱导表达情形。(A)蛋白质电泳结果。(B)蛋白质印迹法结果；泳道1：BL21(DE3)/pET29a；泳道2：BL21(DE3)/pET-SUMO-ORF2；泳道3：BL21(DE3)/pET-OPTSUMO-ORF2；泳道4：Rosetta2/pET-SUMO-ORF2；泳道5：BL21(DE3)/pET-SUMO-OPTORF2；泳道6：BL21(DE3)/pET-OPTSUMO-OPTORF2；泳道7：BL21/pBA-OPTSUMO-OPTORF2。
图3显示利用蛋白质电泳观察实施例一所制得的4个表达载体转化至大肠杆菌宿主后的融合蛋白可溶性表达情形。T：总细胞破碎物；S：可溶性蛋白质；IS：不可溶性蛋白质。箭头标示处为目标蛋白质。
图4为蛋白质电泳图，其显示利用固定化金属离子亲和层析法纯化pBA-OPTSUMO-OPTORF2于宿主细胞(E.coli BL21)表达的融合蛋白的结果。泳道1：E.coli BL21(pBA-OPTSUMO-OPTORF2)的总细胞破碎物；泳道2：经纯化的融合蛋白。
图5显示以蛋白质电泳及蛋白质印迹法观察实施例二中重组SUMO蛋白酶(SUMOPH)和重组D-SUMO蛋白酶(DSUMOPH)于宿主细胞【E.coli BL21(DE3)】中的表达情形。(A)蛋白质电泳结果。(B)蛋白质印迹法结果。T：总细胞破碎物；S：可溶性蛋白质；IS：不可溶性蛋白质。箭头标示处为目标蛋白质。
图6为蛋白质电泳图，其显示利用固定化金属离子亲和层析法纯化pET-SUMOPH及pET-D-SUMOPH于宿主细胞【E.coli BL21(DE3)】表达的重组蛋白酶的结果。泳道1：经纯化的SUMO蛋白酶(SUMOPH)；泳道2：经纯化的D-SUMO蛋白酶(DSUMOPH)。
图7为蛋白质电泳图，其显示重组SUMO-ORF2融合蛋白的纯化、剪切及过滤。泳道1：纯化的SUMO-ORF2融合蛋白。泳道2：经剪切的SUMO-ORF2融合蛋白。泳道3：经剪切及过滤(100kDa过滤膜)后取得的ORF2融合蛋白。
图8为穿透式电子显微镜影像，其显示SUMO-ORF2融合蛋白(A)、经蛋白酶剪切的重组SUMO-ORF2融合蛋白(B)、及经蛋白酶剪切再经过滤后所得的ORF2融合蛋白(C)形成类病毒颗粒的影像。
图9为蛋白质电泳图，其显示实施例三的重组猪干扰素的表达情形；T：总细胞破碎物；S：可溶性蛋白质。(A)pET-OPTPIFNAH/E.coli Shuffle；(B)pBA-OPTPIFNAH/E.coli Shuffle；(C)pET-SUMO-OPTPIFNAH/E.coli Shuffle；(D)pET-OPTSUMO-OPTPIFNAH/E.coli Shuffle；(E)pBA-OPTSUMO-OPTPIFNAH/E.coli Shuffle；(F)pET-OPTPIFNRH/E.coli BL21(DE3)；(G)pET-SUMO-OPTPIFNRH/E.coli BL21(DE3)；(H)pET-OPTSUMO-OPTPIFNRH/E.coli BL21(DE3)；(I)pBA-OPTSUMO-OPTPIFNRH/E.coli BL21(DE3)。箭头标示处为目标蛋白质。
图10为蛋白质电泳图，其显示实施例三的重组猪干扰素经表达后的纯化结果。泳道1：E.coli Shuffle(pET-OPTPIFNAH)表达所得的融合蛋白的纯化结果；泳道2：E.coli Shuffle(pBA-OPTSUMO-OPTPIFNAH)表达所得的融合蛋白经D-SUMO蛋白酶【pET-D-SUMOP/E.coli BL21(DE3)细胞破碎物】剪切后再经纯化的结果；泳道3：E.coli BL21(DE3)(pET-OPTSUMO-OPTPIFNRH)表达所得的融合蛋白经D-SUMO蛋白酶【pET-D-SUMOP/E.coli BL21(DE3)细胞破碎物】剪切后再经纯化的结果。
图11为ELISA试验结果，其显示实施例四实验3中各样品于猪只中产生的抗PCV2抗体效价。
图12显示实施例四实验3中各样品降低猪只病毒血症的程度。
图13为ELISA试验结果，其显示实施例四实验4中各样品于猪只中产生的抗PCV2 抗体效价。
图14显示实施例四实验4中各样品降低猪只病毒血症的程度。
图15为ELISA试验结果，其显示实施例四实验5中各样品于猪只中产生的抗PCV2抗体效价。
具体实施方式
如前所述，虽然领域中已尝试通过大肠杆菌表达系统生产猪圆环病毒2型的外鞘蛋白质，然而截至本发明申请时，仍未能克服产量过低的缺点，而阻碍猪圆环病毒2型防疫工作的精进。
本发明方法是采用本发明的申请人于中国台湾发明专利申请案第103146225号案(申请日：公元2014年12月30日)所揭露的阿拉伯糖诱导表达元件来制备猪圆环病毒2型的外鞘蛋白质。前述中国台湾专利第103146225号案的全部内容并入于本案中作为参考文献。
本文中所述“目标蛋白质”是指欲藉由原核细胞表达系统表达的蛋白质。于本发明中，前述目标蛋白质为猪圆环病毒2型的外鞘蛋白质、猪干扰素α、或猪干扰素γ。
本文中所述“目标蛋白质的核苷酸序列”或其他类似的叙述是指一核苷酸序列，其通过活体内或活体外的转录/转译机制后，可形成前述目标蛋白质。据此，本发明所述“猪圆环病毒2型的外鞘蛋白质的核苷酸序列”或“猪干扰素的核苷酸序列”亦定义如前。同理，本发明所述“融合伴侣的核苷酸序列”或“标记分子的核苷酸序列”亦定义如前。
本发明所述“融合伴侣”是指为利于提高合成的前述目标蛋白质的水溶性而使用的分子。为上述目的，将融合伴侣的核苷酸序列与前述目标蛋白质的核苷酸序列以遗传工程的方式构建于同一表达载体上，从而使前述目标蛋白质与前述融合伴侣合成为融合蛋白。前述融合伴侣例如但不限于：大肠杆菌MsyB、大肠杆菌YjgD、λ噬菌体D蛋白质、面包酵母菌SUMO蛋白质、或其组合。
本发明所述“标记分子”是指为利于观察前述目标蛋白质的合成或为利于纯化前述目标蛋白质所使用的分子。为上述目的，将标记分子的核苷酸序列与前述目标蛋白质的核苷酸序列以遗传工程的方式构建于同一表达载体上，从而使前述目标蛋白质与前述标记分子合成为融合蛋白。前述标记分子例如但不限于：His标签、Strep II标签、FLAG标签、或其组合。
本发明的第一个方面为一种制备猪圆环病毒2型的外鞘蛋白质、猪干扰素α、或猪干扰素γ的方法。前述方法包含(a)取得阿拉伯糖诱导表达载体；其中前述阿拉伯糖诱导表 达载体包含表达元件及目标蛋白质的核苷酸序列；及(b)将前述阿拉伯糖诱导表达载体转化至大肠杆菌宿主中进行目标蛋白质的表达。
于一可行实施态样中，前述目标蛋白质为猪圆环病毒2型的外鞘蛋白质。于一可行实施态样中，前述目标蛋白质为猪干扰素α或猪干扰素γ。
于一较佳实施态样中，前述表达元件如本发明的申请人于中国台湾发明专利申请案第103146225号案(申请日：公元2014年12月30日)中所述者。具体而言，前述表达元件包含：启动子；T7噬菌体转译增强元件；及核糖体结合部位。举例来说，前述表达元件为中国台湾发明专利申请案第103146225号案中所述araB-M11表达元件。
在一较佳实施态样中，前述T7噬菌体转译增强元件具有SEQ ID NO：01所示序列。在一较佳实施态样中，前述核糖体结合部位具有SEQ ID NO：02所示序列。在一较佳实施态样中，前述启动子的-16部位具有SEQ ID NO：03所示序列。在一较佳实施态样中，前述表达元件具有SEQ ID NO：04所示序列。
于一可行实施态样中，其中前述步骤(b)之后进一步包含步骤(c)：纯化前述目标蛋白质。当于本发明方法中使用His标签作为前述标记分子时，可采用固定化金属离子亲和层析法(immobilized-metal ion affinity chromatography)纯化前述目标蛋白质。
于一可行实施态样中，当于本发明方法中使用SUMO蛋白质作为前述融合伴侣时，于前述步骤(c)之后进一步包含步骤(d)：以SUMO蛋白酶处理前述目标蛋白质。前述“处理”是指使前述SUMO蛋白酶剪切前述SUMO融合伴侣，以使前述目标蛋白质与前述SUMO蛋白质分离。
于一可行实施态样中，前述SUMO蛋白酶是通过T7表达载体而制得。于一较佳实施态样中，于前述处理中，前述目标蛋白质与前述SUMO蛋白酶的重量比值为4至20。
于一较佳实施态样中，前述方法不包含前述猪干扰素的折叠步骤。所属领域技术人员当可理解于原核细胞表达系统中的“折叠步骤”是指通过使用尿素或盐酸胍(guanidine hydrochloride)溶解包涵体，再经透析等步骤使所产出的肽折叠形成三级结构或四级结构的过程。从而所属领域技术人员当可理解本发明所述“不包含前述猪干扰素的折叠步骤”是指本发明方法中所制得的肽可自行折叠为所欲蛋白质，而无需前述使用尿素或盐酸胍、及透析等人为步骤。
于一可行实施态样中，前述宿主为大肠杆菌。较佳地，前述大肠杆菌为BL21、BL21(DE3)、Rosetta2、或Shuffle。
本发明的第二个方面为一种防治猪圆环病毒2型感染的组合物，其包含：猪圆环病 毒2型的外鞘蛋白质、猪干扰素α、猪干扰素γ、及医药可接受的载剂。
于一较佳实施态样中，前述防治猪圆环病毒2型感染的组合物包含：2.5至250μg/mL的猪圆环病毒2型的外鞘蛋白质；2.5至25μg/mL的猪干扰素α；2.5至25μg/mL的猪干扰素γ；及医药可接受的载剂。又于另一更佳实施态样中，前述防治猪圆环病毒2型感染的组合物包含：3.5至170μg/mL的猪圆环病毒2型的外鞘蛋白质；5至20μg/mL的猪干扰素α；5至20μg/mL的猪干扰素γ；及医药可接受的载剂。
于一较佳实施态样中，前述猪圆环病毒2型的外鞘蛋白质是按照本发明方法所制得的。于一较佳实施态样中，前述猪干扰素α、及/或前述猪干扰素γ是按照本发明方法所制得的。
本发明所述“医药可接受的载剂”是指从医学/药学的观点而论，不对前述猪圆环病毒2型的外鞘蛋白质、前述猪干扰素α、及/或前述猪干扰素γ于前述组合物防治猪圆环病毒2型感染的目的上产生负面影响的物质。于一可行实施态样中，前述医药可接受的载剂例如，但不限于：水、磷酸缓冲食盐水、醇、甘油、甲壳素、海藻酸盐、软骨素、维生素E、矿物质、或其组合。
