CN114480378A

CN114480378A - Construction method and application of novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome

Info

Publication number: CN114480378A
Application number: CN202210137107.5A
Authority: CN
Inventors: 袁万哲; 郝雪飘; 赵款; 雷白时; 薛拥志; 张武超
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2022-02-15
Filing date: 2022-02-15
Publication date: 2022-05-13
Anticipated expiration: 2042-02-15
Also published as: CN114480378B

Abstract

The invention provides a construction method and application of a novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome, belonging to the technical field of viruses. The invention clones the complete genome of a novel goose parvovirus SD strain to a plasmid pBluescript II SK by a sectional amplification method to obtain a recombinant plasmid; simultaneously, the NcoI site in the genome is mutated by utilizing an overlapping PCR technology and is used as a genetic marker for identifying wild viruses and saving the viruses; and then mixing the recombinant plasmid with a transfection reagent, and inoculating SPF duck embryos through a yolk sac and allantoic cavity way to obtain the rescued viruses. The novel goose parvovirus reverse genetic operation system established by the invention can be used for the research of virulence analysis, cross-species propagation mechanism and the like of the novel goose parvovirus, and simultaneously lays a foundation for the research of novel vaccines of duck short beak and dwarfism syndrome.

Description

Construction method and application of novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome

Technical Field

The invention belongs to the technical field of viruses, and particularly relates to a construction method and application of a novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome.

Background

Since 2015, commercial meat ducks (cherry valley ducks, Beijing ducks and the like) in Jiangsu, Shandong, Hebei and other areas of China have suffered from diseases which are caused by unidentified reasons and are characterized by slow development, atrophy of upper and lower beaks, overhanging, swelling and downward bending of tongues, and are commonly called Short Beak and Dwarfism Syndrome (SBDS) of ducks. The disease incidence rate is 10% -30%, and can reach more than 50% in serious cases, the weight of the sick ducks in slaughtering is reduced by 20% -30% compared with the healthy ducks, and the weight of the sick ducks in slaughtering is only 50% of the normal weight of the sick ducks in serious cases. The feed conversion ratio of infected ducks is obviously increased, the slaughtering qualification rate is reduced, and the coracoid, wing bones, shin bones and the like are easy to fracture in the processes of picking and slaughtering. After the disease occurs, the laboratory and a plurality of domestic laboratories successively verify that the disease is infectious disease, the pathogeny of the disease is waterfowl parvovirus, and the virus is also called duck source goose parvovirus, goose parvovirus variant or Novel Goose Parvovirus (NGPV) because the homology of the virus and gosling plague (goose parvovirus) is very high (92.7% -97.3%); meanwhile, the duck parvovirus has higher homology (81.1% -85.0%) with Muscovy duck parvovirus, is also called novel duck parvovirus and is collectively called NGPV in the research. In 1971 and 1995, the disease was reported to occur in semi-muscovy ducks in france and polish, and the etiology was not finally determined to be NGPV until 2009. At present, the disease still widely occurs and prevails in duck breeding places in China, and meanwhile, clinical manifestations such as abnormal moulting of ducks and the like and poultry infection such as half muscovy ducks (showing short beaks and dwarfies) and ostriches (showing paralysis and found for the first time in the subject group) and the like occur; the disease can be horizontally transmitted through the digestive tract and can also be vertically transmitted through the breeding ducks/hatching eggs, thereby causing great economic loss to the poultry industry in China.

After the onset of epidemic disease, NGPV SD strain was isolated in 2015 from cherry valley duck with SBDS, and the virus proliferated on duck embryo or Duck Embryo Fibroblast (DEF) and also on susceptible goose embryo or Goose Embryo Fibroblast (GEF).

NGPV is combined with Muscovy Duck Parvovirus (MDPV) and Goose Parvovirus (GPV) which belong to the Parvoviridae (Parvoviridae) Parvovirinae (Parvoviridae) and members of the genus Dependovirus (Dependovirus). Under an electron microscope, the NGPV virus particles are divided into a solid form and a hollow form, the diameter is 20-24 nm, no capsule membrane exists, and the regular icosahedron is symmetrical. NGPV is a single-stranded chain DNA virus, with a genome of about 5.1kb in length, containing two Identical Terminal Repeats (ITRs) and two Open Reading Frames (ORFs). ITRs play an important role in viral replication and packaging. The left open reading frame encodes the nonstructural protein NS1 and the NS2 generated by cutting the mRNA, and NS1 and NS2 are jointly involved in the replication and transcription of the virus. The right open reading frame encodes three structural proteins, VP1, VP2, VP3, which constitute the icosahedral capsid in a ratio of 1:1:8, by cleavage using different initiation codons and proteolytic enzymes.

The basic process of the reverse genetic operation technology is to obtain the whole genome of the virus, assemble the genome to a suitable vector, transfect the constructed full-length plasmid to a susceptible cell, and then save the virus with infectivity. Reverse genetics is an effective platform for virus research, and the influence of operations on viruses can be judged by manipulating changes of genomes to influence corresponding apparent changes. The reverse genetic operation system of the novel goose parvovirus is established, on the basis, the virulence, cross-species propagation mechanism and the like of the novel goose parvovirus can be researched, a useful platform can also be provided for the research of related vaccines, and related research and reports are not available at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides a construction method and a rescue method of a novel goose parvovirus SD strain full-length infectious clone, establishes a reverse genetics operating system of the novel goose parvovirus, and provides technical support for virulence research, cross-species propagation mechanism, related vaccine development and the like of the virus in future.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a primer for preparing a novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome, and the specific sequence is shown as SEQ ID No. 1-14.

The invention also provides a molecular marker primer for full-length infectious cloning of the novel goose parvovirus SD strain causing duck short beak and dwarfism syndrome, and the specific sequence is shown as SEQ ID No. 15-16.

The invention also provides a kit containing the primer combination with the gene sequence shown as SEQ ID No. 1-16.

The invention also provides a rescue system for the novel goose parvovirus SD strain aiming at duckling short beak and dwarfism syndrome, which comprises amplification products of seven segments of A, B, C1, C2-1, C2-2 and D, E and two plasmids.

Preferably, the plasmids are pSK-XBSE and pBluescript II SK.

