CN111187782A

CN111187782A - Porcine Delta coronavirus virus-like particle and preparation method and application thereof

Info

Publication number: CN111187782A
Application number: CN202010064111.4A
Authority: CN
Inventors: 严亚贤; 纪立凯; 孙建和; 李莎莎
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2020-05-22
Anticipated expiration: 2040-01-20
Also published as: CN111187782B

Abstract

The invention provides a porcine Delta coronavirus virus-like particle and a preparation method and application thereof, which comprises the steps of firstly designing and amplifying a porcine Delta coronavirus structural protein gene, constructing a recombinant shuttle plasmid by using the structural protein gene, constructing a recombinant bacmid by using the recombinant shuttle plasmid, transfecting the recombinant bacmid to an Sf9 cell to obtain a recombinant baculovirus expressing the porcine Delta coronavirus structural protein, infecting the recombinant baculovirus to an Sf9 cell, and purifying to obtain the porcine Delta coronavirus-like particle; the preparation method disclosed by the invention is prepared by expressing Sf9 cells cultured in a serum-free manner, and combines ultracentrifugation of a sucrose density solution to obtain the PDCoV-VLP virus-like particles, and the PDCoV-VLP virus-like particles have the advantages of integrity, good immunogenicity, high antibody titer generated by immune animals and high safety, can be used for preventing the porcine Delta coronavirus virus, and have good development and application prospects.

Description

Porcine Delta coronavirus virus-like particle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a porcine Delta coronavirus virus-like particle as well as a preparation method and application thereof.

Background

For Porcine Epidemic Diarrhea (PED) which is characterized by high mortality of suckling piglets and developed in China since 2010, epidemic situation reaches hundreds of provinces, and 100 thousands of piglets die. Similarly, PEDs are also frequently prevalent in the united states and so on, and have a serious impact on the development of the world swine industry. Research on PED pathogens shows that new Porcine Delta coronavirus (PDCoV) and Porcine Epidemic Diarrhea Virus (PEDV) are important pathogens for causing the diseases, and the two viruses have a co-infection phenomenon.

Observed under an electron microscope, the PDCoV is spherical or elliptical, the outside of the capsule membrane is provided with the coronary spinous process, and the size of the virus particles is 150-180 nm. The PDCoV genome is a single positive-stranded RNA. According to the PDCoV whole genome sequence which is determined at present, the size of the virus genome is about 25.4kb, the percentage content of G + C is about 43 percent, and the virus genome is known to be a coronavirus containing the minimum genome sequence. The PDCoV genome mainly includes both end non-coding regions (UTRs), i.e., 5 '-UTR and 3' -UTR, and a plurality of Open Reading Frames (ORFs). The PDCoV genome encodes for the most part two overlapping ORFs 1a and 1b encoding the polyproteins pp1a and pp1ab, respectively. The PDCoV genome residual sequence mainly encodes virus structural proteins, including surface fiber protein (Spike, S), Small Membrane protein (Small Membrane, E), Membrane protein (M) and Nucleocapsid protein (N) in sequence. At present, no report is available on the development of a newly discovered vaccine against porcine Delta coronavirus.

Virus-like particle (VLP) vaccines can induce humoral and cellular immune responses, and can greatly enhance the immune protective effect of local mucosa by intranasal vaccination or oral immunization. VLPs, also known as pseudoviruses or pseudoviruses, are hollow particles formed by self-assembly of one or more structural proteins of a certain virus, which are morphologically identical or similar to authentic virions, retain the original antigenic epitopes and spatial conformation of the virus, but do not contain the viral genome and therefore cannot replicate autonomously, and are therefore not pathogenic. Because the vaccine has immunogenicity similar to that of a complete virus and nano-size particles, can induce various types of immune responses, particularly mucosal immunity, and has the characteristics of good immunogenicity, higher safety and the like by activating antigen presenting cells, the vaccine becomes one of potential safe and efficient candidate vaccines for human or animal viral infection diseases.

In conclusion, the development of a method for preparing the porcine Delta coronavirus virus-like particles efficiently, safely and at low cost is urgently needed at present, so as to provide a vaccine for preventing and controlling the infection of the PDCoV.

Disclosure of Invention

Aiming at the problem that no safe and effective vaccine for preventing and controlling PDCoV infection exists in the prior art, the invention mainly aims to provide the virus-like particle of the porcine Delta coronavirus.

The second purpose of the invention is to provide a preparation method of the porcine Delta coronavirus virus-like particle.

The third purpose of the invention is to provide the application of the porcine Delta coronavirus virus-like particle.

In order to achieve the above primary object, the solution of the present invention is:

a porcine Delta coronavirus structural protein gene comprises a structural protein S gene, a structural protein E gene, a structural protein M gene and a structural protein N gene which are amplified respectively;

the nucleotide sequence of the structural protein S gene is shown as SEQ ID NO. 1;

the nucleotide sequence of the structural protein E gene is shown as SEQ ID NO. 2;

the nucleotide sequence of the structural protein M gene is shown as SEQ ID NO. 3;

the nucleotide sequence of the structural protein N gene is shown as SEQ ID NO. 4.