于一较佳实施态样中，前述组合物进一步包含医药可接受的佐剂。本发明所述“医药可接受的佐剂”是指从医学/药学的观点而论，有助于前述猪圆环病毒2型的外鞘蛋白质、前述猪干扰素α、及/或前述猪干扰素γ于前述组合物防治猪圆环病毒2型感染的目的上产生免疫增强效果的物质。于一可行实施态样中，前述医药可接受的载剂例如，但不限于：MONTANIDETM ISA 536 VG佐剂、MONTANIDETM GEL 01佐剂、弗氏完全或不完全佐剂、铝胶、表面活性剂、阴离子型聚合物、肽、油乳液、或其组合。于一较佳实施态样中，前述医药可接受的佐剂为MONTANIDETM ISA 536 VG佐剂、MONTANIDETM GEL 01佐剂、或其组合。
本发明的研究过程将进一步于下列实施例中详述。惟以下内容仅例示性的说明本发明的特征，以利理解。所属领域技术人员自可在不违背本发明精神的前题下参酌下列内容并基于领域中的通常知识加以变化，而仍属于本发明的权利范围。
实施例一：建构猪圆环病毒2型的外鞘蛋白质(PCV2 ORF2)表达载体。
PCV2病毒的分离与基因体定序
由感染PCV2的猪场(云林，中国台湾)取得病弱猪的脾脏与淋巴结等淋巴器官，以灭菌的剪刀剪碎后，再利用灭菌的研磨钵与研磨棒将淋巴器官磨碎，并加入适量的灭菌磷酸缓冲溶液混合均匀，制成乳剂。利用离心(6,000×g，20分钟)方式收取上清液，再经滤网过滤去除组织碎片。利用DNA纯化套组(DNeasy Blood&Tissue kit；Qiagen,USA) 进行病毒DNA的抽取。取100μL的乳剂上清液，加入180μL的ATL Buffer与20μL的蛋白酶K(proteinase K；10mg/mL)，于56℃下作用2小时。之后，加入200μL的绝对酒精并混合均匀。吸取所有溶液至离心分离小管(spin column)，并将离心分离小管放置于收集小管(collection tube)中，于6,000×g下离心1分钟。将离心分离小管放置于一新的收集小管中，再加入500μL的AW1Buffer至分离小管中，于6,000×g下离心1分钟。将离心分离小管放置于一新的收集小管中，再加入500μL的AW2Buffer至离心分离小管中，于20,630×g下离心5分钟。将离心分离小管放入一灭菌微量离心管中，加入适量无菌去离子水引流DNA。
设计引物PCVF(5’-ACCAGCGCACTTCGGCAGC-3’；SEQ ID NO：05)与PCVR(5’-AATACTTACAGCGCACTTCTTTCGT TTTC-3’；SEQ ID NO：06)，并利用聚合酶链式反应(polymerase chain reaction,PCR)扩增PCV2基因体DNA。PCR反应混合物的体积为100μL，其中包括10μL的前述由淋巴器官萃取的DNA，10μL 10X Taq buffer，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物及2.5U DreamTaq DNA聚合酶(Thermo,USA)。PCR反应条件为94℃反应5分钟(1个步骤)；94℃反应30秒、59℃反应30秒、72℃反应1分钟30秒(35个循环)；72℃反应7分钟(1个步骤)。利用DNA电泳确认有无预估大小的DNA片段。
PCR产物经PCR-MTM Clean Up kit(GMbiolab,Taiwan)回收后，利用益生yT&A克隆载体试剂组(Yeastern,Taiwan)进行TA克隆。实验步骤参考厂商提供的yT&A克隆载体试剂组操作手册进行。取5μL回收纯化的PCR产物与2μL yT&A载体、1μL连接缓冲液A(ligation buffer A)、1μL连接缓冲液B和1μL T4DNA连接酶(2unit/μL)混合均匀后，于22℃下作用30分钟。取1μL连接混合物(ligation mixture)转化入大肠杆菌ECOS 9-5(Yeastern,Taiwan)。转化后的菌体加入1mL SOC再生培养液中，于37℃与250rpm的条件下震荡60分钟。之后，取适量菌液涂布于含氨苄西林(ampicillin)(最终浓度为100μg/mL)的固态培养基上，于37℃下培养16小时。
之后，以菌落聚合酶链式反应挑选转化株。菌落聚合酶链式反应实验步骤如下所述。首先，先准备微量离心管加入50μL的2倍Premix反应缓冲液(GMbiolab,Taiwan)、0.5μL的100mM PCVF引物、0.5μL的100mM PCVR引物及49μL的灭菌水，混合均匀后，再将PCR反应液分装于PCR小管(10μL/管)中。以牙签将菌落点至PCR小管后，即可进行PCR反应。PCR反应条件为95℃反应5分钟(1个步骤)；95℃反应30秒、59℃反应30秒、72℃，反应1分钟30秒(25个循环)；72℃反应7分钟(1个步骤)。利用DNA 电泳确认有无预估大小的DNA片段。确认转化株中的重组质粒带有外插DNA(insert DNA)后，再抽取转化株中的质粒并进行DNA定序(源资国际生物科技股份有限公司)，将含有PCV2DNA的质粒命名为pTA-PCV2。
ORF2基因(即外鞘蛋白质的基因)扩增与密码子最佳化
(1)ORF2基因的扩增：
以前述pTA-PCV2作为模板，利用ORF2F/ORF2R引物组合(ORF2F；5’-CAATATGGATCCATGA CGTATCCAAGGAGGCGT TTC-3’；SEQ ID NO：07与ORF2R；5’-GATATAGTCGACTTAGGGT TTAAGTGGGGGGTCTTTAAGATTAA-3’；SEQ ID NO：08)进行ORF2基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pTA-PCV2及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、60℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。利用琼脂糖凝胶电泳确认PCR产物中是否含有预估大小的DNA片段。接着，利用PCR-MTM Clean Up kit进行PCR产物的回收。根据定序结果，前述ORF2基因的序列如SEQ ID NO：09所示。
(2)密码子最佳化ORF2(OPTORF2)基因的合成：
依据大肠杆菌的偏爱密码子(preferred codons)将ORF2的氨基酸序列反向推导为核苷酸序列。依据前述核苷酸序列设计引物：OPTORF2-T1、OPTORF2-T2、OPTORF2-T3、OPTORF2-T4、OPTORF2-T5、OPTORF2-T6、OPTORF2-T7、OPTORF2-T8、OPTORF2-T9、OPTORF2-T10、OPTORF2-T11、OPTORF2-T12、OPTORF2F及OPTORF2R，其序列如下表一所示。
表一：密码子最佳化ORF2(OPTORF2)基因合成所用引物。
Figure PCTCN2015099172-appb-000001
Figure PCTCN2015099172-appb-000002
以OPTORF2-T1～OPTORF2-T12作为模板引物，OPTORF2与OPTORF2R则作为扩增引物。利用重叠延伸聚合酶链式反应(overlapping-extension polymerase chain reaction,OEPCR)大量扩增密码子最佳化的ORF2基因。在50μL的PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM的各引物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。根据定序结果，前述密码子最佳化的ORF2基因的序列如SEQ ID NO：24所示。
SUMO基因的扩增与密码子最佳化
(1)SUMO基因的扩增：
将由日正食品DIY速发酵母所分离的面包酵母(Saccharomyces cerevisiae)接种于YPD(20％peptone,10％yeast extract,20％glucose；pH 6.5)培养基中，于30℃与200rpm的条件下进行振荡培养16小时后，利用YeaStarTM Genomic DNA kit(Zymo Research,USA)进行酵母菌基因体的抽取。取1.5mL隔夜培养的菌液至微量离心管中，离心(2,000×g，5分钟，室温)收集菌体部分，并加入120μL YD Digestion Buffer与5μL R-Zymolase充分混合后，于37℃下作用一小时。接着加入120μL YD Lysis Buffer，缓和混合数次后， 再加入250μL氯仿并进行震荡1分钟。离心(10,000×g，2分钟，室温)收集上清液。将离心分离小管放置于收集小管上，并将上清液置入离心分离小管中，经离心(10,000×g，1分钟，室温)后，倒除滤液。加入300μL DNA清洗缓冲液至离心分离小管中，离心(10,000×g，1分钟，室温)后，倒除滤液，并重复此步骤一次。将离心分离小管放入一灭菌微量离心管中，加入适量的溶离溶液(elution solution)至离心分离小管中并离心(10,000×g，2分钟，室温)以引流基因体DNA。
以前面段落中获得的面包酵母基因体作为模板，利用SUMOF(5’-GATATAGGTACCATGTCGGACTCAGAAGTCAATCAAG-3’；SEQ ID NO：25)/SUMOR(5’-CAATATGGATCCACCACCAATCTG TTCTCTGTGAGC-3；SEQ ID NO：26)引物组合进行SUMO基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，200ng面包酵母基因体及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
(2)密码子最佳化SUMO(OPTSUMO)基因的合成：
