CN116693631A - Porcine circovirus type III recombinant Cap protein, virus-like particles assembled by same and application thereof - Google Patents
Porcine circovirus type III recombinant Cap protein, virus-like particles assembled by same and application thereof Download PDFInfo
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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Abstract
The invention mainly aims at providing a porcine circovirus type III recombinant Cap protein, virus-like particles assembled by the same and application thereof. According to the invention, the N end of the wild porcine circovirus type III Cap protein is subjected to nuclear localization signal sequence removal to obtain the recombinant porcine circovirus type III Cap protein, which can be expressed in a large amount in escherichia coli and can be self-assembled into virus-like particles.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a porcine circovirus type III recombinant Cap protein, virus-like particles assembled by the same and application thereof.
Background
In 2016, a sow farm in the united states developed clinical symptoms of swine dermatitis and nephrotic syndrome (PDNS) and reproductive dysfunction, and porcine circovirus type II PCV2 was detected as negative, followed by detection of porcine circovirus type III (PCV 3) using metagenomic sequencing techniques.
The phylogenetic tree analysis shows that the Cap protein nucleotide similarity of the porcine circovirus type I (PCV 1) and PCV2 is 67%, the Cap protein nucleotide similarity of the PCV1 and PCV3 is lower and is only 24%, and the Cap protein nucleotide similarity of the PCV2 and PCV3 is only 26-37%, so that the existing circovirus vaccine has no cross protection capability on novel PCV 3.
To develop a vaccine that can prevent PCV3, researchers have tried to vaccinate infected pig kidney cells (PK-15) and pig testis cells (ST) with PCV3 positive tissue disease homogenates (PALINSKI et al 2017b,FACCINI et al, 2017), none of these virus isolation assays were successful. No cytopathic effect was observed after inoculation of the passaged cell lines with virus, and proliferation of the viral genome was examined by specific PCR, which found that the viral genome content did not increase after each cell passage (PALINSKI et al 2017b,FACCINI et al, 2017). Thus, the current difficulty in isolation of PCV3 strains severely limits further in-depth investigation of the virus.
The preparation of viral capsid structural proteins and assembly is an important method for obtaining virus-like particles, and chinese patent application No. 201810912587.1 discloses a method for preparing PCV3 Cap proteins using baculoviruses, and the proteins have not been further reported to be capable of self-assembly to produce virus-like particles.
Disclosure of Invention
The invention mainly aims to provide a porcine circovirus type III recombinant Cap protein, virus-like particles assembled by the porcine circovirus type III recombinant Cap protein and application thereof, and aims to provide a recombinant protein which can take escherichia coli as competent cells and can self-assemble and produce virus-like particles.
In order to achieve the above purpose, the invention provides a porcine circovirus type III recombinant Cap protein, wherein the amino acid sequence of the porcine circovirus type III recombinant Cap protein is shown as SEQ ID NO: 1.
In addition, the invention also provides a gene for encoding the porcine circovirus type III recombinant Cap protein, and the sequence of the gene for encoding the porcine circovirus type III recombinant Cap protein is shown as SEQ ID NO: 2.
In addition, the invention also provides a recombinant protein, which comprises an active part and a fusion short peptide, wherein the amino acid sequence of the active part is shown as SEQ ID NO:1, wherein the fusion oligopeptide is connected to the C-terminal end of the active part.
Alternatively, the fusion oligopeptide is selected from the group consisting of SEQ ID NOs: 3, and the polypeptide shown as SEQ ID NO:4 and a polypeptide as set forth in SEQ ID NO:5, and a polypeptide shown in the formula (I).
In addition, the invention also provides a gene for encoding the recombinant protein.
In addition, the invention also provides a recombinant vector, which comprises the coding gene.
In addition, the invention also provides a host, which comprises the recombinant vector.
In addition, the invention also provides application of the porcine circovirus type III recombinant Cap protein, the coding gene thereof, the recombinant vector or the host in any aspect as follows:
preparing virus-like particles; the method comprises the steps of,
a formulation is prepared that can elicit an immune response in an organism against porcine circovirus type III.
In addition, the invention also provides a virus-like particle, which is formed by self-assembly of the proteins.
In addition, the invention also provides the application of the virus-like particle in preparing a preparation which can cause the immune response of organisms to porcine circovirus type III.