As a preferred embodiment of the present invention, the forward primer sequence of the nucleotide sequence of the structural protein S gene is shown in SEQ ID NO.5 during amplification; the reverse primer sequence is shown as SEQ ID NO. 6;

the forward primer sequence of the nucleotide sequence of the structural protein E gene is shown as SEQ ID NO. 7; the reverse primer sequence is shown as SEQ ID NO. 8;

the forward primer sequence of the nucleotide sequence of the structural protein M gene is shown as SEQ ID NO. 9; the reverse primer sequence is shown as SEQ ID NO. 10;

the forward primer sequence of the nucleotide sequence of the structural protein N gene is shown as SEQ ID NO. 11; the sequence of the reverse primer is shown as SEQ ID NO. 12.

As a preferred embodiment of the invention, when cloning the nucleotide sequences of the structural protein S gene, the structural protein M gene and the structural protein N gene, an EcoRI enzyme cutting site sequence is arranged in front of an initiation codon ATG, and a HindIII enzyme cutting site sequence is arranged behind a termination codon;

when cloning the nucleotide sequence of the structural protein E gene, an XhoI enzyme cutting site sequence is arranged in front of an initiation codon ATG, and a KpnI enzyme cutting site sequence is arranged behind a termination codon.

A recombinant shuttle plasmid, which comprises the porcine Delta coronavirus structural protein gene.

A recombinant bacmid comprising the recombinant shuttle plasmid described above.

A recombinant baculovirus comprising the recombinant bacmid described above.

A porcine Delta coronavirus virus-like particle purified from Sf9 cells infected with the recombinant baculovirus described above.

In order to achieve the second objective, the solution of the invention is:

a method for preparing the virus-like particle of the porcine Delta coronavirus comprises the following steps:

(1) designing and amplifying a structural protein gene of the porcine Delta coronavirus;

(2) constructing a recombinant shuttle plasmid of the porcine Delta coronavirus structural protein gene in the step (1);

(3) constructing a recombinant bacmid comprising the recombinant shuttle plasmid of step (2);

(4) infecting the recombinant bacmid obtained in the step (3) with Sf9 cells to obtain recombinant baculovirus;

(5) and (3) infecting Sf9 cells with the recombinant baculovirus obtained in the step (4), and purifying to obtain the porcine Delta coronavirus virus-like particles.

In order to achieve the third object, the solution of the invention is:

application of the porcine Delta coronavirus virus-like particle in a vaccine for preventing porcine diarrhea diseases caused by the porcine Delta coronavirus.

Due to the adoption of the scheme, the invention has the beneficial effects that:

the preparation method is based on a baculovirus-insect cell expression system (Bac-to-Bac), Sf9 cells cultured in serum are used for expression and preparation, and the porcine Delta coronavirus virus-like particles are obtained by combining ultracentrifugation of a sucrose density solution.

Secondly, the Western Blot result in the porcine Delta coronavirus virus-like particle shows that the porcine Delta coronavirus virus-like particle can be specifically combined with the antibody of each structural protein of PDCoV, and the result shows that the recombinant baculovirus can successfully express 4 structural proteins of the porcine Delta coronavirus in insect cells.

Drawings

FIG. 1 is an electrophoretogram of the amplified bands of the porcine Delta coronavirus structural protein (S, E, M, N) gene of example 1 of the present invention; lane M is the nucleic acid molecular weight.

FIG. 2 is an electrophoresis diagram of the restriction enzyme digestion identification nucleic acid of the recombinant vectors of the porcine Delta coronavirus pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M of example 2 in the invention; wherein Lane 1 is an electrophoretogram of pFBD-PDCoV-S recombinant plasmid; lane 2 is an electrophoretogram of pFBD-PDCoV-S recombinant plasmid after restriction endonuclease action; lane 3 is an electrophoretogram of pFBD-PDCoV-E/M recombinant plasmid; lane 4 is an electrophoretogram of pFBD-PDCoV-E/M recombinant plasmid after the restriction endonuclease action of the M gene; lane 5 is an electrophoretogram of pFBD-PDCoV-E/M recombinant plasmid after the restriction endonuclease action of the E gene; lane 6 is an electrophoretogram of pFBD-PDCoV-N recombinant plasmid; lane 7 is an electrophoretogram of pFBD-PDCoV-N recombinant plasmid after restriction endonuclease action; lane M shows different nucleic acid molecular weights.

FIG. 3 is a graph showing the results of the identification of the porcine Delta coronavirus structural protein (S, E, M, N) WesternBlot of example 5 in the present invention;

lanes

1, 3, 6, 8 are negative controls; lane 2 is S protein sample; lane 4 is the E protein sample; lane 5 is N protein sample; lane 7 is an M protein sample; m is the protein standard molecular mass;

lanes

1 and 2 use murine antibodies to protein S;

lanes

3 and 4 use murine antibody to protein E;

lanes

5 and 6 use protein N murine antibody;

lanes

7 and 8 use murine antibody to protein M.