依据大肠杆菌的偏爱密码子将SUMO的氨基酸序列反向推导为核苷酸序列。依据前述核苷酸序列设计引物：OPTSUMO-T1、OPTSUMO-T2、OPTSUMO-T3、OPTSUMO-T4、OPTSUMO-T5、OPTSUMO-T6、OPTSUMO-T7、OPTSUMO-T8、OPTSUMOF及OPTSUMOR，其序列如下表二所示。
表二：密码子最佳化SUMO(OPTSUMO)基因合成所用引物。
Figure PCTCN2015099172-appb-000003
Figure PCTCN2015099172-appb-000004
以OPTSUMO-T1～OPTSUMO-T8作为模板引物，OPTSUMOF与OPTSUMOR则作为扩增引物。利用重叠延伸聚合酶链式反应大量扩增密码子最佳化的SUMO基因。在50μL的PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM的各引物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。根据定序结果，前述密码子最佳化的SUMO基因的序列如SEQ ID NO：37所示。
ORF2融合蛋白表达载体的构建
(1)pET-DRAHIS的构建：
以pET29a作为模板，利用DRAF(5’-GATATACATATGAAAAAAAAATTCGTATCGCATCACCATCACCATCACAGCGGTGGTGGTACCCCAGATCTGGGTACCCTGG-3’；SEQ ID NO：38)/T7terminator(GCTAGTTATTGCTCAGCGG；SEQ ID NO：39)引物组合进行PCR反应。在50μL PCR反应混合物中包含1倍Ex TaqTM缓冲液，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pET29a及1.25U TakaRa Ex TaqTM DNA聚合酶(Takara,Japan)。PCR的反应条件为94℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、72℃反应50秒(35个循环)；72℃反应7分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以NdeI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化入大肠杆菌XL1-blue(Protech,Taiwan)中。随机挑选转化株进行DNA定序确认。将DNA序列正确无误的质粒命名为pET-DRAHIS。此质粒的起始码后带有下游序列(downstream sequence,DS)AAAAAAAAATTCGTATCG(SEQ ID NO：40)与His标签的DNA序列CATCACCATCACCATCAC(SEQ ID NO：41)。
(2)pET-SUMO-ORF2表达载体的构建：
自面包酵母基因体扩增的SUMO基因以KpnI与BamHI剪切后，利用T4DNA连接 酶将DNA片段接入以相同限制酶剪切的pET-DRAHIS中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMO。
自PCV2云林病毒基因体扩增的ORF2基因以BamHI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET-SUMO中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMO-ORF2，其具有如SEQ ID NO：42所示序列。
(3)pET-OPTSUMO-ORF2表达载体的构建：
将合成的OPTSUMO基因以KpnI与BamHI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET-DRAHIS中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTSUMO。
自PCV2云林病毒基因体扩增的ORF2基因以BamHI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET-OPTSUMO中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTSUMO-ORF2，其具有如SEQ ID NO：43所示序列。
(4)pET-SUMO-OPTORF2表达载体的构建：
将合成的OPTORF2基因以BamHI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET-SUMO中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMO-OPTORF2，其具有如SEQ ID NO：44所示序列。
(5)pET-OPTSUMO-OPTORF2表达载体的构建：
将合成的OPTORF2基因以BamHI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET-OPTSUMO中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外 插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTSUMO-OPTORF2，其具有如SEQ ID NO：45所示序列。
(6)pBA-OPTSUMO-OPTORF2表达载体的构建：
本实验中所构建的pBA-OPTSUMO-OPTORF2是将OPTSUMO-OPTORF2的DNA片段嵌入一新颖的阿拉伯糖诱导表达载体pBCM-araM11所得。pBCM-araM11是采用本发明的申请人于中国台湾发明专利申请案第103146225号案(申请日：公元2014年12月30日)所揭露的阿拉伯糖诱导表达元件及中国台湾发明专利申请案第103142753号案(申请日：公元2014年12月9日)所揭露的载体pBRCMMCS(SEQ ID NO:100)进行构建。有关表达载体的构建过程描述如下。
将pARABM11-GFPT以EcoRI与NdeI剪切后，利用Gel-MTM gel extraction system kit(GMbiolab,Taiwan)回收含有araC与araB-M11表达元件的DNA片段。利用T4DNA连接酶将araC与araB-M11表达元件接入以相同限制酶剪切的pBRCMMCS中。将黏合产物转化入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株并抽取质粒进行DNA定序确认。将序列正确无误的质粒命名为pBCM-araM11，其具有SEQ ID NO：98所示序列。
pET-OPTSUMO-OPTORF2以NdeI与SalI剪切后，利用Gel-MTM gel extraction system kit回收含有OPTSUMO-OPTORF2的DNA片段。利用T4DNA连接酶将OPTSUMO-OPTORF2接入以相同限制酶剪切的pBCM-araM11中。将黏合产物转化入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株并抽取质粒进行DNA定序确认。将序列正确无误的质粒命名为pBA-OPTSUMO-OPTORF2，其具有如SEQ ID NO：46所示序列。
前述含有araB-M11表达元件的DNA片段即本发明的阿拉伯糖诱导表达元件，其包含启动子(其-16部位如SEQ ID NO：03所示)、T7噬菌体转译增强元件(SEQ ID NO：01)、及核糖体结合部位(SEQ ID NO：02)。前述阿拉伯糖诱导表达元件是按照中国台湾发明专利申请案第103146225号案(申请日：公元2014年12月30日)所示，其具有如SEQ ID NO：04所示序列。
小结
综上所述，本实施例共制得5个猪圆环病毒2型的外鞘蛋白质表达载体，分别为：pET-SUMO-ORF2(SEQ ID NO：42)、pET-OPTSUMO-ORF2(SEQ ID NO：43)、pET-SUMO-OPTORF2(SEQ ID NO：44)、pET-OPTSUMO-OPTORF2(SEQ ID NO：45)、及pBA-OPTSUMO-OPTORF2(SEQ ID NO：46)，并请参见图1所示。
实施例二：制备本发明的猪圆环病毒2型的外鞘蛋白质。
如前所述，实施例一所制得的各个载体(SEQ ID NO：42至46)含有外鞘蛋白质ORF2的DNA，可应用于外鞘蛋白质的生产。并且，为了纯化及水溶性表达的需要，该目标蛋白质是与SUMO蛋白质及His标签合成为融合蛋白，本文中将此融合蛋白称为SUMO-ORF2融合蛋白，并不再赘述该融合蛋白是带有His标签。本实施例将利用实施例一中所述的表达载体进行本发明SUMO-ORF2融合蛋白的制备。
大肠杆菌的转化与重组SUMO-ORF2融合蛋白的诱导表达
(1)实验步骤：
将pET-SUMO-ORF2、pET-OPTSUMO-ORF2、pET-SUMO-OPTORF2及pET-OPTSUMO-OPTORF2等表达载体分别转化入E.coli BL21(DE3)(Yeastern,Taiwan)中。将pET-SUMO-ORF2转化至E.coli Rosetta2(EMD Millipore,USA)中。将pBA-OPTSUMO-OPTORF2转化入E.coli BL21(New England Biolabs,USA)中。转化的方法参照厂商提供的操作步骤进行。