The invention also provides a construction method of the novel goose parvovirus SD strain full-length infectious clone causing duck brachiocephalic and dwarfism syndrome, which comprises the following steps:

(1) construction of the intermediate plasmid pSK-XBSE for cloning the C fragment: selecting a vector pBluescript II SK, synthesizing a gene fragment, carrying out multi-cloning site modification to obtain a pBluescript II SK plasmid containing the gene fragment shown as SEQ ID NO.17, then carrying out transformation, culture and identification, carrying out propagation on positive bacteria, extracting the plasmid to serve as an intermediate plasmid, and naming the plasmid as pSK-XBSE;

(2) extracting total DNA of NGPV SD F5 virus genome;

(3) designing and synthesizing six pairs of overlapping primers SegA-F, SegA-R, SegB-F, SegB-R, SegC1-F, SegC1-R, SegC2-F, SegC2-R, SegD-F, SegD-R, SegE-F, SegE-R covering the whole genome of the NGPV SD virus, wherein SegA-F contains a homologous arm with a pBluescript II SK plasmid, SegA-R contains a homologous arm with a pBluescript II SK plasmid and restriction enzyme cutting sites of SphI and Bcl I, SegE-F contains a homologous arm with a pBluescript II SK plasmid, SegE-R contains a homologous arm with a pBluescript II SK plasmid and restriction enzyme cutting sites of SphI, and the sequence of each primer is shown as SEQ ID NO. 1-12; introducing a genetic marker delta Nco I into the C2 fragment, wherein mutation primers are 1-R and 2-F, and the sequence of the mutation primers is shown as SEQ ID NO. 13-14; designing an rSD-F, rSD-R primer for amplifying a fragment containing an NcoI enzyme cutting site, wherein the specific sequence is shown as SEQ ID NO. 15-16;

(4) SegA-F/SegA-R, SegB-F/SegB-R, SegC1-F/SegC1-R, SegD-F/SegD-R, SegE-F/SegE-R are respectively used as primers, and high fidelity enzyme is used

The FastPfu Fly DNA Polymerase performs PCR amplification on the five fragments, detects, purifies and recovers PCR amplification products, and the PCR amplification products are equivalent to A, B, C1 and D, E fragments. Then SegC2-F/1-R and 2-F/SegC2-R are used as primers, and high fidelity enzyme is used

Performing PCR amplification on the C2 fragment by Fastpfu Fly DNA Polymerase, detecting, purifying and recovering PCR amplification products, taking the recovered products as a template, taking SegC2-F/SegC2-R as a primer, and using high fidelity enzyme

Performing overlapping PCR on Fastpfu Fly DNA Polymerase, and detecting, purifying and recovering PCR amplification products to obtain a C2 fragment;

(5) plasmid pBluescript II SK Using XhoPerforming double enzyme digestion on the I and the EcoRI, performing agarose gel electrophoresis, recovering the target product, and using

Performing homologous recombination on the recovered product and the fragment A by using a HiFi DNA Assembly MasterMix, transforming the ligation product into JM110 competent cells, treating and culturing, selecting a colony with a positive PCR detection result, performing sequencing to confirm that the ligation is successful, performing propagation, extracting a Plasmid by using a QIAGEN Plasmid Mini Kit, and naming the obtained Plasmid as pSK-A; the plasmid pBluescript II SK was digested with EcoRI and Not I, subjected to agarose gel electrophoresis, and the desired product was recovered and used

Homologous recombination is carried out on the recovered product and the E fragment by a HiFi DNA Assembly Master Mix, the connection product is transformed into a DH5 alpha competent cell, the cell colony which is positive by a PCR detection result is selected after treatment and culture, the cell colony is propagated after the successful connection is confirmed by sequencing, a Plasmid is extracted by a small Plasmid extraction Kit QIAGEN Plasmid Mini Kit, and the obtained Plasmid is named as pSK-E;

(6) performing double enzyme digestion on plasmid pSK-A and B fragment products by using Sph I and Bcl I respectively, performing agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony which is positive in a PCR detection result, performing amplification after the successful connection is verified by sequencing, extracting a plasmid, naming the obtained plasmid as pSK-AB, transforming the plasmid into JM110, selecting a colony which is positive in a PCR detection result, performing amplification after the successful connection is verified by sequencing, extracting the plasmid, and naming the plasmid as pSK-AB (JM 110); performing double enzyme digestion on plasmid pSK-E and a D fragment product respectively by using Sph I and EcoR I, performing agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony which is positive in a PCR detection result, performing amplification propagation after the successful connection is verified by sequencing, extracting a plasmid, and naming the obtained plasmid as pSK-DE;

(7) carrying out double enzyme digestion on plasmid pSK-XBSE and a C1 fragment product respectively by using Bcl I and Sph I, carrying out agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into DH5 alpha competent cells for culture, selecting a colony with a positive PCR detection result, carrying out amplification propagation after the successful connection is verified by sequencing, extracting a plasmid, and naming the obtained plasmid as pSK-C1; carrying out double enzyme digestion on plasmid pSK-C1 and a fragment product of C2 by using Sph I and EcoR I respectively, carrying out agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into JM110 competent cells for culture, selecting a colony with a positive PCR detection result, carrying out amplification after the successful connection is verified by sequencing, extracting a plasmid, and naming the obtained plasmid as pSK-C;

(8) respectively carrying out double enzyme digestion on plasmids pSK-AB (JM110) and pSK-C by using Xho I and Bcl I, carrying out agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony with a positive PCR detection result, carrying out amplification after the successful connection is verified by sequencing, extracting a plasmid, and naming the obtained plasmid as pSK-ABC;

(9) carrying out double enzyme digestion on plasmids pSK-ABC and pSK-DE by using EcoR I and Not I respectively, carrying out agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony which is positive in PCR detection result, carrying out propagation on the colony after the successful connection is verified by sequencing, extracting the plasmid to obtain the plasmid containing the complete clone, and naming the obtained plasmid as pSK-SD; the construction of the full-length plasmid is completed and the gene sequencing is carried out, and the sequence is shown as SEQ ID NO. 1.

The invention also provides a novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome, and the cloned gene sequence is shown as SEQ ID NO. 18.

The invention also provides a rescue method of the novel goose parvovirus SD strain causing duck brachiocephalic and dwarfism syndrome, which comprises the steps of firstly mixing the recombinant plasmid pSK-SD with a transfection reagent, then transfecting the mixed reagent by 0.3 mL/piece of plasmid through the chorioallantoic cavity and the allantoic cavity, wherein the inoculation plasmid amount of each duck embryo is 3.0 mu g, then obtaining allantoic fluid after inoculation for 120h, then inoculating 0.3 mL/piece of duck embryo through the same way for passage, and obtaining the allantoic fluid after 120h, thus obtaining the rescued virus.

The specific rescue process is as follows: the plasmid was mixed with the transfection reagent at a ratio of 1:2.5(μ g: μ L) using Lipofectamine 2000 as the transfection reagent. Diluting the plasmid (the plasmid amount is 20 mu g) in 1mL of Opti-DMEM medium, and gently mixing; after the Lipofectamine 2000 is shaken up lightly, 50 mu L of Lipofectamine is taken to be mixed with 950 mu L of Opti-DMEM culture medium evenly, and the mixture is incubated for 5 minutes at room temperature; mixing the diluted plasmid with the diluted Lipofectamine 2000, gently mixing uniformly, and standing at room temperature for 20 minutes; the transfection mixing reagent is transfected by the allantoic cavity and the allantoic cavity in an amount of 0.3 mL/piece, and the amount of the inoculated plasmid of each duck embryo is 3.0 mu g. And (3) obtaining allantoic fluid after 120h of inoculation, then inoculating 0.3 mL/duck embryo for passage through the same way, and obtaining the allantoic fluid after 120h to obtain the rescued virus.