FIG. 4 is an electron micrograph of porcine Delta coronavirus virus-like particle PDCoV-VLP of example 6 in the present invention.

Detailed Description

The invention provides a porcine Delta coronavirus virus-like particle as well as a preparation method and application thereof.

< porcine Delta coronavirus Virus-like particle >

(structural protein Gene)

The swine Delta coronavirus (PDCoV) structural protein gene comprises a structural protein S gene, a structural protein E gene, a structural protein M gene and a structural protein N gene which are respectively amplified; wherein, the nucleotide sequence of the structural protein S gene is shown as SEQ ID NO. 1; the nucleotide sequence of the structural protein E gene is shown as SEQ ID NO. 2; the nucleotide sequence of the structural protein M gene is shown as SEQ ID NO. 3; the nucleotide sequence of the structural protein N gene is shown as SEQ ID NO. 4.

Wherein the sequence of an amplification forward primer PDCoV-S-PF of the nucleotide sequence of the swine Delta coronavirus structural protein S gene is shown as SEQ ID NO.5, and the sequence of a reverse primer PDCoV-S-PR is shown as SEQ ID NO. 6; the forward primer PDCoV-E-PF sequence of the amplification of the nucleotide sequence of the structural protein E gene is shown as SEQ ID No.7, and the reverse primer PDCoV-E-PR sequence is shown as SEQ ID No. 8; the sequence of an amplification forward primer PDCoV-M-PF of the nucleotide sequence of the structural protein M gene is shown as SEQ ID No.9, and the sequence of a reverse primer PDCoV-M-PR is shown as SEQ ID No. 10; the forward primer PDCoV-N-PF sequence of the amplification of the nucleotide sequence of the structural protein N gene is shown as SEQ ID NO.11, and the reverse primer PDCoV-N-PR sequence is shown as SEQ ID NO. 12.

Actually, when cloning the nucleotide sequence of the structural protein (S, N, M) gene of the swine Delta coronavirus, an EcoRI enzyme cutting site sequence is arranged in front of an initiation codon ATG, and a HindIII enzyme cutting site sequence is arranged behind a termination codon; an XhoI enzyme cutting site sequence is arranged in front of the start codon ATG of the nucleotide sequence of the structural protein E gene, and a KpnI enzyme cutting site sequence is arranged behind the stop codon.

(recombinant shuttle plasmid)

The recombinant shuttle plasmids pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M are obtained by inserting structural protein (S, E, M, N) genes of the swine Delta coronavirus into a pFastBacTMDU vector.

(recombinant bacmid)

The rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M recombinant bacmid is obtained by transforming pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M recombinant plasmids into DH10Bac competent cells for transposition.

(recombinant baculovirus)

Respectively transfecting recombinant bacmids of rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M to Sf9 cells to obtain recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M for expressing structural proteins (S, E, M, N) of the porcine Delta coronavirus.

(porcine Delta coronavirus virus-like particle)

And co-infecting Sf9 cells with recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M, and purifying to obtain the porcine Delta coronavirus virus-like particles PDCoV-VLP.

< preparation of porcine Delta coronavirus virus-like particles >

The preparation method of the virus-like particle of the porcine Delta coronavirus comprises the following steps:

(1) amplifying the gene of the structural protein (S, E, M, N) of the swine Delta coronavirus;

(2) and (2) constructing 3 recombinant shuttle plasmids by using the structural protein gene in the step (1): pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M;

(3) and (3) constructing 3 recombinant bacmid by using the recombinant shuttle plasmid in the step (2): rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M;

(4) respectively transfecting the 3 recombinant bacmids in the step (3) with Sf9 cells to obtain recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M for expressing the structural protein (S, N, E/M) of the swine Delta coronavirus;

(5) and (3) co-infecting Sf9 cells with the 3 recombinant baculoviruses obtained in the step (4), and purifying to obtain the porcine Delta coronavirus virus-like particles PDCoV-VLP.

< use of porcine Delta coronavirus virus-like particles >

The porcine Delta coronavirus virus-like particle can be applied to vaccines for preventing and controlling porcine diarrhea caused by PDCoV infection.

The present invention will be further described with reference to the following examples.