According to the invention, the nuclear localization signal sequence is removed from the N end of the wild porcine circovirus type III Cap protein to obtain the recombinant porcine circovirus type III Cap protein, so that the expression level in escherichia coli is increased, and virus-like particles can be self-assembled.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the detection of amplified fragments in example 1;
FIG. 2 is a diagram showing the PCR detection results of the recombinant plasmid of example 1;
FIG. 3 is a diagram showing the recombinant protein expression test of example 1;
FIG. 4 is a graph showing the recombinant protein expression test after purification in example 1;
FIG. 5 is an electron microscope image of the recombinant protein purified in example 1;
FIG. 6 is a diagram showing detection of amplified fragment of comparative example 1;
FIG. 7 is a diagram showing the PCR detection result of the recombinant plasmid of comparative example 1;
FIG. 8 is a graph showing the recombinant protein expression test after purification of comparative example 1;
FIG. 9 is a graph showing the detection of VLPs produced by self-assembly during purification of comparative example 1;
FIG. 10 is a diagram showing detection of amplified fragments in examples 2 to 4;
FIG. 11 is a graph showing the PCR detection results of recombinant plasmids of examples 2 to 4;
FIG. 12 is a view showing the detection of VLPs produced by self-assembly in example 2;
FIG. 13 is a view showing the detection of VLPs produced by self-assembly during purification in example 3;
FIG. 14 is a view showing the detection of VLPs produced by self-assembly during purification in example 4;
FIG. 15 is a diagram showing the detection of an antibody in example 5.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
The specific conditions were not specified in the examples, and the examples were conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In view of the fact that the existing preparation of porcine circovirus type III Cap proteins cannot use E.coli as a vector and self-assemble into virus-like particles. The invention provides a porcine circovirus type III recombinant Cap protein, wherein the amino acid sequence of the porcine circovirus type III recombinant Cap protein is shown as SEQ ID NO: 1.
According to the invention, a nuclear localization signal sequence is removed from the N end of a wild porcine circovirus III type Cap protein, and a short peptide is fused with the C end, so that the recombinant porcine circovirus III type Cap protein is obtained, the expression quantity in escherichia coli is increased, and virus-like particles can be self-assembled.
In addition, the invention also provides a gene for encoding the porcine circovirus type III recombinant Cap protein, and the sequence of the gene for encoding the porcine circovirus type III recombinant Cap protein is shown as SEQ ID NO: 2. By optimizing the coding gene, the expression of SEQ ID NO:1, and the soluble expression content can be further improved.
In addition, the recombinant protein comprises an active part and a fusion short peptide, wherein the amino acid sequence of the active part is shown in SEQ ID NO:1, wherein the fusion oligopeptide is connected to the C-terminal end of the active part. The content of virus-like particles obtained by self-assembly can be further improved by adding fusion short peptide.
Specifically, in some embodiments, the sequence of the fusion oligopeptide is selected from the group consisting of SEQ ID NOs: 3, and the polypeptide shown as SEQ ID NO:4 and a polypeptide as set forth in SEQ ID NO:5, and the self-assembly yield of the virus-like particles can be improved by any one of the polypeptides shown in the formula (5). And the research shows that when the short peptide is shown as SEQ ID NO:3, the content of virus-like particles is further increased.
In addition, the invention also provides a gene for encoding the recombinant protein. Specifically, if the sequence of the fusion oligopeptide is selected from the group consisting of SEQ ID NOs: 3, the corresponding coding gene of the polypeptide is shown as SEQ ID NO:6 is shown in the figure; if the sequence of the fusion oligopeptide is selected from the group consisting of SEQ ID NO:4, and the corresponding coding gene is shown as SEQ ID NO: shown in figure 7; if the sequence of the fusion oligopeptide is selected from the group consisting of SEQ ID NO:5, and the corresponding coding gene is shown as SEQ ID NO: shown at 8. By adopting the optimized coding gene, the assembly efficiency of the virus-like particle can be further improved.
In addition, the invention also provides a recombinant vector, which comprises a coding gene of the corresponding recombinant protein.
It should be noted that the recombinant vector of the present invention may be prepared by the following method:
after cloning the coding gene by using a primer, introducing the coding gene into a vector plasmid by using an enzyme digestion and enzyme ligation method.