将E.coli BL21(DE3)转化株接种于含有康那霉素(kanamycin)(最终浓度为30μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，菌液以1：100的比例接种至含有康那霉素(kanamycin)(最终浓度为30μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.1mM异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)进行蛋白质的诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分并以蛋白质电泳与蛋白质印迹法观察SUMO-ORF2融合蛋白的表达情形。蛋白质印迹法中所使用的一级抗体与二级抗体分别为兔抗His标签多株抗体(rabbit anti-6×His polyclonal antibody；Protech,Taiwan)与碱性磷酸酶共轭山羊抗兔抗体【alkaline phosphatase-conjugated goat anti-rabbit IgG(H+L)】；所使用的呈色剂为NBT/BCIP(Thermo,USA)。另亦针对菌体进行可溶性蛋白质与不可溶性蛋白质的划分(即，可溶性与否)并以蛋白质电泳观察SUMO-ORF2融合蛋白的可溶性表达情形。
将E.coli Rosetta2转化株接种于含有氯霉素(chloramphenicol)(最终浓度为34μg/mL)与康那霉素(最终浓度为30μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，菌液以1：100的比例接种至含有氯霉素(最终浓度为34μg/mL)与康那霉素(最终浓度为30μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.1mM  IPTG进行蛋白质的诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分并以蛋白质电泳与蛋白质印迹法观察SUMO-ORF2融合蛋白的表达情形。另亦针对菌体进行可溶性蛋白质与不可溶性蛋白质的划分并以蛋白质电泳观察SUMO-ORF2融合蛋白的可溶性表达情形。
将E.coli BL21转化株接种于含氯霉素(25μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，将菌液以1：100的比例接种至含有氯霉素(25μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.2％阿拉伯糖进行蛋白质诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分并以蛋白质电泳与蛋白质印迹法观察SUMO-ORF2融合蛋白的表达情形。另亦针对菌体进行可溶性蛋白质与不可溶性蛋白质的划分并以蛋白质电泳观察SUMO-ORF2融合蛋白的可溶性表达情形。
蛋白质电泳胶片经扫描后，使用Image Quant TL 7.0(GE Healthcare Life Sciences,USA)软件估算重组SUMO-ORF2融合蛋白的表达百分比，并进一步计算融合蛋白的产量。
(2)实验结果：
结果显示，将pET-SUMO-ORF2与pET-OPTSUMO-ORF2转化入E.coli BL21(DE3)中并进行诱导，重组SUMO-ORF2融合蛋白完全不表达(图2)。将pET-SUMO-ORF2转化入可产生对应罕见密码子tRNA的E.coli Rosetta2中并进行诱导。结果显示，重组SUMO-ORF2融合蛋白可表达(图2)，且主要为可溶性蛋白质(图3)；可溶性重组SUMO-ORF2融合蛋白的产量为46.81mg/L。上述ORF2基因无法于E.coli BL21(DE3)中表达的情形说明ORF2所带有的密码子严重影响SUMO-ORF2融合蛋白于大肠杆菌中的表达。
将带有密码子最佳化ORF2基因的pET-SUMO-OPTORF2转化入E.coli BL21(DE3)中并进行诱导。结果显示，重组SUMO-ORF2融合蛋白可顺利被表达(图2)，且主要为可溶性蛋白质(图3)；可溶性重组SUMO-ORF2融合蛋白的产量为54.62mg/L。此结果说明，ORF2密码子经最佳化后，可提升SUMO-ORF2融合蛋白于E.coli BL21(DE3)中的表达。
将带有密码子最佳化的ORF2全长基因与密码子最佳化SUMO基因的pET-OPTSUMO-OPTORF2表达载体转化入E.coli BL21(DE3)中并进行诱导。结果显示，重组SUMO-ORF2融合蛋白可顺利被表达(图2)，且主要为可溶性蛋白质(图3)；可溶性重组SUMO-ORF2融合蛋白的产量为81.66mg/L。此结果说明，融合伴侣基因的密码子经最佳化后，亦可进一步提升ORF2融合蛋白于大肠杆菌中的表达。过去的研究未有针对SUMO基因密码子进行最佳化以提升融合蛋白表达量的实例。本发明证实SUMO基因 的密码子经最佳化后，可提升SUMO-ORF2融合蛋白的产量。
将带有下游序列-His标签DNA-密码子最佳化的SUMO基因-密码子最佳化ORF2基因的DNA片段嵌入阿拉伯糖诱导表达载体pBCM-araM11中并转化入E.coli BL21中进行重组SUMO-ORF2融合蛋白的生产。结果显示，利用阿拉伯糖诱导表达系统亦可生产重组SUMO-ORF2融合蛋白(图2)，且主要为可溶性蛋白质(图3)。利用此表达载体进行SUMO-ORF2融合蛋白的生产，可获得最高的产量(103.04mg/L)；与T7表达系统的最高产量(81.66mg/L)相较之下，产量约可提升1.27倍。本发明实施例一的各表达载体于此实验中表达可溶性SUMO-ORF2融合蛋白的产量统整于下表三。
表三：可溶性SUMO-ORF2融合蛋白的产量。
Figure PCTCN2015099172-appb-000005
利用固定化金属离子亲和层析进行重组SUMO-ORF2融合蛋白的纯化
利用重组SUMO-ORF2融合蛋白的N端带有His标签能与镍或钴离子形成配位共价键的特性，采用固定化金属离子亲和层析法进行蛋白质纯化。纯化的方式是利用蛋白质液相层析系统
Figure PCTCN2015099172-appb-000006
prime plus(GE Healthcare,Sweden)搭配5mL HiTrapTM Ni excel管柱(GE Healthcare,Sweden)进行。
将菌体悬浮于Lysis buffer(50mM Tris-HCl,500mM NaCl,pH 8.0)中，并利用超声波破碎仪将菌体破碎后，离心(8,000×g,15分钟)收集上清液部分。以25mL Lysis buffer平衡管柱后，将破菌上清液注入HiTrapTM Ni excel管柱。待样品注入完成后，以100mL清洗缓冲液(50mM Tris-HCl,500mM NaCl,30mM imidazole,pH 8.0)洗除非特异性结合的蛋白质。最后以150mL Elution buffer(50mM Tris-HCl,500mM NaCl,250mM imidazole,pH 8.0)洗脱树脂上的重组蛋白质，其是藉助高浓度的imidazole与重组蛋白质竞争树脂结合部位，致使重组SUMO-ORF2融合蛋白自树脂上被洗脱下来。利用蛋白质电泳观察重组SUMO-ORF2融合蛋白的纯化情形。实验结果如图4中所示。
以SUMO蛋白酶剪切本发明的SUMO-ORF2融合蛋白
本实验利用SUMO蛋白酶剪切由大肠杆菌表达系统所制得的ORF2融合蛋白。经剪 切后，将可获得带有His标签的SUMO融合伴侣片段与外鞘蛋白质片段。于本实验中将通过大肠杆菌表达系统生产SUMO蛋白酶并应用于前述用途。所属领域技术人员亦可采用其他方式取得的SUMO蛋白酶进行此步骤。
(1)重组SUMO蛋白酶表达载体pET-SUMOPH的构建：
以面包酵母基因体作为模板，利用SUMOPF(5’-CAATATGGATCCCTTGTTCCTGAATTAAATGAAAAAGACG-3’；SEQ ID NO：47)/SUMOPENZHISR(5’-GATATACTCGAGTTAGTGATGGTGATGGTGATGACCACTGCCGCTACCTTTTAAAGCGTCGGTTAAAATCAAATG-3；SEQ ID NO：48)引物组合进行SUMO蛋白酶基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，200ng面包酵母基因体及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
自酵母菌基因体扩增的SUMO蛋白酶基因以BamHI与XhoI剪切后，利用T4DNA连接酶将DNA片段接入以BamHI与SalI剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMOPH，其具有SEQ ID NO：49所示序列。
(2)重组D-SUMO蛋白酶表达载体pET-D-SUMOPH的构建：
以λ噬菌体DNA(Promega,USA)作为模板，利用DF(5’-GATATAGGTACCATGACGAGCAAAGAAACCTTTACC-3’；SEQ ID NO：50)与DR(5’-CAATATGGATCCAACGATGCTGATTGCCGTTC-3’；SEQ ID NO：51)引物组合进行D蛋白质基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ngλ噬菌体DNA及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
自λ噬菌体DNA扩增的D蛋白质基因以KpnI与BamHI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组 质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-D，其具有SEQ ID NO：99所示序列。
自酵母菌基因体扩增的SUMO蛋白酶基因以BamHI与XhoI剪切后，利用T4DNA连接酶将DNA片段接入以BamHI与SalI剪切的pET-D中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-D-SUMOPH，其具有SEQ ID NO：52所示序列。