The invention also provides an identification method for rescuing viruses by the novel goose parvovirus SD strain causing duck brachiocephalic and dwarfism syndrome, which comprises the steps of firstly designing a genetic marker delta Nco I primer rSD-F, rSD-R, wherein the specific sequence is shown as SEQ ID NO. 15-16; then extracting DNA for rescuing virus; carrying out PCR amplification on A, B, C, D, E five fragments by adopting an rSD-F/rSD-R primer, detecting, purifying and recovering a PCR amplification product, and carrying out enzyme digestion reaction by using NcoI restriction endonuclease; to rescue the virus, Nco I could not be cleaved to recover the product.

The invention also provides application of the primer or the kit or the rescue system or the full-length infectious clone of the novel goose parvovirus SD strain in preparation of a novel goose parvovirus SD strain diagnostic reagent and/or vaccine causing duck short beak and dwarfism syndrome.

Compared with the prior art, the invention has the following technical effects:

(1) the invention provides a novel goose parvovirus SD strain full-length infectious clone, which can successfully save viruses, and introduces a genetic marker in the full-length infectious clone, and the marker can still stably exist after passage and can be used as a reliable genetic marker for identifying wild viruses and saving the viruses.

(2) The invention constructs the full-length infectious clone of the novel goose parvovirus SD strain by a segmented amplification method, ensures the integrity of parvovirus gene sequences in obtained plasmids, constructs an intermediate plasmid by a homologous recombination method, leads the construction steps to be simpler, and simultaneously facilitates the transformation of a series of gene loci in the later period by the constructed intermediate plasmid.

(3) The novel goose parvovirus reverse genetics operating system established by the invention can also be used for virulence analysis, cross-species propagation, related vaccine development and the like of the novel goose parvovirus.

Drawings

FIG. 1 is a schematic diagram of the construction strategy of the full-length infectious clone of the novel goose parvovirus SD strain in example 1;

FIG. 2 is a schematic diagram of the NGPV genome and its cleavage sites in example 1;

FIG. 3 is a gel electrophoresis chart of the sectional amplification product of SD strain in example 1;

FIG. 4 is a gel electrophoresis chart of the amplification of each gene segment of pSK-SD plasmid in example 1;

FIG. 5 is a gel electrophoresis chart of the restriction enzyme identification of pSK-SD plasmid in example 1;

FIG. 6 is a gel electrophoresis image of the identification of genetic markers for rescuing viruses in example 2;

FIG. 7 is a graph comparing the sequences of rescued viruses and parental viruses of example 2;

FIG. 8 is a Western Blot analysis of allantoic fluid F3 rescued virus in example 2;

FIG. 9 is the F3 generation allantoic fluid indirect immunofluorescence assay chart of the rescued virus in example 2.

Detailed Description

The experimental methods in the present examples are all conventional methods unless otherwise specified. The following are merely illustrative of several embodiments of the present invention. It is obvious that the present invention is not limited to the following embodiments, but many variations are possible. Therefore, modifications and improvements based on the disclosure of the present invention should be made by those skilled in the art within the scope of the claims of the present invention.

The technical solution of the present invention is further illustrated in detail by the following examples.

EXAMPLE 1 construction of full-Length infectious clone of novel goose parvoVirus SD Strain

A schematic diagram of the construction strategy of the full-length infectious clone of the novel goose parvovirus SD strain is shown in FIG. 1. The specific process is as follows:

1.1 Strain and Duck embryo

NGPV SD was isolated and stored by the North river university veterinary biologics laboratory, and the entire genomic sequence determined had been uploaded to NCBI (GenBank accession number: KY511124), and passage 5 virus used in this study.

1.2 construction of the intermediate plasmid pSK-XBSE

The NGPV SD genome structure is shown in FIG. 2, and 1-189bp is marked as an A fragment, 190-585bp is marked as a B fragment, 586-4470bp is marked as a C fragment, 4471-4868bp is marked as a D fragment, and 4869-5053bp is marked as an E fragment. The sphI cleavage site was chosen as an intermediate cloning site for the separate cloning of ITRs in order to allow complete cloning of ITRs. However, because the C fragment is longer and the middle position of the C fragment also has a SphI restriction site, an intermediate plasmid pSK-XBSE containing the SphI restriction site is constructed to help clone the C fragment.

The specific implementation method comprises the following steps:

synthesizing gene segments, wherein the gene sequence is shown as SEQ ID NO.17, carrying out double enzyme digestion on pBluescript II SK plasmid by using Xho I and EcoRI restriction enzyme, connecting T4 DNA Ligase with corresponding segments, transforming into JM110 competent cells for culture, selecting a single colony for culture, extracting plasmid, carrying out enzyme digestion identification by using Sph I restriction enzyme, carrying out propagation on positive bacteria, and extracting plasmid.

1.3 extraction of Total DNA of viral genome of NGPV SD generation F5

Extracting total DNA of NGPV SD F5 allantoic fluid with DNA extraction kit, and storing in a refrigerator at-20 deg.C.

1.4 primer design and Synthesis

And designing a corresponding primer according to the selected enzyme cutting site according to the NGPV SD whole gene sequence. The selection of the Sph I restriction enzyme sites results in the need to construct and then combine the 3 'and 5' ITRs separately, as well as the C fragment. SegA-F contains the homologous arm with pBluescript II SK plasmid, SegA-R contains the homologous arm with pBluescript II SK plasmid and SphI, Bcl I restriction enzyme cutting site, A segment is connected into pBluescript II SK plasmid by using homologous recombination technology to construct PSK-A intermediate plasmid, and SphI, Bcl I restriction enzyme cutting site is introduced at the same time; SegE-F contains the homology arm with pBluescript II SK plasmid, SegE-R contains the homology arm with pBluescript II SK plasmid and sphI restriction enzyme cutting site, PSK-E intermediate plasmid is constructed, and sphI cutting site is introduced at the same time; the SegB-F, SegB-R, SegC1-F, SegC1-R, SegC2-F, SegC2-R, SegD-F, SegD-R primer can completely amplify corresponding fragments and has protective bases, so that the enzyme digestion probability is increased; the sequences of the primers are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12; meanwhile, in order to introduce a genetic marker delta NcoI into a C2 fragment, mutation primers are designed to be 1-R and 2-F, the 3807 th base C of a genome can be mutated into a base A, both the mutated codon ACA and the mutated codon ACC encode the same amino acid (T), the mutation belongs to the same sense mutation, the gene expression is not influenced, and the NcoI restriction endonuclease recognition sequence of CCATGG is removed after point mutation. The sequence of the mutation primer is shown as SEQ ID NO.13 and SEQ ID NO. 14; designing rSD-F, rSD-R primers for amplifying a fragment containing the restriction enzyme site of Nco I, wherein the sequences of the primers are shown as SEQ ID NO.15 and SEQ ID NO. 16; the primer was synthesized by Shanghai Biotechnology Ltd, and was used by diluting it to 10. mu.M with nuclease-free water. Specific information on the sequences of the primers is shown in Table 1.