In the present invention, the choice of the vector plasmid is not limited as long as it can be ligated to the desired gene. In some embodiments, the vector plasmid pET28a.
In the present invention, the primer is not limited in terms of its choice in the case where the coding gene can be cloned.
For example, in some embodiments, the coding gene is as set forth in SEQ ID NO:2, the sequence of the primer is shown as SEQ ID NO:9 to 10; the coding gene is shown as SEQ ID NO:6, the sequence of the primer is shown as SEQ ID NO:11 to 12; the coding gene is shown as SEQ ID NO:7, the sequence of the primer is shown as SEQ ID NO:13 to 14; the coding gene is shown as SEQ ID NO:8, the sequence of the primer is shown as SEQ ID NO:15 to 16. By the above primer design, amplification efficiency can be further improved.
In addition, the invention also provides a host, which comprises the recombinant vector. On the premise of expressing the porcine circovirus type III recombinant Cap protein, the host selection is not selected. In some embodiments, the host is E.coli.
The escherichia coli expression system is used as a common prokaryotic expression system, has the advantages of simple culture procedure, clear genetic background and the like, and moreover, the escherichia coli is utilized to express a large amount of soluble and unlabeled Cap proteins, and the proteins can form a large amount of VLPs in a neutral buffer solution.
Further, the host is ClearColiTMBL21 (DE 3); since this host is used for an outer membrane agonist of hTLR4/MD-2 activation, lipopolysaccharide (LPS) is eliminated from the source.
The host is obtained by transduction of competent cells with the recombinant vector described above.
In addition, in the present invention, the preparation method of two recombinant proteins comprises the steps of:
step S10: culturing the host to obtain a culture host;
step S20: and (3) carrying out induced expression on the culture host, separating protein in the expression, and purifying to obtain the recombinant DNA.
In the step S10, the culture medium appropriate for the host cells is selected, and the host is subjected to the expansion culture to obtain a cultured host.
If the host is E.coli, the step S20 includes:
step S201, performing induction expression on a host by adopting IPTG, and collecting the host after induction expression;
step S202: crushing a host, performing solid-liquid separation, and collecting supernatant;
and step 203, performing column chromatography on the supernatant to obtain recombinant protein.
By the above-described operations, the host protein can be isolated, and a target protein of high purity can be obtained.
Further, in the step of performing column chromatography in step S203, the chromatographic column is eluted with an eluent, wherein the eluent includes:
when the eluent is adopted, the high-purity expressed protein can be obtained, and the self-assembly of the protein can be carried out to generate the recombinant protein to generate virus-like particles.
In addition, the invention also provides the application of the recombinant protein, the coding gene, the expression cassette, the recombinant vector, the host or the preparation method in any aspect of the following:
preparing virus-like particles; the method comprises the steps of,
a formulation that can elicit an immune response in an organism against porcine circovirus type III.
The verification shows that the protein prepared by the invention can cause the immune response of pigs to the porcine circovirus type III after being compounded into a vaccine.
At present, for the problem of obtaining porcine circovirus type III virus-like particles, recombinant capsid proteins are adopted for reassembling, which is an effective mode, but the current production process is complex, for example, chinese patent with application number 201910516978.6 can be obtained after long construction of recombinant baculovirus containing porcine circovirus type III recombinant Cap proteins, infection of High Five cells. The operation of the process of constructing viruses and the process of infecting by adopting the constructed viruses has a certain complexity.
Thus, the present invention also provides a virus-like particle which is self-assembled from any one of the two recombinant proteins described above.
It was verified that the provided virus-like particles can elicit an immune response against porcine circovirus type III and the route of acquisition is simple.
It should be noted that the self-assembly process in the present invention can be completed in the purification stage, and when the recombinant protein includes an active portion as shown in SEQ ID NO. 1 and a fusion short peptide as shown in SEQ ID NO. 3, the content of virus-like particles generated by self-assembly is increased.
Thus, the present invention also provides the use of the above virus-like particle for the preparation of a formulation which can elicit an immune response in an organism against porcine circovirus type III.
Further, the above virus-like particles are used alone or in combination with an active protein comprising a first protein and/or a second protein to form an immunogenic component.