Example 1 design and Synthesis of the porcine Delta coronavirus structural protein (S, E, M, N) Gene

According to the whole genome sequence of the porcine Delta coronavirus, primers of structural protein (S, E, M, N) genes of the virus are respectively designed, and EcoRI enzyme cutting sites are respectively added in front of the initiation codons ATG of the nucleotide sequences of the structural protein S, M and N genes: gaattc, HindIII cleavage site added after stop codon: aagctt; adding XhoI restriction site before the start codon ATG of the nucleotide sequence of the structural protein E gene: ctcgag, addition of a KpnI cleavage site after the stop codon: ggtacc. Wherein the nucleotide sequence of the structural protein S gene of the swine Delta coronavirus is shown as SEQ ID No.1, the amplification forward primer PDCoV-S-PF sequence of the nucleotide sequence of the structural protein S gene is shown as SEQ ID No.5, and the reverse primer PDCoV-S-PR sequence is shown as SEQ ID No. 6. The nucleotide sequence of the structural protein E gene is shown as SEQ ID NO.2, the amplification forward primer PDCoV-E-PF sequence of the nucleotide sequence of the structural protein E gene is shown as SEQ ID NO.7, and the reverse primer PDCoV-E-PR sequence is shown as SEQ ID NO. 8. The nucleotide sequence of the structural protein M gene is shown as SEQ ID No.3, the amplification forward primer PDCoV-M-PF sequence of the nucleotide sequence of the structural protein M gene is shown as SEQ ID No.9, and the reverse primer PDCoV-M-PR sequence is shown as SEQ ID No. 10. The nucleotide sequence of the structural protein N gene is shown as SEQ ID No.4, the amplification forward primer PDCoV-N-PF sequence of the nucleotide sequence of the structural protein N gene is shown as SEQ ID No.11, and the reverse primer PDCoV-N-PR sequence is shown as SEQ ID No. 12. The RNA extracted by the porcine Delta coronavirus is used for reverse transcription to obtain cDNA serving as a template, and the gene segments of 4 structural proteins are obtained by amplification and purification by using a PCR (polymerase chain reaction) technology, as shown in figure 1.

Example 2 construction and identification of recombinant shuttle plasmids pFBD-PDCoV-S, pFBD-PDCoV-N, pFBD-PDCoV-E/M

Double digestion of purified product of porcine Delta coronavirus structural protein (S, M, N) gene and pFastBac using restriction endonucleases EcoRI and HindIII^TMReacting for 2h at 37 ℃ for Dual plasmid, separating by using 1% agarose gel electrophoresis, recovering Cap protein gene fragment and linearized pFastBacTMDial, adding 1 uL of each of T4DNA ligase, T4Buffer and linearized pFastBacTMDial into 7 uL of S (or M or N) protein gene fragment, mixing gently, adding the mixture into freshly thawed Trans5 α competent cells, incubating for 30min on ice, thermally shocking for 90S at 42 ℃, immediately freezing for 2min, activating for 1h at 37 ℃, uniformly coating on an LB culture plate containing ampicillin (100 mu g/mL), culturing for 12h at 37 ℃ in an inverted way, obtaining recombinant plasmid shuttle BD-PDCoV-S, pFBD-PDCoV-N and pF-PDCoV-M colonies, picking single colonies, inoculating in a liquid LB culture medium containing ampicillin (100 mu g/mL), culturing for 12h at 37 ℃, extracting a small amount of plasmid by using a shake culture kit, and carrying out enzyme digestion and identification on the plasmid DNA plasmid.

The method comprises the steps of carrying out double digestion on a purified product of successfully constructed recombinant shuttle plasmids pFBD-PDCoV-M and E genes by using restriction endonucleases XhoI and KpnI, reacting for 2h at 37 ℃, separating by using 1% agarose gel electrophoresis, recovering Cap protein gene fragments and linearized pFastBac-TMDial, adding 1 mu L of T4DNA ligase, T4Buffer and linearized pFastBac-TMDial into 7 mu L of E protein gene fragments, gently mixing uniformly, adding the mixture into freshly melted Trans5 α competent cells, incubating for 30min on ice, incubating for 42 ℃ for 90s, immediately carrying out 2min on ice, uniformly coating on an LB culture plate containing ampicillin (100 mu g/mL) after being activated for 1h at 37 ℃, carrying out inverted culture for 12h at 37 ℃, obtaining recombinant shuttle plasmids pFBBD-PDCoV-E/M, obtaining single colonies, inoculating and carrying out double digestion on a pFBG-PDCoV-E colony, carrying out double digestion, identifying, and obtaining a recombinant shuttle plasmid clone by using a heat shock PCR-based on a plasmid identification kit.

Example 3 construction of porcine Delta coronavirus structural proteins (S, E, M, N) recombinant bacmids and extraction of recombinant bacmids

Respectively taking 50ng of recombinant shuttle plasmids pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M in example 2, respectively adding the recombinant shuttle plasmids pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E/M into DH10Bac competent cells which are just melted on ice, gently mixing the recombinant shuttle plasmids and the ice for 30min, thermally shocking the recombinant shuttle plasmids and the ice for 90s at 42 ℃, immediately cooling the recombinant shuttle plasmids and the ice for 2min, shaking and activating the recombinant shuttle plasmids and the ice for 4h at 37 ℃ by using SOC culture medium without antibiotics, uniformly coating 100 mu L of the recombinant shuttle plasmids and the ice on LB blue white spot screening flat plates containing kanamycin (50 mu g/mL), tetracycline (10 mu g/mL), gentamycin (7 mu g/mL), X-Gal (100 mu g/mL) and IPTG (40 mu g/mL), inversely culturing the recombinant shuttle plasmids and the white colonies for 48h at 37 ℃, carrying out PCR identification, marking the bacterial liquid containing a, selecting white colonies for PCR identification to obtain rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M recombinant vectors, and selecting bacterial liquid only containing a target band for amplification culture.