TABLE 1 details of amplification primer, mutation primer and molecular marker primer sequences

1.5 PCR amplification of each Gene fragment of NGPV SD

NGPV SD F5 generation DNA is taken as a template, SegA-F/SegA-R, SegB-F/SegB-R, SegC1-F/SegC1-R, SegC2-F/1-R and 2-F/SegC2-R, SegD-F/SegD-R, SegE-F/SegE-R are respectively taken as primers, and high fidelity enzyme is used

The FastPFu Fly DNA Polymerase performs PCR amplification on the five fragments, detects and purifies and recovers the PCR amplification yield to obtain A, B, C1, C2-1, C2-2 and D, E seven fragments which respectively correspond to the 1-189bp fragment (A), 190-585bp fragment (B), 586-2359bp fragment (C1), 2360-3806bp fragment (C2-1), 3807-4470bp fragment (C2-2), 4471-4868bp fragment (D) and 4869-5053bp fragment (E) in the graph 2. The amplification system is

FastPfu Fly Buffer 10μL、2.5mM dNTPs 5μL、Forward Primer(10mM)2μL、Reverse Primer(10mM)2μL、

Fastpfu Fly DNA Polymerase 1. mu.L, template 5. mu. L, ddH2O 25. mu.L; the amplification conditions were: 3min at 95 ℃ for 1 cycle; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃ for 1 cycle. Detecting PCR amplification product by 1% agarose gel electrophoresis after amplification, the electrophoresis pattern is shown in FIG. 3, wherein M is DL2000 Marker; 1 is the A fragment (253bp) amplified from SegA-F/SegA-R; 2 is a B fragment (416bp) amplified from SegB-F/SegB-R; 3 is a C1 fragment (1800bp) amplified by SegC1-F/SegC 1-R; 4 is a C2-1 fragment (1703bp) amplified from SegC 2-F/1-R; 5 is a C2-1 fragment (691bp) amplified from 2-F/SegC 2-R; 6 is a D fragment (407bp) amplified from SegD-F/SegD-R; 7 is the E fragment (237bp) amplified from SegE-F/SegE-R.

The fragments of interest were recovered using a gel recovery kit. The products recovered from C2-1 and C2-2 areTemplate, SegC2-F/SegC2-R as primer, using high fidelity enzyme

Overlap PCR was performed with Fastpfu Fly DNA Polymerase, and the amplification procedure was: taking a PCR tube, adding

FastPfu Fly Buffer 10μL、2.5mM dNTPs 5μL、

Recovery products of Fastpfu Fly DNA Polymerase 1. mu. L, C2-1 and C2-2 were 5. mu. L, ddH2 and 2O 20. mu.L each; performing PCR, wherein the PCR program comprises: 3min at 95 ℃ for 1 cycle; 10 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; after 1 cycles at 72 ℃ for 10min, the PCR tube was removed and 2. mu.L of Forward Primer (10mM) and 2. mu.L of Reverse Primer (10mM) were added; performing PCR, wherein the PCR program comprises: 3min at 95 ℃ for 1 cycle; 30s at 95 ℃, 30s at 55 ℃ and 1min at 72 ℃ for 20 cycles; 10min at 72 ℃ for 1 cycle. After amplification, the PCR amplification product was detected by 1% agarose gel electrophoresis. The target fragment was recovered using a gel recovery kit to obtain a fragment C2.

1.6 construction of pSK-A plasmid

Subjecting plasmid pBluescript II SK to double digestion with Xho I and EcoR I, performing agarose gel electrophoresis, recovering the desired product, and using

And (3) carrying out homologous recombination on the recovered product and the fragment A by using a HiFi DNA Assembly Master Mix, constructing a reaction system with the total volume of 20 mu L according to the instruction, and reacting for 1h at 50 ℃. Transforming all the ligation products into 100 uL JM110 competent cells, gently mixing uniformly, placing on an ice box for ice bath for 30min, thermally shocking in a 42 ℃ water bath kettle for 1min, immediately placing on the ice box for ice bath for 3min, adding non-resistant LB 500 uL, shaking and culturing at 37 ℃ and 200r/min for 1h, centrifuging at 4000rpm for 1min, discarding about 500 uL of supernatant, blowing, sucking, mixing uniformly, sucking out all the precipitate, uniformly coating on an LB/Amp + solid flat plate by using a coating rod, after completely absorbing the precipitate, culturing the inverted culture dish at 37 ℃ overnightAnd (4) a box. Individual colonies were picked in LB/Amp + liquid medium and identified with M13 primer. Selecting the colony which is positive by the PCR detection result, extracting the plasmid and sequencing. After the successful connection is verified by sequencing, carrying out propagation on the Plasmid, extracting the Plasmid by using a Plasmid miniprep Kit QIAGEN Plasmid Mini Kit, and naming the obtained Plasmid as pSK-A;

1.7 construction of pSK-AB plasmid

And (3) performing single enzyme digestion on the plasmid pSK-A and B fragment products by using Sph I, performing agarose gel electrophoresis, recovering a target fragment, performing single enzyme digestion on the plasmid pSK-A and B fragment products by using Bcl I, performing agarose gel electrophoresis, and recovering a target product. Constructing a 10 mu L T4 DNA Ligase connection system of the target product, connecting at 16 ℃ overnight, transforming all the connection products into 100 mu L HB101 competent cells for culture, picking a single colony in LB/Amp + liquid medium, and identifying by using M13 primer. Selecting colonies which are positive in PCR detection result, extracting plasmids, naming the obtained plasmids as pSK-AB, then transforming the plasmids to JM110, selecting colonies which are positive in PCR detection result, carrying out amplification after successful connection through sequencing verification, and extracting plasmids, naming the plasmids as pSK-AB (JM 110);

1.8 construction of pSK-C plasmid

And (3) performing single enzyme digestion on the plasmid pSK-XBSE and the fragment product of C1 by using Sph I, performing agarose gel electrophoresis, recovering a target fragment, performing single enzyme digestion on the plasmid pSK-XBSE and the fragment product of C1 by using Bcl I, performing agarose gel electrophoresis, and recovering a target product. Constructing a 10 mu L T4 DNA Ligase connection system of the target product, connecting at 16 ℃ overnight, transforming all the connection products into 100 mu LDH5 alpha competent cells for culture, picking a single colony in LB/Amp + liquid culture medium, and identifying by using M13 primer. Selecting a colony which is positive in PCR detection result, extracting a plasmid, and naming the obtained plasmid as pSK-C1; the plasmid pSK-C1 and the fragment product C2 were subjected to double digestion with Sph I and EcoRI, respectively, to carry out agarose gel electrophoresis, and the desired product was recovered. Constructing a 10 mu L T4 DNA Ligase ligation system of the target product, ligating at 16 ℃ overnight, transforming all the ligation products into 100 mu L JM110 competent cells for culture, picking single colonies in LB/Amp + liquid medium, and identifying by using M13 primer. Selecting the colony which is positive by the PCR detection result, extracting the plasmid and sequencing. After the successful connection is verified by sequencing, the plasmid is propagated, extracted and named pSK-C.

