CN118773105A - Duck short beak and dwarfism syndrome virus-like particle, and preparation method and application thereof - Google Patents
Duck short beak and dwarfism syndrome virus-like particle, and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a duck short beak and dwarfism syndrome virus-like particle, and a preparation method and application thereof. According to the invention, through constructing an engineering strain for expressing the structural protein VP2 of the Short Beak and Dwarfism (SBDS), the molecular chaperone protein and the structural protein are co-expressed, so that the soluble expression of the structural protein VP2 of the SBDS in an escherichia coli system is realized, and the structural protein VP2 with immunogenicity is prepared. And then, carrying out induced culture, purification and self-assembly on the engineering strain to obtain the SBDS virus-like particles. The SBDS virus-like particles are further prepared into vaccines, and the vaccines can be proved to be effective in protecting ducklings from NGPV attacks.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a VLPs vaccine, and in particular relates to a duck short beak and dwarfism syndrome virus-like particle, a preparation method and application thereof.
Background
Short Beak and Dwarfism (SBDS) is an infectious disease caused by a novel goose parvovirus (Novel goose parvovirus, NGPV for short) in which duckling is retarded in development, and upper and lower beaks are atrophic or shortened. The disease is frequently generated in meat ducks with ages of less than 30 days, and the incidence rate is 5-10%. Most of the ducks in the slaughter house have 20-30% lower weight than healthy ducks, and the serious ducks only have 50% of normal weight, so that great economic loss is caused to the breeding industry.
NGPV can proliferate on Duck Embryo Fibroblasts (DEF) or SPF duck embryos, but cannot proliferate on Chicken Embryo Fibroblasts (CEF) and SPF chicken embryos, death and obvious lesions of embryos do not appear at the same time when the proliferation titer of the duck embryos is low (such as 10 4EID50/mL), in addition, the allantoic fluid harvest (about 5 mL) of the duck embryos at 9 days old is obviously less than that of chicken embryos at the same day old (about 8 mL), and the proliferation amount of DEF is less and CPE does not appear, so that the development and application of the novel goose parvovirus disease conventional vaccine are limited. The virus-like particle (VLPs) vaccine does not need to culture viruses, has the advantages of high safety, excellent immune effect and the like, and is a better choice for developing the virus-like particle vaccine based on NGPV structural proteins. At present, the research of SBDS virus-like particle vaccines by using a baculovirus system has been reported by students, however, the baculovirus system has complex operation, high economic cost and unsatisfactory yield. The escherichia coli expression system has the advantages of simplicity, high growth speed, low cost and the like, is widely applied to the production of recombinant protein and virus-like particle vaccines, but no report on the preparation of SBDS virus-like particles based on the escherichia coli expression system exists at present, and the reason is that: recombinant proteins overexpressed in E.coli cannot acquire the correct conformation and are easily bound to each other to form insoluble aggregates. The formation of virus-like particles depends on the natural higher structure of the monomeric protein, while the inclusion body of E.coli expresses foreign proteins, which cause the loss of the higher structure of the monomeric protein, which is a major problem in the preparation of virus-like particles. It has been found that this problem cannot be solved by using both the pro-dissolution tag and the induction condition optimisation.
Disclosure of Invention
Aiming at the problem that the subsequent development of virus-like particles cannot be carried out due to the loss of protein high-level structure of foreign protein expressed by an escherichia coli inclusion body in the prior art, the invention realizes the soluble expression of the duck short beak and dwarf syndrome structural protein VP2 in an escherichia coli system by constructing an engineering strain for expressing the duck short beak and dwarf syndrome structural protein VP2, successfully assembles the virus-like particles with good morphology, and further prepares the virus-like particles into the duck short beak and dwarf syndrome virus-like particle vaccine.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
In a first aspect, the invention provides an engineering strain for expressing structural proteins VP2 of short beak and dwarfism of ducks, wherein the engineering strain takes escherichia coli as a host cell and comprises coding genes of combination of coding genes and molecular chaperones of structural proteins VP2 of short beak and dwarfism of ducks.
According to the invention, through constructing an engineering strain for expressing the structural protein VP2 of the short beak and dwarfism of the duck, the molecular chaperone protein and the structural protein are expressed together, so that the soluble expression of the structural protein VP2 of the short beak and dwarfism of the duck in an escherichia coli system is realized, and the structural protein VP2 with immunogenicity is prepared.
Preferably, the amino acid sequence of the structural protein VP2 is shown as SEQ ID NO. 1;
the nucleotide sequence of the structural protein VP2 is shown in GenBank accession number KY 511124.1;
The structural protein VP2 is expressed by constructing a recombinant plasmid pET-32a-VP2 containing a coding gene of the structural protein VP2 and a linearization vector.
Preferably, the combination of chaperones includes TF, groES and groEL;
wherein the amino acid sequence of TF is shown as SEQ ID NO. 2;
The amino acid sequence of groES-groEL is shown as SEQ ID NO. 3.
The genes encoding TF, groES and groEL are expressed from the PZT-1 promoter in plasmid pG-Tf 2; plasmid pG-Tf2 of the application was purchased from Bao Bio Inc. under the accession number 3340.