After the bacterial liquid is shake-cultured for 12h at 37 ℃ and 220rpm, the bacterial liquid is centrifuged for 1min at 4000rpm and 4 ℃ to collect the thalli, the thalli is re-suspended and precipitated by using solution I (50mmol/L glucose, 25mmol/L Tris-HCl pH 8.0 and 10mmol/L EDTA pH 8.0), solution II (0.2mol/L NaOH and 1% SDS) is added to slightly reverse and crack the thalli, solution III (60% 5mol/L potassium acetate and 11.5% glacial acetic acid) is added to slightly reverse the upper and the lower parts of the thalli, 12000rpm and 4 ℃ are centrifuged for 10min to discard the precipitate, the supernatant is transferred to a new EP tube, equal amount of absolute ethyl alcohol is added to slightly reverse and mix, the mixture is stood at 20 ℃ for 20min, 12000rpm and 4 ℃ is centrifuged for 10min to discard the supernatant, 70% absolute ethyl alcohol is added to slightly reverse and centrifuged to discard the supernatant, and the process is repeated. And drying the precipitate by using sterile wind to be semitransparent, adding a proper amount of sterile water, standing and naturally dissolving to obtain the recombinant bacmid rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M.

Example 4 rescue of recombinant baculoviruses rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M expressing the structural protein of the porcine Delta coronavirus

Sf9 cells are inoculated in a 6-well plate, when the cells adhere to the wall and the confluency reaches more than 50 percent, the double Grace-free insect cell culture medium is used for replacing the original culture medium (

II). Taking 8 μ L

II transfection reagent was added to 100. mu.L of double Grace-free insect cell culture and gently mixed.

Separately adding 3 μ g of recombinant bacmid rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E/M in example 3 into 100 μ L of double non-Grace insect cell culture, and gently mixing; the diluted recombinant bacmids are added into the diluted transfection reagent, mixed evenly, incubated at room temperature for 20min, and the mixture is evenly dropped into a 6-well plate. After 4h the transfection mixture was discarded and replaced by

II complete culture medium. The incubation was continued at 27 ℃ and continued until signs of viral infection were observed.

When the cells are fallen off or cracked, collecting culture supernatant, centrifuging at 4000rpm for 5min at 4 ℃ to remove macromolecular substances in the supernatant, and filtering by using a 0.22 mu M low-protein combination filter to obtain filtrate, namely first generation recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M, namely recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M for respectively expressing the structural proteins (S, N, E/M) of the porcine Delta coronavirus. The recombinant baculovirus was blindly transferred to the third generation and stored at-80 ℃.

Example 5Western Blot identification of recombinant baculovirus rBV-PDCoV-S, rBV-PDCoV-N and rBV-PDCoV-E/M protein expression

The remaining cells from example 4 were lysed by adding an appropriate amount of lysate on ice for 30min to prepare a protein sample. Protein samples are transferred onto PVDF membranes by 10% SDS-PAGE and semidry method, 5% skim milk is sealed for 1h at room temperature, murine antibodies of 4 structural proteins of the porcine Delta coronavirus are respectively used for incubation for 12h at 4 ℃, the PVDF membranes are washed for 10min multiplied by 3 times by TBST, goat anti-mouse IgG marked by HRP is added for incubation for 1h at room temperature, the PVDF membranes are washed for 10min multiplied by 3 times by TBST, ECL chemiluminescence liquid is uniformly dripped on the PVDF membranes, Tanon2500 is used for exposure, and the structural proteins (S, E, M, N) WesternBlot of the recombinant porcine Delta coronavirus are identified as shown in figure 3.

Example 6 identification of porcine Delta coronavirus-like particles PDCoV-VLPs

Sf9 cells were infected with the recombinant baculovirus of example 4 for 96h as S: E/M: N (5:2: 1). 3500rpm, centrifugation at 4 ℃ for 20min, taking supernatant, filtering by a 0.45-micron low-protein binding filter, using a 20% (M: V) sucrose cushion at 35000rpm, centrifuging at 4 ℃ for 2h, using 2mL PBS to resuspend the precipitate, using PBS to dilute to 40mL, 35000rpm/min, centrifuging at 4 ℃ for 2h, removing sucrose, using ultrapure water to resuspend the precipitate, carrying out 1% phosphotungstic acid negative staining, and observing the structure of the PDCoV-VLP by a transmission electron microscope, as shown in FIG. 4, a PDCoV-VLP electron microscope picture of the porcine Delta coronavirus-like particles, wherein arrows in the picture indicate the porcine Delta coronavirus-like particles, and the porcine Delta coronavirus-like particles with the diameter of about 150nm can be seen by the transmission electron microscope.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> porcine Delta coronavirus virus-like particle and preparation method and application thereof