The sequence of the first protein is shown as SEQ ID NO:1 is shown in the specification; the second protein comprises an active part and a fusion short peptide, and the amino acid sequence of the active part is shown in SEQ ID NO:1, wherein the fusion oligopeptide is connected to the C-terminal end of the active part. Specifically, the sequence of the fusion short peptide is shown as SEQ ID NO:3 is shown in the figure
Since the first protein, the second protein and the virus particles are all immunogenic, the invention has good immune response when used as an immune preparation.
In some embodiments, the concentration of the immunogenic component is from 0.05mg/mL to 0.15mg/mL. When the concentration of the immunogen component is as described above, the immune effect is more excellent.
Example 1
The embodiment provides a preparation method of porcine circovirus type III recombinant Cap protein and virus-like particles assembled by the same, comprising the following steps:
(1) Construction of recombinant plasmid containing cap Gene
The cap gene is made by Beijing Optimu Corp, and the sequence is as follows:
ATGACCTATTATACCAAAAAGTATAGTACCATGAACGTTATTTCCGTTGGCACCCCGCAGAATAATAAACCGTGGCATGCAAATCATTTTATTACCCGCCTGAATGAATGGGAAACCGCAATCAGCTTTGAATATTATAAAATCCTGAAAATGAAAGTTACACTGAGCCCAGTTATTAGCCCAGCACAGCAGACCAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCAATTCTGGCGGGAACTACCAGCGCTCATCCTGGTCAGAGCCTGTTTTTTTTTAGTCGTCCGACCCCTTGGCTGAATACCTATGATCCTACCGTTCAGTGGGGTGCCCTGCTGTGGAGTATTTATGTTCCGGAAAAAACCGGTATGACCGATTTTTATGGTACCAAAGAAGTTTGGATTCGTTATAAAAGCGTTCTG(SEQ ID NO:2)
then, PCR cloning is performed by using a primer F1/R1, and after cleavage by NheI/XhoI, a gene fragment detection diagram is shown in FIG. 1, M is a marker in FIG. 1, and lanes 1 and 2 are cap genes.
Inserted into a vector pET28a (purchased from Wohan vast Biotechnology Co., ltd.) cut by the same enzyme, and transformed into ClearColi after ligation TM BL21 (DE 3) feelThe positive clone of the receptor cell (purchased from Lucigen), kanamycin resistance plate screening, colony PCR verification of the screened monoclonal, sequencing of the correct monoclonal submitted to Beijing engine biotechnology Co., ltd, successful construction of recombinant plasmid after correct sequencing, named pET28a-cap3, the plasmid detection diagram is shown in figure 2, M is marker in figure 2, lanes 1-11 are PCR identification recombinant plasmid, lane 12 is negative control, and the strain named pET28a-cap3/ClearColi TM BL21 (DE 3). The primer F1/R1 sequence is as follows:
(2) Protein expression of recombinant strain containing cap gene
The recombinant strain pET28a-cap3/ClearColiTMBL21 (DE 3) which has been proved to be successful is transferred into LB liquid medium for activation, the strain is cultured for 12 hours at 37 ℃ and 220rpm, the activated strain is transferred into 50ml of LB fermentation liquor in a 250ml triangular flask according to the inoculum size of 2 percent, the strain is cultured at 37 ℃ and 220rpm, 1mmol of IPTG (isopropyl-beta-D-thiopyran galactoside) with the final concentration of 1mmol is added when the bacterial concentration reaches OD600 to 0.6 for induction expression, the induction is stabilized at 23 ℃, the rotating speed is 220rpm, and the bacterial is collected after 9 hours of induction culture.
(3) Cap protein purification
The cells collected by fermentation were subjected to reconstitution with a reconstitution Buffer (20 mM PB, pH 7.4, 150mM sodium chloride), and the cells after reconstitution were centrifuged (12000 rpm, 20 min) to collect the supernatant, which was then examined, as shown in FIG. 3, wherein M is marker, lane 1 is the centrifuged supernatant after sterilization, lane 2 is the centrifuged supernatant after sterilization, the supernatant was collected after centrifugation using the crushed cells, filtered through a 0.45 μm filter, and the supernatant was purified by MMA chromatography using an anion column, and the flow-through was collected.