The invention selects 5 chaperone protein combinations from three chaperones including Trigger Factor (TF), groES/groEL system, dnaK/DnaJ/GrpE system, which are respectively groES-groEL system, dnaK-dnaJ-grpE system, TF, groES-groEL system and TF combined action protein combination, dnaK-dnaJ-grpE system and groES-groEL system. Experiments show that the combined action of the groES-groEL system and TF has the best dissolving promoting effect on recombinant protein VP2, so that the molecular chaperone combination, namely TF2, is selected.
Preferably, the escherichia coli is a BL21 (DE 3) strain; the expression chaperone combinations include the expression of TF, groES and groEL in plasmid pG-Tf 2.
In a second aspect, the present invention provides a method for constructing an engineering strain expressing structural protein VP2 of short beak and dwarfism of duck, the method comprising the steps of:
Firstly, connecting a duck short beak and dwarfism syndrome structural protein VP2 gene with a linearization vector by utilizing a homologous recombination technology, and constructing a recombinant plasmid pET-32a-VP2;
And step two, converting the recombinant plasmid pET-32a-VP2 and the plasmid pG-Tf2 into escherichia coli to obtain a genetic engineering strain for expressing the duck short beak and dwarfism syndrome structural protein VP 2.
Preferably, in the first step, the linearized vector is an engineered pET32a plasmid having no redundant sequence at the N-terminus and carrying only His tag.
Preferably, in the second step, the plasmid pG-Tf2 and the recombinant plasmid pET-32a-VP2 are transformed into E.coli BL21 (DE 3) strain in sequence by a two-step method.
According to the invention, a pET32a expression plasmid is modified, a linearization vector with no redundant sequence at the N end is modified by utilizing a PCR amplification technology, and a homologous recombination technology is utilized to connect a gene sequence of a duck short beak with a His tag at the N end and a dwarfism syndrome structural protein VP2 with the linearization vector, so that a recombinant plasmid pET-32a-VP2 with only the His tag at the N end and without the redundant sequence is constructed. Compared with the recombinant plasmid without the modified vector, the recombinant plasmid with the modified vector has the spatial structure of the recombinant protein expressed by the recombinant plasmid with the modified vector being closer to the natural structure than the recombinant plasmid with the modified vector, and is easier to assemble into virus-like particles.
The plasmid pG-Tf2 is constructed by screening molecular chaperone combinations (namely a groES-groEL system and a TF system, abbreviated as molecular chaperone combination TF 2) with excellent dissolving promoting effect of the structural protein VP 2.
In order to realize the co-expression of molecular chaperone plasmids and recombinant plasmids, the invention constructs recombinant plasmids pET-32a-VP2 and pG-Tf2 respectively, firstly converts the plasmids pG-Tf2 into an escherichia coli BL21 (DE 3) strain, then prepares the molecular chaperone-containing strain into competence again by using a calcium chloride method, and secondarily converts the recombinant plasmids pET-32a-VP2 into prepared competence cells to prepare a double plasmid expression system (namely, a genetic engineering strain for expressing duck short beak and dwarfism syndrome soluble protein VP 2).
In a third aspect, the present invention provides a method for preparing virus-like particles of short beak and dwarfism of duck, the method comprising the steps of: and (3) carrying out induced culture on the engineering strain expressing the duck short beak and dwarfism syndrome soluble protein VP2 to obtain bacterial liquid, and purifying and self-assembling the bacterial liquid to obtain the duck short beak and dwarfism syndrome virus-like particles.
Preferably, the induction agent is at least one of IPTG or tetracycline base;
Preferably, the purification method is column chromatography, and a nickel column affinity chromatography column is selected for purification;
The self-assembled form is in vitro self-assembly.
The invention obtains the virus-like particles of the duck short beak and dwarfism syndrome by purifying and self-assembling bacterial liquid obtained by culturing the engineering strain under a specific inducer.
In a fourth aspect, the invention provides application of the engineering strain expressing the duck short beak and dwarfism structural protein VP2, the engineering strain expressing the duck short beak and dwarfism structural protein VP2 constructed by a construction method of the engineering strain expressing the duck short beak and dwarfism structural protein VP2 or the preparation method of the duck short beak and dwarfism virus-like particles to preparation of the duck short beak and dwarfism virus-like particles.