<141>2020-01-20

<160>12

<170>SIPOSequenceListing 1.0

<210>1

<211>3480

<212>DNA

<213> nucleotide sequence of S gene of porcine Delta coronavirus structural protein (2 Ambystoma lateralxAmbystoma jeffersonanum)

<400>1

atgcagagag ctctattgat tatgacctta ctttgtctcg ttcgagcaaa gtttgctgat 60

gatctactcg atttgctcac cttcccgggt gcacatcgct tcttacataa acccacgagt 120

aattccacca gtctctactc gcgggctaat aactttgatg ttggcgttct tcctggctac 180

cccactaaga acgttaacct cttctcacca cttactaact ccactttgcc cattaatggc 240

cttcatcgga gttaccaacc actcatgctg aattgtctta ctaaaataac taatcacact 300

ctcagcatgt atctcctacc tagtgagata caaacttata gctgcggcgg tgccatggtt 360

aaataccaga cacatgatgc agttcgtatc attttagacc tcactgccac tgaccacatc 420

tctgttgaag tcgttggcca acatggtgaa aattatgtgt ttgtttgtag tgagcagttt 480

aactacacca ctgcattaca caactctacc ttcttctcac ttaattcaga gctttattgc 540

tttactaata acacctactt aggtattctt ccacctgatt taactgactt tacggtctat 600

cgtactggtc agttttatgc taatggttac cttttaggta ctttacctat tacggttaac 660

tatgtaaggt tgtatcgggg tcatctgtcg gccaatagtg cccactttgc cctagcaaac 720

ctaaccgata cactcataac acttaccaat actactatat cgcaaatcac ttattgtgat 780

aagtcagtag ttgattcaat agcatgccag ctctcttctc acgaagtgga ggatgggttt 840

tactccgacc ctaaatctgc cgttagagct aggcaacgta ctattgttac actacctaag 900

ctccctgagc ttgaagtagt gcagttaaat atttctgcac acatggattt tggcgaagcc 960

agacttgaca gcgttaccat caatggtaac acatcctatt gtgtcactaa gccttacttc 1020

aggcttgaaa ctaactttat gtgtacaggt tgcactatga atctgcgcac tgatacctgt 1080

agttttgacc tgtcagcagt aaataatggc atgtcattct ctcaattctg tctaagcact 1140

gaatctggtg cttgtgagat gaaaattatt gttacctatg tatggaatta cttgctaagg 1200

cagcgtttgt atgttactgc tgtagagggc cagactcaca ctggaaccac ttcagtacat 1260

gcaacagaca cttctagtgt aatcactgat gtctgcactg actacactat ctatggagtc 1320

tctggtactg gcattattaa gccatcagat ctcttattgc acaatggcat agcattcacc 1380

tctccaacgg gtgagcttta tgcatttaaa aatataacca ctggaaaaac ccttcaggtc 1440

ttaccgtgtg aaaccccttc tcaactgatt gtgataaata acaccgttgt cggtgctatc 1500

acatccagta actcaactga aaataatagg tttactacta ccattgtcac acctactttc 1560

ttttattcca caaatgccac cactttcaac tgcaccaagc ctgttttgtc ctatggaccc 1620

atcagcgtgt gtagtgatgg tgcaattgcg ggaacatcca cattacagaa tactcgacca 1680

tccatagttt cactatacga tggcgaagtt gaaataccat ctgcattttc tctttctgtt 1740

cagacggagt atttgcaagt tcaagcagag caagttatag ttgattgtcc acagtatgta 1800

tgcaatggca acagccgttg tctacaatta ctggcacaat acacctcagc ttgctctaac 1860

attgaagcag ctctgcattc ctctgcacag ttggatagca gagagattat aaatatgttt 1920

caaacatcaa cacagtcctt gcagttagct aatattacca acttcaaggg tgactacaat 1980

tttagcagca tactaaccac cagacttggt ggcagatctg ctattgaaga ccttcttttt 2040

aataaagttg ttactagtgg ccttggcact gttgatcagg actacaaagc ctgctctaga 2100

gacatggcca tcgctgactt agtttgttcc cagtattaca atggcatcat ggttctacct 2160

ggtgttgttg atgctgagaa aatggcaatg tatactggct ctcttactgg agctatggta 2220

tttggaggac tgactgccgc agcggcaata ccatttgcca cggcagtaca agcccgcctc 2280

aattatgtcg cactgcaaac aaatgtacta caagaaaacc agaaaattct tgcagaatca 2340