(4) SDS-PAGE verification of Cap protein purified samples were pipetted with 80mL and 20mL of 5 Xprotein loading buffer, run on 12% protein electrophoresis running protein gel with loading volume of 10 μl per well, observed after staining and decolorizing, the detection result is shown in FIG. 4, M is marker, lane 1 is centrifugation supernatant after sterilization, lane 2 is MMA flow through.
(5) The purified PCV3 Cap protein was placed on a carbon-coated copper grid, dried with filter paper, negatively stained with 2% phosphotungstic acid, and observed with a transmission electron microscope (HT 7700) at an accelerating voltage of 120 kv, the results of which are shown in fig. 5, showing that the recombinant protein of example 1 was self-assembled to give virus-like particles, and the amino acid sequence of the recombinant protein of example 1 was as shown in SEQ ID NO: 1.
MTYYTKKYSTMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSIYVPEKTGMTDFYGTKEVWIRYKSVL (SEQ ID NO: 1) comparative example 1
This comparative example provides a recombinant Cap protein containing NLS porcine circovirus type III, which operates in substantially the same manner as example 1, except that the cloning templates are:
ATGCGTCGTCTGCTGTTTATTCGTCGTCCGACCGCCGGTACCTATTATACCAAAAAGTATAGTACCATGAACGTTATTTCCGTTGGCACCCCGCAGAATAATAAACCGTGGCATGCAAATCATTTTATTACCCGCCTGAATGAATGGGAAACCGCAATCAGCTTTGAATATTATAAAATCCTGAAAATGAAAGTTACACTGAGCCCAGTTATTAGCCCAGCACAGCAGACCAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCAATTCTGGCGGGAACTACCAGCGCTCATCCTGGTCAGAGCCTGTTTTTTTTTAGTCGTCCGACCCCTTGGCTGAATACCTATGATCCTACCGTTCAGTGGGGTGCCCTGCTGTGGAGTATTTATGTTCCGGAAAAAACCGGTATGACCGATTTTTATGGTACCAAAGAAGTTTGGATTCGTTATAAAAGCGTTCTG
cloning primer F2/R2
| Primer name | Sequence information |
| F2 | ctacatatgatgcgtcgtctgctgtttatt |
| R2 | ccgctcgagcagaacgcttttataacgaatc |
The constructed plasmid was named pET28a-cap3-NLS. The amplified fragment is shown in FIG. 6, M is marker, lane 1 is the amplified fragment of the comparative example, lane 2 is the amplified fragment of the repeated experiment of example 1, the double digestion verification of the recombinant vector is shown in FIG. 7, M is marker, lane 1 is the digestion verification chart of the recombinant vector of the comparative example, and lane 2 is the digestion verification chart of the recombinant vector of the repeated experiment of example 1.
SDS-PAGE verification of Cap proteins purified samples were blotted 80mL with 20mL of 5 Xprotein loading buffer, stained with 12% protein electrophoresis protein gel at a loading volume of 10. Mu.L per well, and observed after staining and dehydration, the results are shown in FIG. 8, M in FIG. 8 is marker, lane 1 is pET28a-Cap3/ClearColi TM BL 21-disrupted supernatant, lane 2 pET28a-cap3/ClearColi TM BL21 bacterial cell pellet, lane 3 pET28a-cap3-NLS/ClearColi TM BL 21-disrupted supernatant, lane 4 pET28a-cap3-NLS/ClearColi TM BL21 bacterial cell pellet, lane 5 pET28/ClearColi TM BL 21-disrupted supernatant, lane 6 pET28/ClearColi TM As shown in FIG. 9, the structural detection chart of VLPs formed by BL21 bacterial disruption precipitation, compared with example 1, the N-segment of the amino acid sequence of the comparative example has an NLS cap-containing amino acid sequence of RRLLFIRRPTAG, and after verification, the expression level is increased and the self-assembly content of VLPs is increased after NLS sequence is removed.