(3)重组蛋白酶的诱导表达与纯化：
将pET-SUMOPH与pET-D-SUMOPH等表达载体分别转化入E.coli BL21(DE3)中。将E.coli BL21(DE3)转化株接种于含有康那霉素(kanamycin)(最终浓度为30μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，菌液以1：100的比例接种至含有康那霉素(最终浓度为30μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.1mM IPTG进行蛋白质的诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分进行可溶性蛋白质与不可溶性蛋白质的划分并以蛋白质电泳与蛋白质印迹法观察重组蛋白酶的可溶性表达情形。蛋白质印迹法中所使用的一级抗体与二级抗体分别为兔抗His标签多株抗体与碱性磷酸酶共轭山羊抗兔抗体；所使用的呈色剂为NBT/BCIP。重组蛋白酶的纯化方法同重组ORF2融合蛋白的纯化方法。
结果显示，SUMO蛋白酶与D-SUMO蛋白酶皆可于E.coli BL21(DE3)中表达(图5)，产量分别为20.55mg/L与46.94mg/L；其中D-SUMO蛋白酶的产量较高，其莫耳数约为SUMO蛋白酶的2.2倍。此结果说明，利用融合表达的策略可增进SUMO蛋白酶于大肠杆菌中的表达量。
接着利用重组蛋白酶的C端带有His标签的特性，采用固定化金属离子亲和层析法进行蛋白质纯化。结果显示，利用固定化金属离子亲和管柱可进行胞内可溶性重组SUMO蛋白酶与D-SUMO蛋白酶的纯化(图6)；其中D-SUMO蛋白酶的纯化产量较高，由1L培养液中可纯化21.50mg的蛋白质，约为SUMO蛋白酶纯化产量(15.33mg)的1.4倍。
(4)剪切重组SUMO-ORF2融合蛋白，并观察类病毒颗粒的形成：
将纯化的重组SUMO-ORF2融合蛋白与重组蛋白酶(SUMO蛋白酶或D-SUMO蛋白酶)以重量比为1：0.05的比例混合(例如1mg重组ORF2融合蛋白与0.05mg重组蛋白酶)，并使混合物于4℃下作用16小时。将剪切后的蛋白质溶液放入Amicon ultra-15  ultracel-100K离心管(Merck Millipore,USA)中，在4℃以2,600×g离心至适当体积。之后利用100kDa的再生纤维素(regeberated cellulose)过滤膜进行剪切蛋白质的过滤。结果显示，利用100kDa的过滤膜可有效去除融合伴侣，毋需利用管柱层析方法进行ORF2与融合伴侣的分离，可有效节省抗原生产成本(图7)。
接着，分别将SUMO-ORF2融合蛋白、经蛋白酶剪切的SUMO-ORF2融合蛋白、及经蛋白酶剪切再经过滤后所得的ORF2融合蛋白置于铜网(copper grid)上，于室温下放置三分钟。然后以滤纸(filter paper)将多余水分吸干后，加入醋酸铀酰(uranyl acetate)染剂进行负染色(negative stain)，染色时间约为40秒至1分钟。之后利用滤纸将多余染剂吸干，以场发射穿透式电子显微镜JEM-2100F(JEOL,Japan)进行类病毒颗粒的观察。
结果显示，SUMO-ORF2融合蛋白无法形成类病毒颗粒，但经蛋白酶剪切的重组SUMO-ORF2融合蛋白、及经蛋白酶剪切再经过滤后所得的ORF2融合蛋白皆可形成类病毒颗粒(图8)。由穿透式电子显微镜图形计算类病毒颗粒的平均粒径约为19nm。
实施例三：制备猪干扰素。
本发明揭露的猪干扰素为特别合适作为猪圆环病毒2型的亚单位疫苗的佐剂。是故本实施例中以大肠杆菌宿主细胞生产本发明的亚单位疫苗所需的猪干扰素α及猪干扰素γ。
重组猪干扰素α(IFN-α)与γ(IFN-γ)基因的合成
(1)IFN-α基因的合成：
依据大肠杆菌的偏爱密码子将成熟猪干扰素α-6的氨基酸序列反向推导为核苷酸序列。依据前述核苷酸序列设计引物：OPTIFNA-T1、OPTIFNA-T2、OPTIFNA-T3、OPTIFNA-T4、OPTIFNA-T5、OPTIFNA-T6、OPTIFNA-T7、OPTIFNA-T8、OPTIFNAF及OPTIFNAR，其序列如下表四所示。
表四：密码子最佳化猪干扰素α-6基因合成所用引物。
Figure PCTCN2015099172-appb-000007
Figure PCTCN2015099172-appb-000008
以OPTIFNA-T1～OPTIFNA-T8作为模板引物，OPTIFNAF与OPTIFNAR则作为扩增引物。利用重叠延伸聚合酶链式反应大量扩增密码子最佳化的IFN-α基因。在50μL的PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM的各引物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、58℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
利用CloneJET PCR Cloning Kit(Thermo,USA)进行基因克隆，并将黏合混合物(ligation mixture)转化入大肠杆菌ECOS 9-5。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pJET-IFNA-6，其具有如SEQ ID NO：63所示序列。经定序验证，密码子最佳化的IFN-α基因具有SEQ ID NO：64所示序列。
(2)IFN-γ的合成：
依据大肠杆菌的偏爱密码子将成熟猪干扰素γ的氨基酸序列反向推导为核苷酸序列。依据前述核苷酸序列设计引物：OPTIFNR-T1、OPTIFNR-T2、OPTIFNR-T3、OPTIFNR-T4、OPTIFNR-T5、OPTIFNR-T6、OPTIFNR-T7、OPTIFNR-T8、OPTIFNRF及OPTIFNRR，其序列如下表五所示。
表五：密码子最佳化猪干扰素γ基因的合成所用引物。
Figure PCTCN2015099172-appb-000009
Figure PCTCN2015099172-appb-000010
以OPTIFNR-T1～OPTIFNR-T8作为模板引物，OPTIFNRF与OPTIFNRR则作为扩增引物。利用重叠延伸聚合酶链式反应大量扩增密码子最佳化的IFN-γ基因。在50μL的PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM的各引物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、57℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
利用CloneJET PCR Cloning Kit进行基因克隆，并将黏合混合物转化入大肠杆菌ECOS 9-5。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pJET-IFNR，其具有如SEQ ID NO：75所示序列。经定序验证，密码子最佳化的IFN-γ基因具有SEQ ID NO：76所示序列。
猪干扰素α与γ表达载体的构建
(1)pET-OPTPIFNAH表达载体的构建：
以pJET-IFNA-6质粒作为模板，利用PIFNANDEIF(5’-CAATATCATATGTGCGATCTGCCGCAAACC-3’；SEQ ID NO：77)/PIFNAHISSALIR(5’-GATATAGTCGACTTATTAGTGATGGTGATGGTGATGTTCCTTTTTACGCAGGCGGTC-3’；SEQ ID NO：78)引物组合进行IFN-α基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液 B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pJET-IFNA-6及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以NdeI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTPIFNAH，其具有SEQ ID NO：79所示序列。
(2)pBA-OPTPIFNAH表达载体的构建：
利用上述PCR扩增的IFN-α基因以NdeI与SalI剪切后，利用T4DNA连接酶将DNA片段分别接入以相同限制酶剪切的pBCM-araM11中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒分别命名为pBA-OPTPIFNAH，其具有SEQ ID NO：80所示序列。
(3)pET-SUMO-OPTPIFNAH表达载体的构建：