tttaaccaag cagttggcaa tatatcactt gcactatctt ctgttaatga tgccatccag 2400

caaacttctg aggctcttaa caccgtagct attgctatta aaaagattca aacagttgtt 2460

aaccagcagg gtgaggcatt atcacacctg actgcacagc tgtcaaacaa tttccaagca 2520

atttcgactt ctattcaaga catttacaac cgtcttgagg aagtagaggc taaccagcaa 2580

gttgaccgtc tcatcacagg acggttggct gcacttaatg catatgttac tcagttactc 2640

aatcagatgt ctcagattag acaatctcga ttgttagctc agcaaaagat taatgagtgt 2700

gtcaaatctc agtcgtccag atacggtttc tgtggaaatg gcacacacat cttctcactt 2760

acacagactg caccaaatgg catatttttt atgcatgcag tgcttgtacc caacaaattc 2820

acacgtgtca acgcttctgc cggcatttgt gtggataata cgagaggcta ctcattgcag 2880

cctcaactta tactctacca gtttaataac tcctggagag ttacacctag aaatatgtat 2940

gaacccagac tgccccggca agctgatttc atacaattaa ctgattgcag cgttactttt 3000

tataacacta ccgctgctaa tcttcccaat attattcctg acgttataga tgtcaatcaa 3060

acagtcagtg atattattga caatttacct acagcaacac ctcctcagtg ggatgttggt 3120

atctataaca acactattct caacctcacc gttgagatta atgatctaca agagcggtct 3180

aaaaacctct cacagattgc agatcgttta caaaattata ttgacaatct taacaatact 3240

ctagttgacc ttgaatggct caacagagta gaaacttacc ttaaatggcc gtggtatata 3300

tggcttgcca ttgccctggc tcttattgca tttgtgacaa tcctcataac aatctttctt 3360

tgtactggtt gttgtggtgg ttgctttggt tgttgtggcg gttgttttgg ccttttctct 3420

aagaagaaaa ggtataccga cgaccaacca acaccgtctt ttaagtttaa ggaatggtag 3480

<210>2

<211>252

<212>DNA

<213> nucleotide sequence of the E gene of the porcine Delta coronavirus structural protein (2 Ambystoma lateralxAmbystoma jeffersonanum)

<400>2

atggtagtcg acgactgggc tgttaccatc cctggacaat atattattgc tatactagtt 60

gtcatctgca ttggtgtggc actacttttt attaacactt gcttagcttg tgttaaatta 120

ttttacaagt gctacctagg ggcagcatat cttgttaggc ctattatagt gtactactcc 180

aagccgaacc ccgtacctga ggatgagttt gtaaaagtac accaatttcc tagaaacact 240

cactatgtct ga 252

<210>3

<211>654

<212>DNA

<213> nucleotide sequence of the M gene of the porcine Delta coronavirus structural protein (2 Ambystoma lateralxAmbystoma jeffersonanum)

<400>3

atgtctgacg cagaagagtg gcaaattatt gttttcattg cgatcatatg ggcgcttggc 60

gttatcctcc aaggaggcta tgccacgcgt aatcgtgtga tctatgttat taaacttatt 120

ctgctttggc tgctccaacc cttcacccta gtggtgacca tttggaccgc agttgacaga 180

tcatctaaga aggacgcagt tttcattgtg tccataattt ttgccgtatt gacctttata 240

tcctgggcca agtactggta tgactcaatt cgtttattaa tgaaaaccag atctgcatgg 300

gcactctcac ctgagagtag actccttgca gggattatgg atccaatggg tacatggagg 360

tgcattccca tcgaccacat ggctccaata ctcacaccag tcgttaagca tggcaagctc 420

aagctacatg ggcaagagct ggccaatggc atatcagtta gaaatccgcc acaggatatg 480

gtgatagtgt caccaagtga cacctttcac tacactttta agaaacctgt ggaatcaaac 540

aacgatccag aattcgctgt tctgatatac cagggtgacc gtgcttcaaa cgctggactt 600

cacactataa ccacttcaaa ggccggtgac gctcgcctgt ataagtatat gtaa 654

<210>4

<211>1029

<212>DNA

<213> nucleotide sequence of the N gene of the porcine Delta coronavirus structural protein (2 Ambystoma lateralxAmbystoma jeffersonanum)