Preparing the obtained short beak and dwarf syndrome virus-like particles into oil vaccine, inoculating NGPV-VLPs vaccine to 1 day old duckling, and attacking 200 μ L SBDSV in 14 days after immunization
(TCID 50:10-5/mL) to evaluate immune effects. The results show that the weight, beak length, beak width, tongue length and health of the vaccinated duckling are not significantly different (P > 0.05), the levels of biochemical indexes such as ALP, blood calcium, blood phosphorus and the like related to bone metabolism are not significantly different from those of the PBS group (P > 0.05), and the tibia density is equivalent to those of the PBS group. The average weight of ducklings without vaccine protection on day 7 after virus challenge is significantly lower than that of PBS group (P < 0.05). The test result shows that the duckling short beak and dwarfism syndrome virus-like particle vaccine provided by the invention can effectively protect duckling from NGPV attacks.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a PCR verification diagram of recombinant plasmid pET32a-VP2 of example 1 of the present invention, wherein M1: DNA MARKER with a molecular weight of 100 to 2000, M2: DNA MARKER with molecular weight of 100-5000; lane1: amplified VP2 gene fragment, lane2: an amplified linearized vector;
FIG. 2 is a PCR verification chart of engineering strain identification in example 1 of the present invention, M in FIG. 2: the protein Marker, lane1-9 is: empty bacteria, bacteria obtained after VP2 induction, bacteria obtained after TF2-VP2 tetracycline induction, bacteria obtained after TF2-VP2 IPTG induction, bacteria obtained after TF2-VP2 induction as full inducer induction, VP2 supernatant, VP2 precipitation, TF2-VP2 supernatant and TF2-VP2 precipitation;
FIG. 3 is a diagram showing PCR verification of expression of engineering strains under different induction systems in example 1 of the present invention, wherein M: protein markers, lane1-8 were: precipitation at IPTG concentration of 0.25M, 0.5M, 0.75M, 1M, supernatant at IPTG concentration of 0.25M, 0.5M, 0.75M, 1M; lane9-16 is in the order: precipitates with tetracycline concentration of 250ng/ml, 175ng/ml, 87.5ng/ml, 43.75ng/ml, supernatants with tetracycline concentration of 250ng/ml, 175ng/ml, 87.5ng/ml, 43.75 ng/ml; lane17-21 is in the order: inducing supernatant at 16deg.C, 20deg.C, 37deg.C, inducing precipitation at 16deg.C, 20deg.C, 37deg.C;
FIG. 4 is a graph showing the results of purification and PCR identification of structural proteins in example 1 of the present invention, wherein M: protein Marker, lane1: SDS-PAGE identification of purified proteins, lane2: a western blot identification result of the induced supernatant; lane3: a western blot identification result of the purified protein;
FIG. 5 is a transmission electron microscope image of NGPV-VLPs in example 1 of the present invention;
FIG. 6 is a statistical chart of the levels of antibodies and toxin expelling levels of different groups in example 2 of the present invention;
fig. 7 is a graph of the duckbill and tongue comparison of example 2 of the present invention, wherein fig. 7 a: healthy ducks (up) and ducks (down) that attack toxins after immunization; in fig. 7 b: healthy ducks (up) and ducks (down) that are not immune and then detoxified; in fig. 7 c: the tongues of PBS group (left), VLPs+ SBDSV group (middle) and SBDSV group (right) are arranged in sequence from left to right;
Fig. 8 is a statistical chart of body weight, beak length, tongue length and beak width of different groups of ducks in example 2 of the present invention, wherein: figure 8a shows the weight of different groups of ducks; figure 8b shows beak width profiles of different groups of ducks; figure 8c shows beak lengths of different groups of ducks; figure 8d shows tongue length of different groups of ducks;
Fig. 9 is a statistical chart of serum indices related to bone development for different groups of ducks in example 2 of the present invention, wherein: FIG. 9a shows serum ALP levels for different groups of ducks; figure 9b is serum Ca levels for different groups of ducks; FIG. 9c shows serum Pi levels for different groups of ducks;
Fig. 10 is a graph showing the comparison of tibia length of different groups of ducks in example 2 according to the present invention;
FIG. 11 is a graph showing the X-ray comparison of the leg bones of different groups of ducks in example 2 of the present invention;
FIG. 12 is a transmission electron microscope image of NGPV-VLPs, NGPV-VLPs 'and NGPV of comparative example 1 of the present invention, in which the electron microscope images of NGPV-VLPs, NGPV-VLPs' and NGPV are shown in the order from left to right;
FIG. 13 is a gel electrophoresis chart of supernatants and precipitations of fermentation broths of different engineering strains in comparative example 2 according to the present invention, wherein M: protein markers; lane 1-12 is in turn: precipitation of groES-groEL and VP2 co-expression, supernatant; precipitation, supernatant of groES-groEL and TF protein combination and VP2 co-expression; precipitation of TF and VP2 co-expression, supernatant; precipitation, supernatant of the combination of dnaK-dnaJ-grpE and groES-groEL proteins and VP2 co-expression; precipitation, supernatant of the coexpression of dnaK-dnaJ-grpE and VP 2; precipitation of VP2, supernatant without chaperone co-expression.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to better illustrate the embodiments of the present invention, the following is further illustrated by examples.
The materials selected in the embodiment of the invention comprise the following materials: high fidelity enzyme Super Pfx DNA Polymerase and nickel column were purchased from Jiangsu kang as century biotechnology company, inc.; chaperpone PLASMID SET available from TaKaRa; chloramphenicol, ampicillin, tetracycline, IPTG, ECL color development solutions were purchased from soribao corporation; DH5 alpha competent cells, BL21 (DE 3) competent cells were purchased from Ding Wan-Messaging Biotech Co., ltd; DNA MARKER (2000 bp, 5000 bp), protein rainbow Marker (130 kDa) were purchased from Takara Bio-engineering (Dalian) Inc.; plasmid miniprep kit, gel recovery kit was purchased from Omega company; the nucleic acid extraction kit was purchased from Hebei platinum biotechnology limited; agarose Beijing Biotechnology Co., ltd; yeast extract and tryptone were purchased from OXOID company, uk; polyacrylamide gels were purchased from Shanghai elease Biomedicine technologies Co.
Example 1
The embodiment provides a preparation method of short beak and dwarfism syndrome-like particles of a duck, which comprises the following steps:
Step one, constructing recombinant plasmid pET-32a-VP2
Construction of recombinant plasmid pET-32a-VP2
The amino acid sequence of the structural protein VP2 of the NGPV SD isolate is shown as SEQ ID NO. 1; the nucleotide sequence of the structural protein VP2 is shown in GenBank accession number: KY511124.1. Designing 1 pair of primers according to the nucleotide sequence, and respectively introducing homologous arms at the 5' ends of the upstream primer and the downstream primer; the plasmid pET32a (+) is used as a template, and a primer reverse amplification vector is designed. The list of primers used in this step is shown in Table 1.