Example 2
This comparative example provides a recombinant Cap protein free of NLS porcine circovirus type III, which operates in substantially the same manner as example 1, except that the cloning templates are:
ATGACCTATTATACCAAAAAGTATAGTACCATGAACGTTATTTCCGTTGGCACCCCGCAGAATAATAAACCGTGGCATGCAAATCATTTTATTACCCGCCTGAATGAATGGGAAACCGCAATCAGCTTTGAATATTATAAAATCCTGAAAATGAAAGTTACACTGAGCCCAGTTATTAGCCCAGCACAGCAGACCAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCAATTCTGGCGGGAACTACCAGCGCTCATCCTGGTCAGAGCCTGTTTTTTTTTAGTCGTCCGACCCCTTGGCTGAATACCTATGATCCTACCGTTCAGTGGGGTGCCCTGCTGTGGAGTATTTATGTTCCGGAAAAAACCGGTATGACCGATTTTTATGGTACCAAAGAAGTTTGGATTCGTTATAAAAGCGTTCTGAATCTCAAGGCTCCTCCACTGAACCCG(SEQ ID NO：6)
cloning primer F1/R3
Compared with example 1, the recombinant protein amino acid sequence of this example has NLKAPPLNP (SEQ ID NO: 3) terminal fusion peptide added to its C-segment.
Example 3
The present example provides a recombinant Cap protein free of NLS porcine circovirus type III, which operates substantially the same as the procedure of example 1, except that the cloning templates are: ATGACCTATTATACCAAAAAGTATAGTACCATGAACGTTATTTCCGTTGGCACCCCGCAGAATAATAAACCGTGGCATGCAAATCATTTTATTACCCGCCTGAATGAATGGGAAACCGCAATCAGCTTTGAATATTATAAAATCCTGAAAATGAAAGTTACACTGAGCCCAGTTATTAGCCCAGCACAGCAGACCAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCAATTCTGGCGGGAACTACCAGCGCTCATCCTGGTCAGAGCCTGTTTTTTTTTAGTCGTCCGACCCCTTGGCTGAATACCTATGATCCTACCGTTCAGTGGGGTGCCCTGCTGTGGAGTATTTATGTTCCGGAAAAAACCGGTATGACCGATTTTTATGGTACCAAAGAAGTTTGGATTCGTTATAAAAGCGTTCTGAATCTCAAGGCTCCTCCACTGAACCCGCAAGTAAAACCTCTCCAACCGAATGTC (SEQ ID NO: 7)
Cloning primer F1/R4
Compared with example 1, the recombinant protein amino acid sequence of this example has a NLKAPPLNPQVKPLQPNV terminal fusion peptide (SEQ ID NO: 4) added to the C-segment thereof.
Example 4
This comparative example provides a recombinant Cap protein containing NLS porcine circovirus type III, which operates in substantially the same manner as example 1, except that the cloning templates are:
ATGACCTATTATACCAAAAAGTATAGTACCATGAACGTTATTTCCGTTGGCACCCCGCAGAATAATAAACCGTGGCATGCAAATCATTTTATTACCCGCCTGAATGAATGGGAAACCGCAATCAGCTTTGAATATTATAAAATCCTGAAAATGAAAGTTACACTGAGCCCAGTTATTAGCCCAGCACAGCAGACCAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCAATTCTGGCGGGAACTACCAGCGCTCATCCTGGTCAGAGCCTGTTTTTTTTTAGTCGTCCGACCCCTTGGCTGAATACCTATGATCCTACCGTTCAGTGGGGTGCCCTGCTGTGGAGTATTTATGTTCCGGAAAAAACCGGTATGACCGATTTTTATGGTACCAAAGAAGTTTGGATTCGTTATAAAAGCGTTCTGAATCTCAAGGCTCCTCCACTGAACCCGCAAGTAAAACCTCTCCAACCGAATGTCACACTGCTACGAGCTCTGCCGAGAGGT(SEQ ID NO：8)
cloning primer F1/R5
Compared with example 1, the recombinant protein amino acid sequence of this example has NLKAPPLNPQVKPLQPNVTLLRALPRG (SEQ ID NO: 5) terminal fusion peptide added to its C-segment.
The amplified fragments of examples 2 to 4 were detected, and the results are shown in FIG. 10, wherein M is a marker, lane 1 is the amplified fragment of the target gene of example 4, lane 2 is the amplified fragment of the target gene of example 3, and lane 3 is the amplified fragment of the target gene of example 2.