<400>4

atggccgcac cagtagtccc tactactgac gcgtcttggt ttcaggtgct caaagctcaa 60

aacaaaaagg ctactcatcc tcagtttcgt ggcaatggag ttccgcttaa ctccgccatc 120

aaacccgctg aaaaccatgg ctactggctg cgttacacta gacaaaagcc aggtggtact 180

ccgattcctc catcctatgc cttttattat actggcacag gtcccagagg aaatcttaag 240

tatggtgaac tccctcctaa tgatacccca gtaaccactc gtgttacttg ggttaagggt 300

ccgggagctg acacttctat taagcctcat gttgccaaac gcaaccccaa caatcctaaa 360

catcagctgc tacctctccg attcccaacc ggagatggcc cagctcaagg tttcagagtt 420

gaccccttca acgctagagg aagacctcag gagcgtggaa gtggcccaag atctcaatct 480

gttaactcca gaggcacagg caatcagccc aggaaacgcg accaatctgc accagctgcg 540

gtacgccgta agacccagca tcaagctccc aagcggactt tacccaaggg taaaaccatt 600

tctcaggtat ttggcaaccg gtctcgtact ggtgccaatg tcggctctgc agacactgag 660

aagacgggta tggctgatcc tcgcatcatg gctctagcca gacatgtgcc tggtgttcag 720

gaaatgcttt tcgctggcca ccttgagagc aactttcagg cgggggcaat tacccttacc 780

ttctcttact caatcacagt caaggagggt tctcctgact atgagagact taaggatgcg 840

ctcaatacgg tcgttaacca gacctatgag ccacccacca aaccaactaa ggacaagaag 900

cctgacaaac aagaccagtc tgctaaaccc aaacagcaga agaaacctaa aaaggtaact 960

ctgccagcag acaaacagga ttgggagtgg gatgatgctt ttgagataaa gcaggaatca 1020

gcagcgtag 1029

<210>5

<211>34

<212>DNA

<213> amplification of nucleotide sequence of porcine Delta coronavirus structural protein S Gene Forward primer PDCoV-S-PF sequence (2 Ambytoma laterals x Ambytoma jeffersonanum)

<400>5

cgaattcatg cagagagctc tattgattat gacc 34

<210>6

<211>34

<212>DNA

<213> amplification reverse primer PDCoV-S-PR sequence of nucleotide sequence of porcine Delta coronavirus structural protein S gene (2 Ambytoma laterale x Ambytoma jeffersonanum)

<400>6

caagcttcta ccattcctta aacttaaaag acgg 34

<210>7

<211>28

<212>DNA

<213> amplification of nucleotide sequence of porcine Delta coronavirus structural protein E gene forward primer PDCoV-E-PF sequence (2 Ambytoma laterals x Ambytoma jeffersonanum)

<400>7

tctcgagatg gtagtcgacg actgggct 28

<210>8

<211>29

<212>DNA

<213> amplification reverse primer PDCoV-E-PR sequence of nucleotide sequence of porcine Delta coronavirus structural protein E gene (2 Ambytoma laterale x Ambytoma jeffersonanum)

<400>8

cggtacctca gacatagtga gtgtttcta 29

<210>9

<211>29

<212>DNA

<213> amplification of nucleotide sequence of porcine Delta coronavirus structural protein M Gene Forward primer PDCoV-M-PF sequence (2 Ambytoma laterals x Ambytoma jeffersonanum)

<400>9

cgaattcatg tctgacgcag aagagtggc 29

<210>10

<211>29

<212>DNA

<213> amplification reverse primer PDCoV-M-PR sequence of nucleotide sequence of porcine Delta coronavirus structural protein M gene (2 Ambytoma laterale x Ambytoma jeffersonanum)

<400>10

caagctttta catatactta tacaggcga 29

<210>11

<211>28

<212>DNA

<213> amplification of nucleotide sequence of porcine Delta coronavirus structural protein N gene forward primer PDCoV-N-PF sequence (2 Ambytoma laterals x Ambytoma jeffersonanum)

<400>11

cgaattcatg gccgcaccag tagtccct 28

<210>12

<211>27

<212>DNA

<213> amplification reverse primer PDCoV-N-PR sequence of nucleotide sequence of swine Delta coronavirus structural protein N gene (2 Ambytoma laterale x Ambytoma jeffersonanum)

<400>12

caagcttcta cgctgctgat tcctgct 27

Claims

1. A porcine Delta coronavirus structural protein gene, which is characterized in that: it comprises a structural protein S gene, a structural protein E gene, a structural protein M gene and a structural protein N gene which are respectively amplified;

2. The porcine Delta coronavirus structural protein gene of claim 1, wherein: during the amplification, the forward primer sequence of the nucleotide sequence of the structural protein S gene is shown as SEQ ID NO. 5; the reverse primer sequence is shown as SEQ ID NO. 6;

3. The porcine Delta coronavirus structural protein gene of claim 1, wherein: when cloning the nucleotide sequences of the structural protein S gene, the structural protein M gene and the structural protein N gene, an EcoRI enzyme cutting site sequence is arranged in front of an initiation codon ATG, and a HindIII enzyme cutting site sequence is arranged behind a termination codon;

4. A recombinant shuttle plasmid characterized by: which comprises the porcine Delta coronavirus structural protein gene of any one of claims 1-3.

5. A recombinant bacmid characterized by: comprising the recombinant shuttle plasmid of claim 4.

6. A recombinant baculovirus, characterized in that: comprising the recombinant bacmid of claim 5.

7. A porcine Delta coronavirus virus-like particle, characterized by: purified from the recombinant baculovirus of claim 6 infected Sf9 cells.

8. A method of preparing the porcine Delta coronavirus virus-like particle of claim 7, wherein: which comprises the following steps:

9. Use of the porcine Delta coronavirus virus-like particle of claim 7 in a vaccine for preventing porcine diarrhea disease caused by porcine Delta coronavirus.