In order to achieve that the recombinant protein structure is closer to the natural structure, the embodiment modifies the pET32a plasmid, and the specific method comprises the following steps: modifying the pET32a plasmid by utilizing a PCR amplification technology, knocking out the 1 st base pair to the 474 th base pair of the N end of an MCS region in the pET32a plasmid, and modifying a linearization vector with no redundant sequence at the N end, namely the plasmid pET32a (+);
TABLE 1 primer list
The DNA of NGPV SD strains is used as a template, VP2-F and VP2-R are used as primers, and VP2 genes are respectively amplified by a PCR technology; wherein the amplification PCR reaction system: 25 mu L Super Pfx DNA Polymerase, 2 mu L of each of the upstream and downstream primers, 4 mu L of the template, and water was added to 50 mu L. Amplification procedure was set up according to Super Pfx DNA Polymerase kit instructions: pre-denaturation at 98℃for 3min, denaturation at 98℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min,30 cycles; final extension at 72℃for 10min.
Reversely amplifying linearization vectors by taking pET-32a plasmid as a template and pET-32a-F and pET-32a-R as primers to obtain amplified products; and (3) identifying the amplification product by using 1.0% agarose gel electrophoresis, and recovering and purifying positive strips by using a gel recovery kit after positive identification, so as to obtain the linearization vector. Wherein, reverse system: 25 mu L Super Pfx DNA Polymerase, 2 mu L of each of the upstream and downstream primers, 4 mu L of the template, and adding water to 50 mu L; amplification procedure: pre-denaturation at 98℃for 3min, denaturation at 98℃for 30s,55℃ annealing for 30s, and extending at 72 ℃ for 6min for 30 cycles; final extension at 72℃for 10min.
And (3) carrying out seamless cloning on the VP2 gene and the linearization vector by utilizing a homologous recombination technology to construct a recombinant plasmid pET32a-VP2, converting a connection product into an escherichia coli DH5 alpha strain, screening positive colonies by taking T7-R and T7-F as primers, and sequencing and identifying. The PCR verification diagram of the recombinant plasmid pET32a-VP2 is shown in FIG. 1, wherein M1: DNA MARKER with a molecular weight of 100 to 2000, M2: DNA MARKER with molecular weight of 100-5000; lane1: amplified VP2 gene fragment, lane2: amplified linearized vector. As can be seen from the verification result of FIG. 1, the VP2 gene and the linearization vector were successfully obtained.
Step two, 10ng of recombinant plasmid pET-32a-VP2 and 10ng of plasmid pG-Tf2 are transformed into 100. Mu.L of strain OD 600 as BL21 (DE 3) by thermal excitation by using a two-step method. Coating the transformation product on a solid LB plate with double resistance of chloramphenicol (the final concentration is 20 mug/mL) and ampicillin (the final concentration is 50 mug/mL), culturing overnight at 37 ℃, picking single colony, selecting 1mL of liquid LB culture medium, and culturing for 4 hours at 37 ℃ to obtain an engineering strain for expressing duck short beak and dwarfism structural protein VP 2;
In the invention, plasmid pG-Tf2 is purchased from Bao Bio Inc. and has the product number of 3340; plasmid pG-Tf2 can express three proteins, TF, groES and groEL, from the PZt-1 promoter;
wherein the amino acid sequence of TF is shown as SEQ ID NO. 2;
The amino acid sequence of groES-groEL is shown as SEQ ID NO. 3.
Step three, purifying structural protein and assembling the duck short beak and dwarfism syndrome virus-like particles
(1) Purification and identification of structural proteins
And screening the solubility identification experiment of different inducers and induction concentrations on the proteins in the engineering bacteria liquid under the conditions that the IPTG concentration is 0.25-1 mM and the tetracycline alkali concentration is 43.75-250 mug/L by a single factor experiment, and calculating the ratio of the target proteins to the total target proteins in the supernatant expressed by the proteins by using Image J software gray analysis. Finally, 0.25mM IPTG and 250. Mu.g/L tetracycline base were selected as inducers.
Inoculating the engineering strain into a liquid LB culture medium containing 0.25mM IPTG and 250 mug/L tetracycline base according to a ratio of 1:1000, performing induction culture for 12 hours at 20 ℃ and a rotating speed of 180r/min, and collecting bacterial liquid;
Centrifuging the bacterial liquid for 10min at the rotating speed of 10000r/min, and discarding the supernatant to obtain bacterial mud;
The bacterial sludge is treated with PBS with pH of 7 according to the weight-volume ratio of 1g: carrying out ultrasonic treatment for 20min after 20mL of heavy suspension, centrifuging the obtained ultrasonic product for 25min under 10000r/min, and collecting supernatant;
and (3) carrying out nickel column affinity chromatography on the supernatant to obtain purified target protein, and identifying the target protein by SDS-PAGE, wherein the expected size of the protein is 72kDa.
SDS-PAGE is carried out on the purified structural proteins and the structural proteins before induced expression, a wet transfer method is adopted to transfer the recombinant proteins onto a PVDF membrane, 5% skim milk powder is used for overnight blocking, rabbit hyperimmune serum (1:5 000) is used as a primary antibody, and goat anti-rabbit (1:6 000) marked by HRP is used as a secondary antibody. And finally, carrying out light-proof color development by using ECL color development liquid, and verifying the reactivity of the recombinant protein.