1.9 construction of pSK-ABC plasmid

Respectively carrying out single enzyme digestion on the fragment products of the plasmids pSK-AB and pSK-C by using Xho I, carrying out agarose gel electrophoresis, recovering a target fragment, respectively carrying out single enzyme digestion on the fragment products of the plasmids pSK-XBSE and C1 by using Bcl I, carrying out agarose gel electrophoresis, and recovering a target product. Constructing a 10 mu L T4 DNA Ligase connection system of the target product, connecting at 16 ℃ overnight, transforming all the connection products into 100 mu L HB101 competent cells for culture, and selecting a single colony in an LB/Amp + liquid culture medium for identification. Selecting a colony which is positive in PCR detection result, extracting a plasmid, and naming the obtained plasmid as pSK-ABC;

1.10 construction of pSK-E plasmid

The plasmid pBluescript II SK was digested with EcoRI and Not I, subjected to agarose gel electrophoresis, and the desired product was recovered and used

Homologous recombination is carried out on the recovered product and the E fragment by the HiFi DNA Assembly Master Mix, the ligation product is transformed into DH5 alpha competent cells, the cells are treated and cultured, colonies with positive PCR detection results are selected, the colonies are propagated after the successful ligation is verified by sequencing, plasmids are extracted, and the obtained plasmids are named as pSK-E;

1.11 construction of pSK-DE plasmid

Performing double enzyme digestion on plasmid pSK-E and D fragment products by using Sph I and EcoR I respectively, performing agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony with a positive PCR detection result, performing amplification after the successful connection is verified by sequencing, extracting a plasmid, and naming the obtained plasmid as pSK-DE;

1.12 construction of pSK-SD plasmid

Carrying out double enzyme digestion on plasmids pSK-ABC (JM110) and pSK-DE by using EcoR I and Not I respectively, carrying out agarose gel electrophoresis, recovering a target product, connecting by using T4 DNA Ligase, transforming into HB101 competent cells for culture, selecting a colony with a positive PCR detection result, carrying out propagation after the successful connection is verified by sequencing, extracting the plasmid to obtain a plasmid containing complete clone, and naming the obtained plasmid as pSK-SD;

after the construction was complete, the plasmid pSK-SD was identified by PCR using SegA-F/SegA-R, SegB-F/SegB-R, SegC1-F/SegC2-R, SegD-F/SegD-R, SegE-F/SegE-R as primers. The identification result is shown in fig. 4, wherein M is DL5000 Marker; 1 is the A fragment (253bp) amplified from SegA-F/SegA-R; 2 is a B fragment (416bp) amplified from SegB-F/SegB-R; 3 is a C fragment (3900bp) amplified by SegC-F/SegC-R; 4 is a D fragment (407bp) amplified from SegD-F/SegD-R; 5 is the E fragment (237bp) amplified from SegE-F/SegE-R.

The results show that: the recombinant plasmid pSK-SD can amplify correct target fragments.

The plasmid pSK-SD is subjected to double enzyme digestion by using Xho I and Not I, and simultaneously is subjected to single enzyme digestion by using Xho I and Not I respectively, and the result is shown in figure 5, wherein M is DL5000 Marker; 1 is a carrier molecule (2891bp) generated by double enzyme digestion of Xho I and Not I and an NGPV SD genome molecule (5060 bp); 2 is a DNA fragment (7951bp) generated by single enzyme digestion of Xho I; 3 is a DNA fragment (7951bp) generated by Not I single digestion.

The results show that: the bands after enzyme digestion are all correct bands.

Example 2: rescue of novel goose parvovirus SD strain infectious DNA

2.1 transfection

The plasmid and the transfection reagent were mixed at a ratio of 1:2.5(μ g: μ L) using Lipofectamine 2000 as the transfection reagent. Diluting the plasmid (the plasmid amount is 20 mu g) in 1mL of Opti-DMEM medium, and gently mixing; after the Lipofectamine 2000 is shaken up lightly, 50 mu L of Lipofectamine is taken to be mixed with 950 mu L of Opti-DMEM culture medium evenly, and the mixture is incubated for 5 minutes at room temperature; mixing the diluted plasmid with the diluted Lipofectamine 2000, gently mixing uniformly, and standing at room temperature for 20 minutes; the transfection mixing reagent is transfected by the allantoic cavity and the allantoic cavity in an amount of 0.3 mL/piece, and the amount of the inoculated plasmid of each duck embryo is 3.0 mu g. And (3) obtaining allantoic fluid after 120h of inoculation, then inoculating 0.3 mL/duck embryo for passage through the same way, and obtaining the allantoic fluid after 120h to obtain the rescued virus.

2.2 restriction enzyme identification and sequencing verification of rescued Virus genetic marker

Collecting F3-generation virus-rescuing allantoic fluid, extracting DNA, amplifying by using rSD-F, rSD-R primer, carrying out agarose gel electrophoresis, recovering a target product, and carrying out single enzyme digestion identification by using Nco I. Simultaneously, NGPV SD F5 virus allantoic fluid DNA was amplified using rSD-F, rSD-R primer, and single-enzyme digestion was performed using Nco I as a control. After the enzyme digestion, 1% agarose gel electrophoresis was performed, and the result is shown in FIG. 6, wherein M is DL2000 Marker; 1 is a DNA fragment generated by single enzyme digestion of Nco I after an NGPV SD wild strain is amplified by an rSD-F/rSD-R primer; 2 is a DNA fragment generated by single enzyme digestion of Nco I after amplification of virus of duck embryo generation F3 for saving virus by rSD-F/rSD-R primer.

The results show that: the NGPV SD wild strain is amplified by an rSD-F/rSD-R primer and then generates two DNA fragments (822 bp and 690bp respectively) after single enzyme digestion by NcoI, while the rescued virus duck embryo passage F3 virus generates a DNA fragment (1490bp) after single enzyme digestion by the NcoI after being amplified by an rSD-F/rSD-R primer. The genetic marker delta Nco I can still stably exist after the passage of the rescued virus and can be used as a reliable genetic marker for identifying the wild virus and the rescued virus.

Meanwhile, the amplification product of the rSD-F, rSD-R primer is sequenced, and the result is shown in FIG. 7.

The results show that: the rescued virus had a sequence identical to the parental virus except for the unique base mutation introduced in the rescued virus as a genetic marker (C → a). Indicating that the virus is a rescued virus and not a result of contamination.

2.3 Western Blot identification of rescued virus

Collecting F3-generation virus-rescuing allantoic fluid, concentrating the allantoic fluid by 2 times, adding SDS sample buffer, boiling for 10min, performing SDS-PAGE electrophoresis, recovering albumin glue containing target fragments, and performing membrane transfer for 2h by using a wet electroblotting membrane transfer method; sealing the transfer membrane with 5% skimmed milk powder overnight, diluting the primary antibody (murine anti-VP 3) with 5% skimmed milk powder by 100 times, incubating at room temperature for 1.5h, diluting the secondary antibody (goat anti-mouse IgG-HRP) with 5% skimmed milk powder by 200 times, incubating at room temperature for 1.5h, and performing light-shielding color development by using a DAB method color development kit, wherein the result is shown in FIG. 8, wherein M is 180kDa Marker; western Blot for saving virus F3 generation allantoic fluid and identifying 1.