The recombinant vectors of examples 2 to 4 were tested, and the results are shown in FIG. 11, wherein M is a marker, lane 1 is the recombinant vector of example 4, lane 2 is the recombinant vector of example 3, and lane 3 is the recombinant vector of example 2.
The VLPs produced by self-assembly in the purification process of examples 2 to 4 were examined, the examination results of example 2 were shown in fig. 12, the examination results of example 3 were shown in fig. 13, the examination results of example 4 were shown in fig. 14, and the results showed that a large number of virus-like particles were formed in examples 2 to 4, and that the particles of example 2 were smaller in size than those of examples 3 to 4, less prone to deagglomeration, and more excellent in morphology, and the particle sizes of examples 3 to 4 were larger.
Example 5
Preparation and application of porcine circovirus 3-type VLPs vaccine
(1) Preparation of porcine circovirus 3-type VLPs vaccine
The complex of the porcine circovirus 3-type Cap protein and the virus-like particle prepared in example 2 and French Sibirk adjuvant GEL01 are mixed according to the volume ratio of 1:1, so that the final concentration of the complex is 0.1mg/mL, and the prepared vaccine is placed on an emulsifying machine for even emulsification. And (3) performing aseptic inspection, viscosity measurement and stability measurement according to the requirements of the appendices of the current Chinese veterinary drug dictionary, and then storing at 2-8 ℃ for later use.
(2) Application of porcine circovirus 3-type VLPs vaccine
10 healthy 21-day-old piglets double-negative with PCV3 antigen antibodies were screened, and randomly divided into 2 groups of 5 piglets each. The porcine circovirus type 3 VLPs vaccine prepared by intramuscular injection of each piglet neck of the first group (immune group) is 2 ml/head; the second group (control group) was given 2 ml/head of a sterilized PBS solution for intramuscular injection to the neck of each piglet. After 14 days, each piglet was immunized a second time in the same manner. After the first immunization, each group of piglets was collected and serum was isolated weekly, and PCV3 antibody levels in serum were detected using a commercial porcine circovirus type 3 antibody detection kit. The detection results are shown in FIG. 15.
The results show that the PCV3 antibody can be rapidly produced after the porcine circovirus type 3 VLPs vaccine is used for immunizing piglets. The antibody starts to turn positive 7 days after the immune, the antibody level (OD 450 value) can reach more than 1.0 after 21 days, and the control group piglet PCV3 antibody is negative. The research result shows that the porcine circovirus type 3 VLPs vaccine prepared by using the genetic engineering strain can effectively stimulate the porcine body to generate PCV3 specific antibodies, and the antibody level generated after immunization is obviously different from that of a control group.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The porcine circovirus type III recombinant Cap protein is characterized in that the amino acid sequence of the porcine circovirus type III recombinant Cap protein is shown as SEQ ID NO: 1.
2. A gene encoding the porcine circovirus type III recombinant Cap protein of claim 1, wherein the sequence of the gene encoding the porcine circovirus type III recombinant Cap protein is as set forth in SEQ ID NO: 2.
3. The recombinant protein is characterized by comprising an active part and a fusion short peptide, wherein the amino acid sequence of the active part is shown as SEQ ID NO:1, wherein the fusion oligopeptide is connected to the C-terminal end of the active part.
4. The recombinant protein according to claim 3, wherein said fusion oligopeptide is selected from the group consisting of SEQ ID NOs: 3, and the polypeptide shown as SEQ ID NO:4 and a polypeptide as set forth in SEQ ID NO:5, and a polypeptide shown in the formula (I).
5. A gene encoding the recombinant protein of claim 3.
6. A recombinant vector comprising the coding gene of claim 2 or 5.
7. A host comprising the recombinant vector of claim 6.
8. Use of a porcine circovirus type III recombinant Cap protein according to claim 1, a coding gene according to claim 2, a recombinant protein according to claim 3, a coding gene according to claim 5, a recombinant vector according to claim 6 or a host according to claim 7 in any of the following:
preparing virus-like particles; the method comprises the steps of,
a formulation is prepared that can elicit an immune response in an organism against porcine circovirus type III.
9. A virus-like particle, characterized in that it is self-assembled from the proteins according to claim 1 and/or 3.
10. Use of a virus-like particle according to claim 9 for the preparation of a formulation capable of eliciting an immune response in an organism against porcine circovirus type III.
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