The PCR verification diagram of engineering strain identification is shown in fig. 2, and M in fig. 2: the protein Marker, lane1-9 is: empty bacteria, bacteria obtained after VP2 induction, bacteria obtained after TF2-VP2 tetracycline induction, bacteria obtained after TF2-VP2 IPTG induction, bacteria obtained after TF2-VP2 induction as full inducer induction, VP2 supernatant, VP2 precipitation, TF2-VP2 supernatant and TF2-VP2 precipitation.
The PCR verification graph of the expression of the engineering strain under different induction systems is shown in FIG. 3, wherein M: protein markers, lane1-8 were: precipitation at IPTG concentration of 0.25M, 0.5M, 0.75M, 1M, supernatant at IPTG concentration of 0.25M, 0.5M, 0.75M, 1M; lane9-16 is in the order: precipitates, supernatants, at tetracycline concentrations of 250ng/ml, 175ng/ml, 87.5ng/ml, 43.75 ng/ml; lane17-21 is in the order: inducing supernatant at 16deg.C, 20deg.C, 37deg.C, and inducing precipitation at 16deg.C, 20deg.C, 37deg.C.
Through Western blot experiments, the purification and identification results of VP2 protein are shown in FIG. 4, M in FIG. 4: protein Marker, lane1: SDS-PAGE identification of purified proteins, lane2: a western blot identification result of the induced supernatant; lane3: and (5) a western blot identification result of the purified protein.
As can be seen from FIGS. 2 to 4, VP2 structural proteins are expressed in the supernatant, and the target proteins were successfully purified after nickel column affinity chromatography.
Gray value analysis by image J software: the supernatant VP2 structural protein accounts for 80% of the total amount; the soluble VP2 structural protein in the supernatant was purified by nickel column affinity chromatography, and the purity of the VP2 structural protein after purification was about 90%.
Wherein, the amino acid sequence of the structural protein VP2 is shown as SEQ ID NO.1, and the specific steps are as follows:
MTAPAKKNTGKLTDHYPVVKKPKLTEEVSAGGGSSVVQDGGATAEGTEPVAASEMAEGGGGAMGDSSGGADGVGNASGNWHCDSQWMGNTVITKTTRTWVLPSYNNHIYKAITSGTSQDANVQYAGYSTPWGYFDFNRFHCHFSPRDWQRLINNHWGIRPKSLKFKIFNVQVKEVTTQDQTKTIANNLTSTIQVFTDDEHQLPYVLGSATEGTMPPFPSDVYALPQYGYCTMHTNQNGARFNDRSAFYCLEYFPSQMLRTGNNFEFTFDFEEVPFHSMFAHSQDLDRLMNPLVDQYLWNFNEVDSNRNAQFKKAVKGAYGTMGRNWLPGPKFLDQRVRAYTGGTDNYANWNIWNNGNKVNLKDRQYLLQPGPVSATHTEGEASSIPAQNILGIAKDPYRSGSTTAGISDIMVTDEQEVAPTNGVGWKPYGRTVTNEQNTTTAPTSSDLDVLGALPGMVWQNRDIYLQGPIWAKIPKTDGKFHPSPNLGGFGLHNPPPQVFIKNTPVPADPPVEYVNQKWNSYITQYSTGQCTVEMVWELRKENSKRWNPEIQFTSNFSNRTSIMFAPNETGGYVEDRLIGTRYLTQNL;
the amino acid sequence of TF is shown in SEQ ID NO.2, and is specifically as follows:
MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQA;
The amino acid sequence of groES-groEL is shown in SEQ ID NO.3, and is specifically as follows:
MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILENGEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLIMSESDILAIVEASAHDTEHTNLRNKDNGSRRKIRRRSCENAARRKRTGRCSESYPRSKRPRSSGIFRCTDHHQRWCFRCSNRTGRQVRKYGCADGERSCLSKRRCRRRYHHCNRTGSGYHHRSESCCCGHEPDGPETWYRQSGYRCSRTESAVRTMLLSDCSGWYHLRLRRNRRTDRSDGQSRRRRYHRRRYRSAGRTGRGRYAVRPWLPVSLLHQQAGNWRSRTGKPVHPAGQENLQHPRNAAGSGSCCQSRQTAADHRRCRRRSAGNSGCHHAWHRESRCGSTGLRRSSSYAAGYRNPDWRYRDLRDRYGAGKSNPGRPGSGTCCDQQRHHHYHRWRGRSCNPGPCCSDPSADRSNFLRPKTAGTRSETGRRRCSYQSGCCYRSNEREKSTRRCPARDPCCGRRRRGCWWWCCADPRSVTGPAWSERRPERGYQSCTACNGSSAASDRIELRRRTVCCCHRRRRRQLRLQRSNRRIRQHDRHGYPGSNQSNSFCSAVRSFCGWPDDHHRMHGYRPAEKRCSLRRCWRYGRHGWHGRHDVIALHLAEINKPPCDFLRQ.
(2) In vitro self-assembly of duck short beak and dwarfism syndrome virus-like particles
And (3) placing the purified structural protein in a buffer solution (300mM NaCl,50mM Tris,50mM,10mm imidazole, pH 8.0) and dialyzing at 4 ℃ for 16 hours to obtain the short beak and dwarf syndrome virus-like particles of the duck, which are marked as NGPV-VLPs. The morphology of NGPV-VLPs was examined by Transmission Electron Microscopy (TEM), and the results of the transmission electron microscopy of NGPV-VLPs are shown in FIG. 5.