The results show that: the primary antibody is murine, anti-VP 3 protein, about 60 kDa.

2.4 Indirect immunofluorescence assay for rescuing viruses

Inoculating F3 generation rescued virus allantoic fluid and parent virus allantoic fluid to Duck Embryo Fibroblast (DEF), and performing indirect immunofluorescence identification 36h after infection by using anti-NGPV rabbit-derived polyclonal antibody as a primary antibody and goat anti-rabbit IgG-FITC as a secondary antibody, wherein the identification result is shown in FIG. 9, and A is the identification result of rescued virus; b is a parental virus identification result; and C is a negative control.

The results show that: the rescued virus and the parental virus can detect green fluorescent signals, which indicates that the virus is rescued successfully.

According to the embodiment, the complete genome of the novel goose parvovirus SD strain is cloned to the plasmid pBluescript II SK by a segmented amplification method to obtain a recombinant plasmid; simultaneously, the NcoI mutation in the genome is used as a genetic marker for identifying wild viruses and saving viruses by utilizing overlapping PCR; and then mixing the recombinant plasmid with a transfection reagent, and inoculating the SPF duck embryo through a yolk sac and allantoic cavity way to obtain the rescued virus. The novel goose parvovirus reverse genetic operation system established by the invention can be used for the research of virulence analysis, cross-species propagation mechanism and the like of the novel goose parvovirus, and simultaneously lays a foundation for the research of novel vaccines of duck short beak and dwarfism syndrome.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> university of agriculture in Hebei

<120> construction method and application of novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome

<130> 2022.01.21

<141> 2022-02-15

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213> Artificial Synthesis ()

<400> 1

gtaccgggcc ccccctcgag cttattggag ggttcgttcg 40

<210> 2

<211> 59

<212> DNA

<213> Artificial Synthesis ()

<400> 2

ccgggctgca ggaattccgg ttccaatgat cattggacat gcatgcgcgt ggtcaacct 59

<210> 3

<211> 24

<212> DNA

<213> Artificial Synthesis ()

<400> 3

acatgcatgc cgcgcggtca gccc 24

<210> 4

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<213> Artificial Synthesis ()

<400> 4

ttccaatgat caatatccga tggtaa 26

<210> 5

<211> 26

<212> DNA

<213> Artificial Synthesis ()

<400> 5

gtccaatgat caagatgtcc ccggtc 26

<210> 6

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<212> DNA

<213> Artificial Synthesis ()

<400> 6

ttttcagcat catcaag 17

<210> 7

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<400> 7

ttgttctcat cagttgctcc a 21

<210> 8

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<212> DNA

<213> Artificial Synthesis ()

<400> 8

tcccggcagc caattgcggc ccattgtgcc 30

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<400> 9

ctgtgaaagg ggcttatggc acaatgggcc 30

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<213> Artificial Synthesis ()

<400> 10

ccggaattct cttttctcag ctc 23

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<212> DNA

<213> Artificial Synthesis ()

<400> 11

ccggaattca aagagatgga acc 23

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<400> 12

acatgcatgc gcgctgtcag ccca 24

<210> 13

<211> 47

<212> DNA

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ataagcttga tatcgaattc cggacatgca tgcgcgtggt caaccta 47

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<211> 40

<212> DNA

<213> Artificial Synthesis ()

<400> 14

tccaccgcgg tggcggccgc cttattggag ggttcgttcg 40

<210> 15

<211> 18

<212> DNA

<213> Artificial Synthesis ()

<400> 15

catcacaaag accaccag 18

<210> 16

<211> 25

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<213> Artificial Synthesis ()

<400> 16

gattttgagt tagatatctg gttcc 25

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<213> Artificial Synthesis ()

<400> 17

ccgctcgagc gggtccaatg atcattggaa acatgcatgc atgtccggaa ttccgg 56

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<211> 5053

<212> DNA

<213> Artificial Synthesis ()

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cttattggag ggttcgttcg ttcgagccag ccaatcaggg gagggggaag tgacgcaagt 60