以面包酵母基因体作为模板，利用SUMOF(SEQ ID NO：25)/SUMOR2(5’-ACCACCAATCTGTTCTCTGTGAGC-3’；SEQ ID NO：81)引物组合进行SUMO基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，200ng面包酵母基因体及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以pJET-IFNA-6质粒作为模板，利用SUMOIFNAF(5’-GCTCACAGAGAACAGATTGGTGGTTGCGATCTGCCGCAAACC-3’；SEQ ID NO：82)/PIFNAHISSALIR(SEQ ID NO 78)引物组合进行IFN-α基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pJET-IFNA-6及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应 30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以上述的两个PCR产物作为模板，利用SUMOF(SEQ ID NO：25)/PIFNAHISSALIR(SEQ ID NO：78)引物组合进行聚合酶链式反应即可获得SUMO-IFN-α融合基因。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng SUMO PCR产物，100ng IFN-αPCR产物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应1分钟(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以KpnI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMO-OPTPIFNAH，其具有SEQ ID NO：83所示序列。
(4)pET-OPTSUMO-OPTPIFNAH表达载体的构建：
以pET-OPTSUMO-ORF2(SEQ ID NO：43)作为模板，利用OPTSUMOF(SEQ ID NO：35)/OPTSUMOR2(5’-GCCGCC GATTTGTTCACGG-3’；SEQ ID NO：84)引物组合进行OPTSUMO基因的扩增。
在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pET-OPTSUMO-ORF2及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以pJET-IFNA-6质粒(SEQ ID NO：63)作为模板，利用OPTSUMOIFNAF(CCGTGAACAAATCGGCGGCTGCGATCTGCCGCAAACC；SEQ ID NO：85)/PIFNAHISSALIR(SEQ ID NO：78)引物组合进行IFN-α基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP， 1μM扩增引物，100ng pJET-IFNA-6及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以上述的两个PCR产物作为模板，利用OPTSUMOF(SEQ ID NO：35)/PIFNAHISSALIR(SEQ ID NO：78)引物组合进行聚合酶链式反应即可获得OPTSUMO-IFN-α融合基因。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng OPTSUMO PCR产物，100ng IFN-αPCR产物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应1分钟(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以KpnI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTSUMO-OPTPIFNAH，其具有SEQ ID NO：86所示序列。
(5)pBA-OPTSUMO-OPTPIFNAH表达载体的构建：
pET-OPTSUMO-OPTPIFNAH以NdeI与SalI剪切后，利用Gel-MTM gel extraction system kit回收含有OPTSUMO-IFN-α融合基因的DNA片段。利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pBCM-araM11中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pBA-OPTSUMO-OPTPIFNAH，其具有SEQ ID NO：87所示序列。
(6)pET-OPTPIFNRH表达载体的构建：
以pJET-IFNR质粒作为模板，利用PIFNRNDEIF(5’-CAATATCATATGCAAGCCCCGTTTTTCAAAGAA-3’；SEQ ID NO：88)/PIFNRHISSALIR(5’-GATATAGTCGACTTATTAGTGATG GTGATGGTGATGTTTGCTGGCACGCTGACC-3’；SEQ ID NO：89)引物组合进行IFN-γ基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pJET-IFNR及1U GDP-HiFi  DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以NdeI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTPIFNRH，其具有SEQ ID NO：90所示序列。
(7)pET-SUMO-OPTPIFNRH表达载体的构建：
以面包酵母基因体作为模板，利用SUMOF(SEQ ID NO：25)/SUMOR2(SEQ ID NO：81)引物组合进行SUMO基因的扩增。扩增条件与PCR回收方式如前所述。
以pJET-IFNR质粒(SEQ ID NO：75)作为模板，利用SUMOIFNRF(5’-GCTCACAG AGAACAGATTGGTGGTCAAGCCCCGTTTTTCAAAGAA-3’；SEQ ID NO：91)/PIFNRHISSALIR(SEQ ID NO：89)引物组合进行IFN-γ基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pJET-IFNR及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以上述的两个PCR产物作为模板，利用SUMOF(SEQ ID NO：25)/PIFNRHISSALIR(SEQ ID NO：89)引物组合进行聚合酶链式反应即可获得SUMO-IFN-γ融合基因。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng SUMO PCR产物，100ng IFN-γPCR产物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应1分钟(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以KpnI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反 应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-SUMO-OPTPIFNRH，其具有SEQ ID NO：92所示序列。
(8)pET-OPTSUMO-OPTPIFNRH表达载体的构建：
以pET-OPTSUMO-ORF2(SEQ ID NO：43)作为模板，利用OPTSUMOF(SEQ ID NO：35)/OPTSUMOR2(SEQ ID NO：84)引物组合进行OPTSUMO基因的扩增。扩增条件与PCR回收方式如前所述。
以pJET-IFNR质粒(SEQ ID NO：75)作为模板，利用OPTSUMOIFNRF(5’-CCGTGAACAAATCGGCGGCCAAGCCCCGTTTTTCAAAGAAATC-3’；SEQ ID NO：93)/PIFNRHISSALIR(SEQ ID NO：89)引物组合进行猪干扰素γ基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng pJET-IFNR及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用Gel-MTM gel extraction system kit进行PCR产物的回收。
以上述的两个PCR产物作为模板，利用OPTSUMOF(SEQ ID NO：35)/PIFNRHISSALIR(SEQ ID NO：89)引物组合进行聚合酶链式反应即可获得OPTSUMO-IFN-γ融合基因。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，100ng OPTSUMO PCR产物，100ng猪干扰素γPCR产物及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应2分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应1分钟(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
PCR产物以KpnI与SalI剪切后，利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pET29a中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-OPTSUMO-OPTPIFNRH，其具有SEQ ID NO：94所示序列。
(9)pBA-OPTSUMO-OPTPIFNRH表达载体的构建：
pET-OPTSUMO-OPTPIFNRH以NdeI与SalI剪切后，利用Gel-MTM gel extraction  system kit回收含有OPTSUMO-IFR-γ融合基因的DNA片段。利用T4DNA连接酶将DNA片段接入以相同限制酶剪切的pBCM-araM11中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pBA-OPTSUMO-OPTPIFNRH，其具有SEQ ID NO：95所示序列。
重组猪干扰素的表达及纯化