As can be seen from fig. 5, the purified VP2 was successfully assembled into virus-like particles of the morphology of the pseudo-natural virus.
Example 2 application of Duck short beak and dwarfism Virus-like particles
The present example uses white oil to prepare NGPV-VLPs into oil seedlings, and specifically includes the steps of mixing NGPV-VLPs prepared with Tween 80 according to a volume ratio of 100:5, mixing and preparing into a water phase, and placing on ice for standby; preparing an oil phase of 94% of white oil and 6% of span 80 in volume ratio, shaking uniformly after high pressure for 2 minutes, and observing that the oil phase is white and milky at the moment; mixing the water phase and the oil phase in a volume ratio of 1:1, and emulsifying for 10 minutes to 30 minutes by using an emulsifier to prepare NGPV-VLPs vaccine.
Dripping 10 mu L of emulsified vaccine into water, and observing that the droplets are not dispersed to indicate that the vaccine is water-in-oil type; the aspirated fraction was centrifuged at 12000r/min for 5min. If the emulsion is not layered after centrifugation, the vaccine has good stability, the vaccine is marked as SBDS vaccine, the prepared SBDS vaccine is used for immunizing ducklings, and the SBDS vaccine is evaluated by methods of detoxification detection, antibody detection, clinical morbidity condition monitoring, bone development condition monitoring and the like. The specific contents are as follows:
(1) Attack toxin and immunity
Thirty ducklings were randomly divided into 3 groups: SBDSV (14-day-old challenge), vlps+ SBDSV (1-day-old challenge, 14-day-old challenge), PBS (1-day-old and 14-day-old intramuscular injection of PBS at pH 7). The method comprises the steps of carrying out toxin expelling and immunization in an intramuscular injection mode, wherein the toxin expelling dosage is 0.2 mL/dose (TCID 50:10-5/mL); the immunization dose was 100. Mu.g/dose. Three groups collect anal swabs 1,3, 5, 7, 9, 13, 14 days after challenge; serum was collected 7, 14, 21 days after challenge and serum levels of ALP, serum calcium and serum phosphorus were measured. Body weight and beak length were measured at 0, 4, 7, 14, 21 after challenge; serum was collected 5, 7, 14 days after immunization.
(2) Toxin expelling detection and antibody detection
The collected anal swab is placed in 600 mu L of physiological saline for standing for 2 hours at the temperature of 4 ℃, and QPCR detection is carried out after virus DNA is extracted by using a nucleic acid extraction kit, wherein the primers are as follows:
QPCR-F：5'-TGCCGATGGAGTGGGTAAT-3'；
QPCR-R：5'-GAAGTGGCAGTGGAAGCGATT-3'。
ELISA experiments were performed on the collected sera to detect specific antibodies: 0.5. Mu.g/mL of VP2 protein bicarbonate buffer (pH 9.6) was coated onto a 96-well microtiter plate and incubated overnight at 4 ℃; adding tween-20 with the volume ratio of 20% into PBS solution with the pH of 7 to prepare PBS-T solution; after washing the VP2 protein incubated overnight as described above three times with PBS-T solution, blocking with PBS-T solution containing 5% BSA at 37deg.C for 2h; after the PBS-T solution is washed for 3 times, 100 mu L (dilution ratio: 1:100) of serum to be tested is diluted by adding the PBS-T solution, and the mixture is incubated for 1h at 37 ℃; then adding goat anti-duck secondary antibody with HRP (dilution ratio: 1:5000), and incubating at 37 ℃ for 1 hour; the reaction was washed 3 times and colorimetrically reacted with 100. Mu.L of tetramethylbenzidine substrate solution (Sangon BiotechCo., ltd., china) at 37℃for 15 min. The development was stopped with 50. Mu.L of 2M H 2SO4 and the OD was read at 450 nm. The collected serum was subjected to IFA experiments to detect neutralizing antibodies: DEF cells were spread on 96-well plates and the serum to be tested was diluted in the ratio 10 mu L NGPV according to volume 1:1, 96 wells were added and IFA assay was performed after 36 h. The antibody level and the detoxification level results are shown in fig. 6, wherein fig. 6a shows the specific IgG antibody level of the immunized duck, and fig. 6b shows the statistical diagram of the detoxification.
As can be seen from FIG. 6a, ELISA detected specific IgG level in the immunized group, and OD 450nm after immunization was significantly higher than that in the control group (P < 0.001). After the challenge, the level of the anal swab was examined every other day, and as shown in fig. 6b, the level of the vlps+ SBDSV group (protective group) was significantly lower than that of SBDSV group (virus model group) (P < 0.001), only 1 out of 10 vlps+ SBDSV group showed the challenge, and the SBDSV group ducks began to expel the virus on day 3 of the challenge, consistently for 14 days.