cccggtcacg tgcttccggt gacgcacatc cggtgacgtg cttcctgtca cgtgacttcc 120

ggtcatgtga cttccggtca tgtgacttcc ggtgacgtgt ttccggctgt taggttgacc 180

acgcgcatgc cgcgcggtca gcccaatagt taagccggaa acacgtcacc ggaagtcaca 240

tgaccggaag tcacatgacc ggaagtcacg tgacaggaag cacgtcaccg gatgtgcgtc 300

accggaagca cgtgaccggg acttgcgtca cttccccctc ccctgattgg ctggctcgaa 360

cgaacgaacc ctccaataag actcaaggac aagaaaatat tttgcgcgcc aggaagtgac 420

gtgcaatgct accctatata agccaggaag cttccggttt agttcattcg ttactctgct 480

ctcagagaga acggacctca ggtcggagag atggcacttt ctaggcctct tcagatttct 540

tccgataaat tctatgaagt tattattaga ttaccatcgg atattgatca agatgtcccc 600

ggtctgtctc ttaattttgt agaatggctt tctaccggag tttgggagcc cacgggcacc 660

tggaacatgg agcatgtgaa tttaccgatg gtgaccttgg cagagaagat caaaaacatt 720

ttcatacaaa gatggaatca gttcaaccag gacgaaacgg acttcttctt tcaactggaa 780

gaaggcagtg agtacattca tcttcattgc tgtattgccc agggcaatgt acggtctttt 840

gttctcggga gatatatgtc tcagataaaa gactctatca taagagatgt atatgaaggg 900

agacaaatca agatccccga ttggttttcc attactaaaa ccaagagggg aggacaaaat 960

aagaccgtga ctgcagcata catactgcat tacctgattc ctaaaaagca acctgaactg 1020

cagtgggcct ttaccaatat gcctttattt actgctgctg ctctctgtct gcaaaagcgg 1080

caagaattgc tggatgcatt tcaagagagt gatttggctg cccctttacc cgatcctcaa 1140

gcatcaactg tggcaccgct tatttccaac agagcggcaa agaactatag caaccttgtt 1200

gattggctca ttgaaatggg cataacatct gagaagcaat ggctcactga gaaccgagac 1260

agctacagaa gctttcaagc aacttcttca aataatagac aagtgaaagc tgcactggag 1320

aatgcccgtg ctgaaatgct attaacaaag actgcaactg attacctgat aggaaaagac 1380

cctgtcctgg atataactaa gaacagggtc tatcaaattc tgaaaatgaa taactacaac 1440

cctcaataca taggaagtat cctgtgcggc tgggtgaaga gagagttcaa caaaagaaac 1500

gccatatggc tctacggacc tgccaccacc gggaagacca acattgcaga ggctattgtc 1560

catgctgtac ccttctatgg ctgcgttaac tggactaatg agaactttcc ttttaatgat 1620

tgtgttgata agatgctgat ttggtgggag gagggaaaaa tgactaacaa ggttgttgaa 1680

tctgctaaag caattttggg agggtctgct gttcgggtag accagaaatg taaaggatct 1740

gtttgtattg aacctactcc tgtaattatt accagtaata ctgatatgtg tatgattgtt 1800

gatggcaact ctactacaat ggaacacaga ataccattag aggagcgcat gtttcaaatt 1860

gtcctatcac ataaattgga gccttctttt ggaaaaatat ctaaaaagga aatcagagaa 1920

tttttcaaat gggccaacga caatttagtt cctgttgtgt ctgagttcaa agtccgaacg 1980

aatgaacaaa ccaacttacc aaggcccgtt cctgaacgag cgaacgagcc agaggagcct 2040

cctaaaatct gggctcctcc tactagggag gagttagaag agcttttaag agccagccca 2100

gaattgttct catcagttgc tccaattcct gtgactcctc agaactcccc tgagcctaag 2160

agaagcaaga acacttacca ggtacggtgt gctttgcata cttatgacaa ttctatggat 2220

gtgtttcaat gtatggaatg tgagaaggca aattttcctg aatttcaacc tctgggagaa 2280

aattactgtt atgaacatgg gtggtatgat tgtgctatat gtacagaatt gaaagatgaa 2340

cttgcagaaa ttgagcatgc gtttgagctt gatgatgctg aaaatgaaca ataaagatga 2400

ttcaaaacag atatgtctac ttttttagat tcttttgaag agtggtatga gactgcagcc 2460

gcctcgtggc ggaatctgaa agctggagcc cctcagccaa aaccaaacca gcagtctcag 2520

tctgtgtctc cagacagaga acccgaacga agagataata atcggggctt tgtacttcct 2580

ggctataagt atcttgggcc tggtaacggc cttgataaag gcccacctgt caacaaggcg 2640

gacagcgtcg cgcttgaaca cgacaaggcc tacgacctac agcttaaagc gggagacaat 2700

ccatatataa aattcaatca cgctgaccag gactttatag atagcctcca acacgaccac 2760

tcatttggag gtaatcttgg aaaggctgta ttccaggcca aaaaacgtat cttagagcca 2820

ttcggcctag tagaagagcc tatcaacacg gctcctgcaa aaaaaaatac agggaagctt 2880

actgaccatt acccagtagt taagaagcct aaactcaccg aggaagtcag tgcgggaggt 2940

ggtagcagtg tcgtacaaga cggaggagcc accgcggagg gcaccgaacc tgtggcagca 3000

tctgaaatgg cagagggagg aggcggagct atgggcgact cttcaggggg tgccgatgga 3060

gtgggtaatg cctcgggaaa ttggcattgc gattcccaat ggatgggaaa cacagtcatc 3120

acaaagacca ccagaacctg ggtcctgcca agctacaaca atcacatcta caaagcaatt 3180

accagtggaa cctctcaaga tgcaaatgtc cagtatgctg gatacagtac cccctggggg 3240

tactttgatt tcaatcgctt ccactgccac ttctccccta gagactggca gagacttatc 3300

aacaaccact ggggaatcag gcccaagtct cttaaattca agatcttcaa tgttcaagtc 3360

aaggaagtca caacgcagga tcagacaaag accattgcaa acaatctcac ctcaacaatc 3420

caagttttta cggatgatga gcaccaactc ccgtatgtcc tgggctcggc tacggaaggg 3480

accatgccgc cgttcccgtc ggatgtatat gccctgccgc agtacgggta ctgcacaatg 3540

cacaccaacc agaatggagc acggttcaat gaccgtagcg cattctactg cttagagtac 3600

ttccctagtc agatgctgag aacaggtaac aactttgagt tcacatttga ctttgaagaa 3660

gttcctttcc acagcatgtt cgctcattca caggacttag acaggcttat gaacccccta 3720

gtggatcaat acctctggaa tttcaatgag gtagacagca acagaaatgc tcaatttaaa 3780

aaagctgtga aaggggctta tggcaccatg ggccgcaatt ggctgccggg acctaaattc 3840

ctggatcaga gagttagggc ctacacagga ggaacagaca attatgcaaa ctggaacatc 3900

tggaataatg ggaacaaggt gaatttaaag gacaggcagt atctcctaca acccggacct 3960

gtgtcagcta ctcacacaga aggggaggct tccagcatcc cagctcagaa tattttaggg 4020

atagctaaag atccatacag atctggcagc actacagcag gaataagtga tattatggtc 4080

acggacgagc aggaagtagc acccacgaat ggagtagggt ggaaaccata tggtaggact 4140

gtaacgaatg aacaaaacac tactacagct cctacaagtt cagatctgga tgttcttgga 4200

gctttaccag gaatggtgtg gcagaacaga gatatatatc tgcagggacc tatttgggca 4260

aaaataccga agactgatgg caaattccat ccttctccaa atctcggagg atttggcctg 4320

cacaatccac caccacaggt cttcatcaag aatacaccag tacctgcaga ccctccagta 4380

gaatatgtga accagaagtg gaactcctac ataactcaat actctacagg ccagtgtaca 4440

gtagaaatgg tgtgggagct gagaaaagag aattcaaaga gatggaaccc agaaatccag 4500

ttcaccagca atttcagtaa cagaactagt ataatgtttg cacctaatga aactggtgga 4560

tatgtagaag atagattgat tggaaccaga tatctaactc aaaatctgta aaatctgtgt 4620

aaaaattcaa ataaagcact tcctggcgcg caaaatattt tcttgtcctt gagtcttatt 4680

ggagggttcg ttcgttcgag ccagccaatc aggggagggg gaagtgacgc aagtcccggt 4740

cacgtgcttc cggtgacgca catccggtga cgtgcttcct gtcacgtgac ttccggtcat 4800

gtgacttccg gtcatgtgac ttccggtgac gtgtttccgg cttaactatt gggctgacag 4860

cgcgcatgcg cgtggtcaac ctaacagccg gaaacacgtc accggaagtc acatgaccgg 4920

aagtcacatg accggaagtc acgtgacagg aagcacgtca ccggatgtgc gtcaccggaa 4980

gcacgtgacc gggacttgcg tcacttcccc ctcccctgat tggctggctc gaacgaacga 5040

accctccaat aag 5053

Claims

1. A primer for preparing a novel goose parvovirus SD strain full-length infectious clone causing duck short beak and dwarfism syndrome is characterized in that a specific sequence is shown as SEQ ID No. 1-14.

2. A molecular marker primer for carrying out full-length infectious cloning on a novel goose parvovirus SD strain causing duck short beak and dwarfism syndrome is characterized in that a specific sequence is shown as SEQ ID No. 15-16.

3. A kit containing a primer combination with a gene sequence shown as SEQ ID No. 1-16.

4. A rescue system of a novel goose parvovirus SD strain aiming at duckling brachiocephalic and dwarfism syndrome is characterized by comprising amplification products of seven fragments of A, B, C1, C2-1, C2-2 and D, E and two plasmids.