(1)重组猪干扰素的表达：
将pET-OPTPIFNAH(SEQ ID NO：79)、pBA-OPTPIFNAH(SEQ ID NO：80)、pET-SUMO-OPTPIFNAH(SEQ ID NO：83)、pET-OPTSUMO-OPTPIFNAH(SEQ ID NO：86)及pBA-OPTSUMO-OPTPIFNAH(SEQ ID NO：87)分别转化至E.coli Shuffle(NEB,USA)中；将pET-OPTPIFNRH(SEQ ID NO：90)、pET-SUMO-OPTPIFNHR(SEQ ID NO：92)、pET-OPTSUMO-OPTPIFNRH(SEQ ID NO：94)及pBA-OPTSUMO-OPTPIFNRH(SEQ ID NO：95)分别转化入E.coli BL21(DE3)中。将转化株接种于含有康那霉素(最终浓度为30μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，菌液以1：100的比例接种至含有康那霉素(最终浓度为30μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.1mM IPTG于25℃与180rpm的条件下进行蛋白质的诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分并以蛋白质电泳观察重组猪干扰素的表达情形。另亦针对菌体进行可溶性蛋白质与不可溶性蛋白质的划分并以蛋白质电泳观察重组猪干扰素的可溶性表达情形。
请参见图9中(A)至(E)的实验结果。结果显示本发明藉由E.coli Shuffle宿主成功生产可溶性重组猪IFN-α与SUMO-IFN-α融合蛋白，可免除折叠步骤，以避免折叠效率不佳影响生物活性的问题。在SUMO-IFN-α融合蛋白表达部分，将SUMO基因的密码子经最佳化后，可提升SUMO-IFN-α融合蛋白的产量。不同表达系统对SUMO-IFN-α融合蛋白表达的影响结果显示，利用突变型阿拉伯糖诱导表达系统生产SUMO-IFN-α的产量较高(155.07mg/L；图9中(E))。又请参见图9中(F)至(I)的实验结果。结果显示利用SUMO融合的策略可增进重组猪IFN-γ的可溶性。SUMO基因的密码子经最佳化后，可提升SUMO-IFN-γ融合蛋白的产量。不同表达系统对SUMO-IFN-γ融合蛋白表达的影响结果显示，利用T7诱导表达系统及突变型阿拉伯糖诱导表达系统生产SUMO-IFN-γ的产量皆相当理想。
(2)重组SUMO蛋白酶表达载体pET-D-SUMOP的构建与表达：
为了剪切前述段落中所述于大肠杆菌表达系统中表达的猪干扰素，以取得不带有SUMO蛋白质片段的猪干扰素，本实验中将通过大肠杆菌表达系统生产SUMO蛋白酶。所属领域技术人员亦可采用其他方式取得的SUMO蛋白酶进行此步骤。
以面包酵母基因体作为模板，利用SUMOPF(SEQ ID NO：47)/SUMOPENZR(5’-GATATACTCGAGTTATTTTAAAGCGTCGGT TAAAATCAAATG-3；SEQ ID NO：96)引物组合进行SUMO蛋白酶基因的扩增。在50μL PCR反应混合物中包含1倍GDP-HiFi PCR缓冲液B，200μM的dATP、dTTP、dGTP与dCTP，1μM扩增引物，200ng面包酵母基因体及1U GDP-HiFi DNA聚合酶。PCR反应条件为96℃反应5分钟(1个步骤)；94℃反应30秒、55℃反应30秒、68℃反应30秒(35个循环)；68℃反应5分钟(1个步骤)。PCR反应结束后，利用琼脂糖凝胶电泳确认有无预估大小的DNA片段。然后利用PCR-MTM Clean Up system kit进行PCR产物的回收。
自酵母菌基因体扩增的SUMO蛋白酶基因以BamHI与XhoI剪切后，利用T4DNA连接酶将DNA片段接入以BamHI与SalI剪切的pET-D中。将黏合产物转化进入大肠杆菌ECOS 9-5中。以菌落聚合酶链式反应挑选转化株。利用DNA电泳确认转化株中的重组质粒带有外插DNA后，抽取转化株中的质粒并进行DNA定序。将DNA序列正确无误的质粒命名为pET-D-SUMOP，其具有SEQ ID NO：97所示序列。
将pET-D-SUMOP(SEQ ID NO 97)转化入E.coli BL21(DE3)中。将E.coli BL21(DE3)转化株接种于含有康那霉素(最终浓度为30μg/mL)的LB培养基中，于37℃与180rpm的条件下进行振荡培养。经隔夜培养后，菌液以1：100的比例接种至含有康那霉素(最终浓度为30μg/mL)的LB培养基中。于37℃与180rpm的条件下进行振荡培养。将细菌培养至以分光光度计量测细胞浓度达OD600约0.4～0.6，加入0.1mM IPTG于28℃与180rpm的条件下进行蛋白质的诱导表达。诱导4小时后，离心(8,000×g，30分钟，4℃)收集菌体部分。
(3)重组猪干扰素的剪切与纯化：
将分别带有SUMO-猪干扰素融合蛋白表达载体与SUMO蛋白酶表达载体的转化株经诱导表达后，离心(8,000×g，30分钟，4℃)收集菌体部分。将收集的菌体悬浮于适量的Lysis buffer(20mM sodium phosphate,500mM NaCl,pH 7.4)中，使其于600nm下的吸光值达50。利用超声波破碎仪将菌体破碎后，离心(8,000×g，15分钟，4℃)收集上清液部分。将纯化的重组SUMO-猪干扰素融合蛋白与重组蛋白酶(SUMO蛋白酶)以 重量比值为4的比例混合，于4℃静置16小时；此阶段中，SUMO-猪干扰素融合蛋白会被SUMO蛋白酶剪切成SUMO蛋白质与C端带有His标签的猪干扰素。
接着采用固定化金属离子亲和层析法进行蛋白质纯化。纯化的方式是利用蛋白质液相层析系统
Figure PCTCN2015099172-appb-000011
prime plus搭配5mL HiTrapTM Ni excel管柱进行。以25mL Lysis buffer平衡管柱后，将融合蛋白剪切溶液注入HiTrapTM Ni excel管柱。待样品注入完成后，以100mL清洗缓冲液(20mM sodium phosphate,500mM NaCl,30mM imidazole,pH 7.4)洗除非特异性结合的蛋白质。最后以150mL Elution buffer(20mM sodium phosphate,500mM NaCl,250mM imidazole,pH 7.4)洗脱树脂上的重组猪干扰素并利用蛋白质电泳观察纯化情形(如图10中所示)。
实施例四：本发明的防治猪圆环病毒2型感染的组合物的制备及应用。
本实施例是使用前述实施例二及实施例三所制得的ORF2、SUMO-ORF2融合蛋白、及猪干扰素制备防治猪圆环病毒2型感染的组合物。在许多样品中，该组合物进一步包含MONTANIDETM ISA 563 VG佐剂(SEPPIC,France)及/或MONTANIDETM GEL 01佐剂(SEPPIC,France)。将各项成分依下列各实验设计混合，再接种于仔猪，以观察诱发的免疫反应或是否产生不良反应(如，呕吐、发抖、精神抑郁、呼吸急促及施打部位肿胀情形；若出现3种以上的前述症状且症状出现的比例高于50％，则判定该组合物的安全性较低)。
(1)实验1：猪干扰素含量对本发明组合物的安全性的影响：
选择3周龄田间仔猪14头，以随机方式进行分组，共分为A～G七组，每组猪只数目为2头。各组进行1次肌肉注射免疫，免疫剂量为2mL。有关各疫苗成分如下表六。于施打疫苗当天与隔天进行观察并记录不良临床反应出现比例。
表六：实验1的实验设计。
Figure PCTCN2015099172-appb-000012
实验结果显示(表七)，经接种V-001样品的猪只呈现精神抑郁的临床症状，但无其他不良反应。经接种V-002样品的猪只出现呕吐与发抖的症状。此外，V-003样品的安全性较具疑虑，而经接种V-004样品、V-005样品、或V-006样品的猪只的不良反应较为轻微。据此后续试验中猪干扰素的含量将维持于每剂量(2mL)为25μg。
表七：实验1的实验结果。
Figure PCTCN2015099172-appb-000013
(2)实验2：佐剂含量对本发明组合物的安全性的影响：
选择3周龄田间仔猪73头，以随机方式进行分组，共分为A与B两组；A组猪只数目为38头，B组则为35头。各组进行1次肌肉注射免疫，免疫剂量为2mL。有关各疫苗成分如下表八。于施打疫苗当天与隔天进行观察并记录不良临床反应出现比例。
表八：实验2的实验设计。
Figure PCTCN2015099172-appb-000014
实验结果显示(表九)，V-009样品的安全性较高，但V-008样品的安全性亦可接受。
表九：实验2的实验结果。
Figure PCTCN2015099172-appb-000015
(3)实验3：不同佐剂对本发明组合物的免疫诱发效果的影响：
本实验于家畜卫生试验所动物用药品检定分所基因改造产品(genetically modified organisms,GMOs)动物舍中进行。选择4周龄无特定病原猪只11头，以随机方式进行分组，共分为A～E五组；A～D为实验组，每组猪只数目为2头，E组为对照组(Control)，猪只头数为3头。A～D组的猪只于4与6周龄各进行1次肌肉注射免疫，免疫剂量为2mL； E组不进行免疫处理。有关各疫苗成分如下表十。
表十：实验3的实验设计。
Figure PCTCN2015099172-appb-000016
各组猪只于8周龄时，进行PCV2攻毒，并于攻毒后4周，全数进行剖检。于猪只免疫前(4周龄)、免疫后(6与8周龄)及攻毒后(9、10、11、12周龄)进行血清与血浆样品的收集。利用市售ELISA套组(BioCheck,Netherlands)测定血清中抗PCV2抗体的效价。利用即时定量聚合酶链式反应测定血浆中的病毒含量。