(3) Clinical onset of disease
After NGPV-VLPs are prepared into oil seedlings, 1-day-old duckling is inoculated, and the duckling is challenged at 14-day-old, and whether NGPV-VLPs can protect the duckling is examined. During the trial, different groups of duckbill and tongue comparison figures are shown in fig. 7, wherein healthy ducks (upper) and post-immune toxin-counteracting ducks (lower) in fig. 7 a; in fig. 7b, healthy ducks (upper) and non-immune post-challenge ducks (lower); the tongues of PBS group (left), vlps+ SBDSV group (middle) and SBDSV group (right) are shown in fig. 7c, in order from left to right; the statistical graphs of the weights, beak lengths, tongue lengths and beak widths of the different groups are shown in fig. 8, wherein fig. 8a shows the weights of the different groups of ducks; figure 8b shows beak lengths of different groups of ducks; fig. 8c shows tongue length for different groups of ducks; figure 8d shows beak width profiles for different groups of ducks. The test period shows that the ingestion rule of the immune group ducks is good in mental state; the weight of the VLPs+ SBDSV group is not different from that of the PBS group (P is less than 0.05), the weight difference appears between the SBDSV group and the PBS group at 7 days of challenge, and the difference is obviously increased at 14 days of challenge and 21 days of challenge (14 days of challenge: P is less than 0.01; 21 days of challenge: P is less than 0.001); as shown in FIG. 7b, the beak lengths of the VLPs+ SBDSV group and the PBS group are not different (P < 0.05), the beak lengths of the SBDSV group ducks are obviously shortened (P < 0.05) on the 4 th day of virus attack, and the difference is further increased with the increase of the age of the days. As can be seen from FIG. 8d, the beak width of the VLPs+ SBDSV group is not different from that of the PBS group (P < 0.05), and the beak length of the SBDSV group ducks is obviously shortened on day 7 (P < 0.05). 35 days after the challenge, the duck tongues were collected by killing each group of ducks, and FIG. 7c shows that SBDSV groups of ducks had a tongue length shorter than the other two groups (P < 0.001), and the tongue length of VLPs+ SBDSV ducks was not different from that of healthy ducks (P > 0.05).
(4) Bone development
Bone dysplasia is one of symptoms caused by SBDS, and the invention detects 3 serum biochemical indexes (ALP, ca, pi) related to bone development, and detects the tibia length of each group of duckling, so as to compare the development condition of each group of duckling bone. The bone development condition of each group of duckling is shown in fig. 9-11, wherein fig. 9 is a statistical chart of serum indexes related to bone development of different groups of duckling, and the statistical chart comprises the following steps: FIG. 9a shows serum ALP levels for different groups of ducks; figure 9b is serum Ca levels for different groups of ducks; FIG. 9c shows serum Pi levels for different groups of ducks; fig. 10 is a graph comparing tibia lengths of different groups of ducks; fig. 11 is a graph of the X-ray comparison of the leg bones of different groups of ducks. As can be seen from fig. 9, the serum of the ducks in the vlps+ SBDSV group and the serum of the ducks in the healthy group have no significant difference in ALP, calcium and phosphorus (P > 0.05), and compared with the ducks without vaccine protection, the serum of the ducks in the vlps+ SBDSV group has significantly lower ALP, calcium and phosphorus levels than those of the ducks in the other two groups (P < 0.05) after 7 days of challenge. As shown in fig. 10, the SBDSV groups of ducks had significantly lower tibia length than the vlps+ SBDSV groups of ducks (P < 0.01) 7 days after challenge, and the vlps+ SBDSV groups were indistinguishable from the PBS group (P > 0.05). As shown in fig. 11,5 ducks were randomly extracted from each group for X-ray examination, 3 ducks in SBDSV groups had significantly lower metatarsal density than PBS group, and vlps+ SBDSV group was not significantly different from PBS group for X-ray imaging.
Comparative example 1
The present comparative example provides a preparation method of virus-like particles of short beak and dwarfism of duck, which is basically the same as that provided in example 1, except that: recombinant plasmid pET-32a-VP2' is constructed by using unmodified pET32a plasmid as a vector, and duck short beak and dwarfism syndrome virus-like particles (recorded as NGPV-VLPs ') are prepared by using the recombinant plasmid pET-32a-VP2 '. The remaining parameters in the preparation method of this example were the same as those of example 1.
A transmission electron microscope image of NGPV-VLPs, NGPV-VLPs 'and NGPV is shown in FIG. 12, and an electron microscope image of NGPV-VLPs, NGPV-VLPs' and NGPV is shown in FIG. 12, in this order from left to right.
As can be seen from FIG. 12, the modified vector with the redundant sequences removed had good morphology and uniform size of VLPs assembled, and was similar to the morphology of the natural virus. Particles assembled by the unmodified vector are irregular and different in size and have no good virus-like morphology.
Comparative example 2
The comparative example provides a construction method of different engineering bacteria, and different engineering bacteria are constructed under the conditions of no molecular chaperones, single component molecular chaperones, dnaK-dnaJ-grpE and groES-groEL protein combination and dnaK-dnaJ-grpE selection in sequence. The method for constructing different engineering bacteria in this comparative example is basically the same as that in example 1, except that the selected molecular chaperones are different. Further, the engineering bacteria provided in example 1 and the engineering bacteria constructed in this comparative example were subjected to fermentation culture, cell collection, ultrasonic disruption, and sds-page detection of the supernatant and precipitate, and the resulting gel electrophoresis chart is shown in FIG. 13. In fig. 13, the bands are respectively: m: protein markers; lane 1-12 is in turn: precipitation of groES-groEL and VP2 co-expression, supernatant; precipitation, supernatant of groES-groEL and TF protein combination and VP2 co-expression; precipitation of TF and VP2 co-expression, supernatant; precipitation, supernatant of the combination of dnaK-dnaJ-grpE and groES-groEL proteins and VP2 co-expression; precipitation, supernatant of the coexpression of dnaK-dnaJ-grpE and VP 2; precipitation of VP2, supernatant without chaperone co-expression.