5. The rescue system of claim 4, wherein the plasmids are pSK-XBSE and pBluescript II SK.

6. A construction method of a novel goose parvovirus SD strain full-length infectious clone causing duck brachiocephalic and dwarfism syndrome is characterized by comprising the following steps:

(2) extracting total DNA of NGPV SD F5 virus genome;

(3) designing and synthesizing primers covering the whole genome: designing corresponding primers according to the selected enzyme cutting sites according to the NGPV SD whole gene sequence, wherein the primers comprise six pairs of amplification primers SegA-F, SegA-R, SegB-F, SegB-R, SegC1-F, SegC1-R, SegC2-F, 1-R, 2-F, SegC2-R, SegD-F, SegD-R, SegE-F, SegE-R, a pair of mutation primers 1-R and 2-F and a molecular marker primer rSD-F, rSD-R for synthesizing the Nco I mutation sites; the amplification primer sequence is shown as SEQ ID No. 1-12, the mutation primer sequence is shown as SEQ ID No. 13-14, and the molecular marker primer sequence is shown as SEQ ID No. 15-16;

(4) PCR amplification of each gene fragment:

firstly, taking the NGPV SD whole gene as a template, performing PCR amplification by using primers SegA-F/SegA-R, SegB-F/SegB-R, SegC1-F/SegC1-R, SegC2-F/1-R, 2-F/SegC2-R, SegD-F/SegD-R, SegE-F/SegE-R, and detecting, purifying and recycling PCR amplification products to obtain seven fragments of A, B, C1, C2-1, C2-2 and D, E;

then, the recovered products of C2-1 and C2-2 are used as templates, SegC2-F/SegC2-R are used as primers to carry out overlap PCR amplification, and the target product is recovered to obtain a C2 fragment.

(5) The recombinant plasmid pSK-SD is constructed in stages:

construction of pSK-A plasmid: carrying out double enzyme digestion on the plasmid pBluescript II SK by using Xho I and EcoRI to obtain a target product, then carrying out homologous recombination on the target product and the fragment A, transforming the target product into JM110 competent cells, processing and culturing the competent cells to obtain a positive colony, carrying out propagation on the competent cells after the successful connection is verified by sequencing, and extracting a plasmid which is named as pSK-A;

(ii) construction of pSK-E plasmid: carrying out double enzyme digestion on the plasmid pBluescript II SK by using EcoRI and Not I to obtain a target product, then carrying out homologous recombination on the target product and the E fragment, transforming the target product into DH5 alpha competent cells, processing and culturing the cells to obtain a positive colony, carrying out amplification after the successful connection is verified by sequencing, and extracting a plasmid which is named as pSK-E;

construction of pSK-AB plasmid: performing double enzyme digestion on the plasmid pSK-A and the fragment product B by using Sph I and Bcl I respectively to obtain a target product, transforming the target product into HB101 competent cells for culture, selecting a positive colony, performing amplification after the successful connection is verified by sequencing, extracting a plasmid, and naming the plasmid as pSK-AB;

construction of plasmid pSK-AB (JM 110): transforming the pSK-AB plasmid into JM110 for culture, selecting positive colonies, carrying out sequencing to verify that the connection is successful, carrying out propagation on the positive colonies, extracting the plasmid and naming the plasmid as pSK-AB (JM 110);

fifthly, constructing the pSK-DE plasmid: performing double enzyme digestion on the plasmid pSK-E and the fragment product D by using Sph I and EcoRI respectively to obtain a target product, transforming the target product into HB101 competent cells for culture, selecting a positive colony, performing amplification after the successful connection is verified by sequencing, extracting a plasmid, and naming the plasmid as pSK-DE;

sixthly, constructing the pSK-C1 plasmid: carrying out double enzyme digestion on the plasmid pSK-XBSE and the fragment product of C1 by using Bcl I and Sph I respectively to obtain a target product, transforming the target product into DH5 alpha competent cells for culture, selecting a positive colony, carrying out amplification after the successful connection is verified through sequencing, extracting a plasmid, and naming the plasmid as pSK-C1;

and (C) constructing a pSK-C plasmid: carrying out double enzyme digestion on the plasmid pSK-C1 and the fragment product of C2 by using SphI and EcoRI respectively to obtain a target product, transforming the target product into JM110 competent cells for culture, selecting a positive colony, carrying out propagation on the positive colony after the successful connection is verified through sequencing, extracting a plasmid, and naming the plasmid as pSK-C;

construction of plasmid pSK-ABC: xho I and Bcl I are used for respectively carrying out double enzyme digestion on plasmids pSK-AB and pSK-C to obtain target products, the target products are transformed into HB101 competent cells for culture, positive colonies are selected, after the connection is verified by sequencing, the positive colonies are propagated, and plasmids are extracted and named as pSK-ABC;

ninthly, constructing the pSK-SD plasmid: and carrying out double enzyme digestion on plasmids pSK-ABC and pSK-DE by using EcoRI and Not I respectively to obtain a target product, transforming the target product into HB101 competent cells for culture, selecting positive colonies, carrying out propagation on the positive colonies after the successful connection is verified by sequencing, and extracting plasmids to obtain the plasmids containing complete clones, wherein the plasmids are named as pSK-SD.

7. A novel goose parvovirus SD strain full-length infectious clone causing duck brachiocephalic and dwarfism syndrome, prepared by the construction method of claim 6, wherein the gene sequence of the clone is shown as SEQ ID No. 18.

8. A method for rescuing novel goose parvovirus SD strain causing duck short beak and dwarfism syndrome by using the construction method of claim 6 is characterized in that firstly, recombinant plasmid pSK-SD is mixed with a transfection reagent, then the transfection reagent is transfected by a chorioallantoic cavity and an allantoic cavity in an amount of 0.3 mL/piece, the inoculation plasmid amount of each duck embryo is 3.0 mu g, then allantoic fluid is harvested after inoculation for 120h, then the allantoic fluid is seeded by 0.3 mL/piece of duck embryo for passage through the same way, and the allantoic fluid is harvested after 120h, so that the rescued virus is obtained.

9. A method for identifying a novel goose parvovirus SD strain rescued virus causing duck short beak and dwarfism syndrome is characterized in that a genetic marker delta Nco I primer is designed, and the specific sequence is shown as SEQ ID NO. 15-16; then extracting DNA for rescuing virus; carrying out PCR amplification on A, B, C, D, E five fragments by adopting a genetic marker delta Nco I primer, detecting, purifying and recovering a PCR amplification product, and carrying out enzyme digestion reaction by using Nco I restriction endonuclease; to rescue the virus, Nco I could not be cleaved to recover the product.

10. Use of the primer according to any one of claims 1 to 2, or the kit according to claim 3, or the rescue system according to any one of claims 4 to 5, or the full-length infectious clone of the novel goose parvovirus SD strain according to claim 7, in the preparation of a diagnostic reagent and/or vaccine for the novel goose parvovirus SD strain causing duck brachiocephalic and dwarfism syndrome.