实验结果显示V-009、V-010、V-011、及V-012皆能诱发抗ORF2抗体产生(图11)，且能降低实验猪只的病毒血症(图12)。从实验结果亦可观察到每剂量(2mL)含有67μg ORF2的含量即可产生足够的免疫效果(V-011及V-012)。
(4)实验4：采用SUMO-ORF2融合蛋白或OFR2对本发明组合物的免疫诱发效果的影响：
本实验于家畜卫生试验所动物用药品检定分所基因改造产品动物舍中进行。选择4周龄无特定病原猪只16头，以随机方式进行分组，共分为A～E五组；A～D为实验组，每组猪只数目为3头，E组为对照组，猪只头数为4头。A～D组的猪只于4与6周龄各进行1次肌肉注射免疫，免疫剂量为2mL；E组不进行免疫处理。有关各疫苗成分如下表十一。
表十一：实验4的实验设计。
Figure PCTCN2015099172-appb-000017
各组猪只于8周龄时，进行PCV2攻毒，并于攻毒后5周，全数进行剖检。在特定时间点下进行血清与血浆样品的收集。利用市售ELISA套组测定血清中抗PCV2抗体的效价。利用即时定量聚合酶链式反应测定血浆中的病毒含量。
实验结果显示各样品都能诱发猪只产生抗PCV2抗体，其中又以V-013样品(含27μg的ORF2融合蛋白)的效果最好(图13)。此外，所有样品都能降低猪只的病毒血症(图14)。
(5)实验5：猪干扰素α与猪干扰素γ对本发明组合物的免疫诱发效果的影响：
本实验于病原污染程度较低且无PCV2感染的牧场中进行。选用无感染PCV2的4周龄1代SPF猪只20头。以随机方式进行分组，共分为A～E五组，每组猪只数目为4头；A～D为实验组，E组为对照组。A～D组的猪只于4与7周龄各进行1次肌肉注射免疫，免疫剂量为2mL；E组不进行免疫处理。有关各疫苗成分如下表十二。在特定时间点下进行血清样品的收集。利用市售ELISA套组测定血清中抗PCV2抗体的效价。
表十二：实验5的实验设计。
Figure PCTCN2015099172-appb-000018
实验结果显示，单独添加IFN-α(V-018)或IFN-γ(V-019)于本发明组合物中对于诱发免疫反应均有增强的效果；其中添加IFN-α的效果优于添加IFN-γ。另一方面，于本发明组合物中同时添加IFN-α与IFN-γ(V-020)可诱发更好的免疫反应，显示在本发明组合物中，IFN-α与IFN-γ对于增强免疫反应具有协同效果(图15)。

Claims (19)

  1. 一种表达蛋白质的方法，其包含：
    (a)取得阿拉伯糖诱导表达载体；其中前述阿拉伯糖诱导表达载体包含表达元件及目标蛋白质的核苷酸序列；
    其中前述表达元件包含：
    启动子；
    T7噬菌体转译增强元件，其具有SEQ ID NO：01所示序列；及
    核糖体结合部位，其具有SEQ ID NO：02所示序列；
    (b)将前述阿拉伯糖诱导表达载体转化至大肠杆菌宿主中，并进行目标蛋白质的诱导表达；
    其中前述目标蛋白质为：猪圆环病毒2型的外鞘蛋白质或猪干扰素。
  2. 如权利要求1所述的方法，其中前述启动子的-16部位具有SEQ ID NO：03所示序列。
  3. 如权利要求1所述的方法，其中前述表达元件具有SEQ ID NO：04所示序列。
  4. 如权利要求1所述的方法，其中前述阿拉伯糖诱导表达载体进一步包含融合伴侣的核苷酸序列、及/或标记分子的核苷酸序列。
  5. 如权利要求4所述的方法，其中前述融合伴侣为：大肠杆菌MsyB、大肠杆菌YjgD基因、λ噬菌体D蛋白质、面包酵母菌SUMO蛋白质、或其组合。
  6. 如权利要求4所述的方法，其中前述标记分子为：His标签、Strep II标签、Flag标签、或其组合。
  7. 如权利要求1所述的方法，其中前述目标蛋白质为猪圆环病毒2型的外鞘蛋白质，且其核苷酸序列具有SEQ ID NO：09或SEQ ID NO：24所示序列。
  8. 如权利要求7所述的方法，其中前述阿拉伯糖诱导表达载体具有SEQ ID NO：46所示序列。
  9. 如权利要求1所述的方法，其中前述猪干扰素为猪干扰素α或猪干扰素γ。
  10. 如权利要求9所述的方法，其中前述目标蛋白质为猪干扰素，且其核苷酸序列具有SEQ ID NO：64或SEQ ID NO：76所示序列。
  11. 如权利要求10所述的方法，其中前述阿拉伯糖诱导表达载体具有SEQ ID NO：80、SEQ ID NO：87、或SEQ ID NO：95所示序列。
  12. 如权利要求11所述的方法，其不包含前述猪干扰素的折叠步骤。
  13. 如权利要求8或11所述的方法，其中前述步骤(b)之后进一步包含步骤(c)：纯化前述目标 蛋白质。
  14. 如权利要求13所述的方法，其中前述步骤(c)之后进一步包含步骤(d)：以一SUMO蛋白酶处理前述目标蛋白质。
  15. 如权利要求14所述的方法，其中前述步骤(d)的处理中，前述目标蛋白质与前述SUMO蛋白酶的重量比值为4至20。
  16. 一种防治猪圆环病毒2型感染的组合物，其包含：
    2.5至250μg/mL的猪圆环病毒2型的外鞘蛋白质；
    2.5至25μg/mL的猪干扰素α；
    2.5至25μg/mL的猪干扰素γ；及
    医药可接受的载剂。
  17. 如权利要求16所述的组合物，其进一步包含医药可接受的佐剂。
  18. 如权利要求17所述的组合物，其中前述医药可接受的佐剂为：MONTANIDETM ISA 536VG佐剂、MONTANIDETM GEL 01佐剂、弗氏完全或不完全佐剂、铝胶、表面活性剂、阴离子型聚合物、肽、油乳液、或其组合。
  19. 如权利要求16所述的组合物，其包含：
    3.5至170μg/mL的猪圆环病毒2型的外鞘蛋白质；
    5至20μg/mL的猪干扰素α；
    5至20μg/mL的猪干扰素γ；及
    医药可接受的载剂。
PCT/CN2015/099172 2015-12-28 2015-12-28 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物 WO2017113050A1 (zh)

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DK15911682.1T DK3399040T3 (da) 2015-12-28 2015-12-28 Fremgangsmåde til fremstilling af porcine circovirus type 2 capsid-protein og farmaceutisk sammensætning omfattende samme
JP2018534063A JP6629975B2 (ja) 2015-12-28 2015-12-28 ブタシルコウイルス２型キャプシドタンパク質の調製方法及びそれを含む医薬組成物
KR1020187018139A KR102055215B1 (ko) 2015-12-28 2015-12-28 돼지 써코바이러스 2형 캡시드 단백질 및 이를 포함하는 약학적 조성물의 제조 방법
RU2018127469A RU2701816C1 (ru) 2015-12-28 2015-12-28 Способ получения капсидного белка цирковируса свиней 2 типа и содержащая его фармацевтическая композиция
CA3009903A CA3009903C (en) 2015-12-28 2015-12-28 Method of preparing porcine circovirus type 2 capsid protein and pharmaceutical composition comprising same
EP15911682.1A EP3399040B1 (en) 2015-12-28 2015-12-28 Method of preparing porcine circovirus type 2 capsid protein and pharmaceutical composition comprising same
ES15911682T ES2881625T3 (es) 2015-12-28 2015-12-28 Método de preparación de la proteína de la cápside del circovirus porcino tipo 2 y composición farmacéutica que la comprende
BR112018013007-9A BR112018013007B1 (pt) 2015-12-28 Método para expressar uma proteína, e composição para prevenir infecção por circovírus suíno tipo 2 (pcv2)
CN201580082991.5A CN108026538B (zh) 2015-12-28 2015-12-28 猪圆环病毒2型的外鞘蛋白质的制备方法及含该外鞘蛋白质的医药组合物
US15/778,062 US10767185B2 (en) 2015-12-28 2015-12-28 Method of preparing porcine circovirus type 2 capsid protein and pharmaceutical composition comprising same
PH12018501183A PH12018501183A1 (en) 2015-12-28 2018-06-04 Method of preparing porcine circovirus type 2 capsid protein and pharmaceutical composition comprising same

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