As shown in FIG. 13, the ratio of the target protein (72 KD) in the supernatant and the precipitate is the highest in the engineering strain constructed by combining the groES-groEL and TF proteins and VP2, in other words, the combined action of the groES-groEL system and TF has the best dissolving promoting effect on the recombinant protein VP2, so that the chaperone combination, namely the chaperone TF2, is selected.
In summary, the invention firstly optimizes molecular chaperone combination and reforms structural protein VP2 expression plasmid vector, then constructs two recombinant plasmids pET-32a-VP2 and pG-Tf2, and successfully realizes structural protein VP2 soluble expression by utilizing molecular chaperone protein through a double-plasmid co-expression method. Further explores that under the condition that IPTG and tetracycline alkali are used as inducers, the constructed engineering strain for producing the structural protein VP2 of the short beak and dwarfism of the duck can well and soluble express the structural protein VP2. Gray analysis is carried out by Image-J software, and the supernatant structural protein VP2 accounts for 80% of the total amount under the optimal condition; the soluble structural protein VP2 in the supernatant was purified by nickel column affinity chromatography, and the purity of the purified structural protein VP2 was about 90%. And then Western blot experiments are carried out, and the result of the Western blot experiments not only verifies the soluble expression of the structural protein VP2 in the supernatant and the successful purification of the structural protein VP2, but also proves that the antigen has good reactivities. The purified structural protein is assembled into the duck short beak and dwarfism virus-like particles, and the duck short beak and dwarfism virus-like particles are further prepared into vaccines, so that the vaccines can be proved to be capable of effectively protecting duckling from virus attack.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (10)
1. An engineered strain expressing structural protein VP2 of short beak and dwarfism of duck, characterized in that: the engineering strain takes escherichia coli as a host cell and comprises a coding gene of a combination of coding genes and molecular chaperones of duck short beak and dwarfism syndrome structural protein VP 2.
2. The engineered strain expressing structural protein VP2 of the short beak and dwarfism of duck of claim 1, wherein the engineered strain is characterized by: the amino acid sequence of the structural protein VP2 is shown as SEQ ID NO. 1;
The structural protein VP2 is expressed by constructing a recombinant plasmid pET-32a-VP2 containing a coding gene of the structural protein VP2 and a linearization vector.
3. The engineered strain expressing structural protein VP2 of the short beak and dwarfism of duck of claim 1, wherein the engineered strain is characterized by: the combination of chaperones includes TF, groES and groEL;
the amino acid sequence of TF is shown as SEQ ID NO. 2;
The amino acid sequence of groES-groEL is shown as SEQ ID NO. 3.
4. The engineered strain expressing structural protein VP2 of the short beak and dwarfism of duck of claim 1, wherein the engineered strain is characterized by: the escherichia coli is BL21 (DE 3) strain; and/or
The expression chaperone combinations include the expression of TF, groES and groEL in plasmid pG-Tf 2.
5. A method of constructing an engineered strain expressing structural protein VP2 of the short beak and dwarfism of duck as claimed in any one of claims 1 to 4, wherein: the construction method comprises the following steps:
Firstly, connecting a duck short beak and dwarfism syndrome structural protein VP2 gene with a linearization vector by utilizing a homologous recombination technology, and constructing a recombinant plasmid pET-32a-VP2;
and step two, converting the recombinant plasmid pET-32a-VP2 and the plasmid pG-Tf2 into escherichia coli to obtain an engineering strain for expressing the structural protein VP2 of the short beak and dwarfism of the duck.
6. The method for constructing an engineering strain expressing structural protein VP2 of short beak and dwarfism of duck according to claim 5, wherein the method comprises the steps of: in the first step, the linearization vector is an engineered pET32a plasmid with only a His tag at the N end.
7. The method for constructing an engineering strain expressing structural protein VP2 of short beak and dwarfism of duck according to claim 4, wherein the method comprises the steps of: in the second step, the plasmid pG-Tf2 and the recombinant plasmid pET-32a-VP2 are sequentially transformed into an E.coli BL21 (DE 3) strain by a two-step method.
8. A preparation method of short beak and dwarfism syndrome-like particles of ducks, which is characterized by comprising the following steps: the preparation method comprises the following steps: and (3) carrying out induced culture on the engineering strain expressing the duck short beak and dwarfism structural protein VP2 to obtain bacterial liquid, and purifying and self-assembling the bacterial liquid to obtain the duck short beak and dwarfism virus-like particles.
9. The method for preparing the duck short beak and dwarfism virus like particle as claimed in claim 8, wherein: the self-assembled form is in vitro self-assembly.
10. The use of the engineered strain expressing the structural protein VP2 of the short beak and dwarf syndrome according to any one of claims 1 to 4, the engineered strain expressing the structural protein VP2 of the short beak and dwarf syndrome constructed by the construction method of the engineered strain expressing the structural protein VP2 of the short beak and dwarf syndrome according to any one of claims 5to 7, or the preparation method of the virus-like particles of the short beak and dwarf syndrome according to claim 8 or 9, to prepare the virus-like particles of the short beak and dwarf syndrome.
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