CN114395568A

CN114395568A - Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

Info

Publication number: CN114395568A
Application number: CN202111274202.1A
Authority: CN
Inventors: 李燕华; 周弇扬; 李晨曦; 任次成; 胡晶博
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-04-26

Abstract

The invention provides a construction method and application of porcine epidemic diarrhea virus infectious cDNA clone, belonging to the field of biotechnology. The construction method comprises the following steps: amplifying F1, F2, F3, F4, F5, F6 and F7 fragments by taking a reverse transcription product of the genome RNA of the porcine epidemic diarrhea virus as a template; based on an in-yeast homologous recombination mechanism, mixing a linearized pYES1L vector and F1-F7 fragments, and transforming the mixture into yeast competent cells, thereby realizing one-step seamless connection of a virus cDNA fragment and an original vector to obtain a porcine epidemic diarrhea virus infectious cDNA clone plasmid; based on CRISPR/Cas9 gene editing technology, the coding region of virus ORF3 is replaced by green fluorescent protein gene to construct recombinant porcine epidemic diarrhea virus expressing green fluorescent protein. The method is rapid, simple and convenient, has high success rate, can be used for saving the porcine epidemic diarrhea virus and other coronavirus, and provides a reliable technical platform for researching the replication mechanism and the pathogenic mechanism of the virus and developing a novel vaccine.

Description

Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of porcine epidemic diarrhea virus infectious cDNA clone.

Background

Porcine Epidemic Diarrheic (PED) is an acute and highly contagious Porcine intestinal disease caused by Porcine Epidemic Diarrheic Virus (PEDV), with clinical symptoms similar to Porcine Transmissible Gastroenteritis Virus (TGEV), characterized by vomiting, Diarrhea and anorexia, and finally death due to wasting and severe dehydration. All ages can be infected, wherein the clinical symptoms of infected piglets are particularly serious, and the death rate is close to 100 percent. Besides infecting suckling piglets, PEDV can also reduce the growth performance of fattening pigs. In recent years, the emergence and widespread dissemination of highly pathogenic variant strains of PEDV has caused enormous economic losses to the swine industry. In addition to biosafety, vaccination is the most effective method for the control of porcine epidemic diarrhea. However, currently commercial vaccines offer limited protection and the development of a new generation of vaccines is still imminent. PEDV belongs to the order of the nested viruses, the subfamily of coronaviruses, the genus A coronavirus. It is a membrane-enveloped single-stranded positive-strand RNA virus with a full-length genome of about 28kb, comprising a 5 'end cap structure and a non-coding region, a 3' end non-coding region and a poly (A) tail, and 7 Open Reading Frames (ORFs) including the genes ORF1a, ORF1b, S, ORF3, E, M and N. Two polyproteins (pp1a and pp1ab) encoded by ORF1a and ORF1b at the 5 'end, and 5 ORFs at the 3' end sequentially encode spike protein (S), ORF3 protein, small envelope glycoprotein (E), membrane glycoprotein (M), and nucleocapsid protein (N). It has been shown that ORF3 is a non-essential element for PEDV replication and can be substituted for the expression of foreign genes.

The reverse genetic operation system is an important genetic engineering technology for directionally transforming and modifying virus genomes, and the technology becomes an essential technical platform for virology basic research and vaccine development. Currently, the PEDV reverse genetic manipulation system mainly includes the following three types: techniques based on targeted RNA recombination, infectious cDNA clones based on bacterial artificial chromosomes, infectious cDNA clones based on in vitro ligation, etc. The PEDV reverse genetic operation system can be divided into an RNA transfection system and a DNA transfection system according to different virus rescue modes. In order to simplify the construction process of PEDV infectious cDNA clone, the invention uses a PEDV HM strain of a gene II type as a parent strain, constructs a reverse genetic operation platform of the CMV-initiated porcine epidemic diarrhea virus based on a yeast Transformation Associated Recombination (TAR) technology, and constructs the PEDV marker virus expressing green fluorescent protein by virtue of a CRISPR/Cas9 gene editing technology. The invention lays a foundation for the research of PEDV pathogenic mechanism and genetic engineering vaccine thereof.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a construction method of infectious cDNA clone of porcine epidemic diarrhea virus, the construction method is simple and convenient, good in stability and high in efficiency, after the virus rPEDV and rPEDV-EGFP rescued by the infectious clone infects Vero CCL-81 cells, cytopathic effect can be caused, the growth characteristic is basically consistent with that of parent virus, and the genetic stability is kept.

The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:

a method for constructing porcine epidemic diarrhea virus infectious cDNA clone comprises the following steps:

(1) using cDNA of porcine epidemic diarrhea virus as a template and SEQ ID No.: 1-14 is used as a primer, and the porcine epidemic diarrhea virus biological genome is divided into 7 segments to be amplified;

(2) pYES1L-vector as template, SEQ ID No.: 15-16 is used as a primer, and a linearized vector pYES1L is obtained by amplification, wherein the nucleotide sequence of pYES1L-vector is shown in SEQ ID No.: 18 is shown in the figure;

(3) mixing a linearization vector pYES1L with the 7 genome fragments obtained in the step (1), adding the mixture into MaV203 yeast competence, transforming the mixture into yeast cells by using a lithium acetate transformation method, inoculating the yeast cells on a tryptophan (Trp) defect type plate, screening positive clones by using a colony PCR method, transforming the yeast lysate of the positive clones into DH10B electric shock competence, and obtaining recombinant plasmids, namely the porcine epidemic diarrhea virus infectious cDNA clone.

Preferably, the porcine epidemic diarrhea virus has a genomic sequence as set forth in SEQ ID No.: shown at 17.

The porcine epidemic diarrhea virus infectious cDNA clone is constructed by the construction method of the porcine epidemic diarrhea virus infectious cDNA clone.

The method for constructing the porcine epidemic diarrhea virus expressing green fluorescent protein based on CRISPR/Cas9 gene editing technology comprises the following steps:

(4) EGFP gene as template, SEQ ID No.: performing PCR amplification by taking the sequence shown by 19-20 as a primer to obtain an EGFP gene segment;

(5) cleaving the porcine epidemic diarrhea virus infectious cDNA clone obtained in claim 1 with Cas9 Nuclear and two guide RNAs, the sequence of which is as set forth in SEQ ID No.: 21-22, obtaining a porcine epidemic diarrhea virus infectious cDNA cloning framework lacking ORF 3;

(6) EGFP and the porcine epidemic diarrhea virus infectious cDNA cloning framework segment lacking ORF3 are mixed, in vitro homologous recombination is carried out, and the mixture is transformed into DH10B electric shock competence to obtain recombinant plasmid, namely the porcine epidemic diarrhea virus expressing green fluorescent protein.

The porcine epidemic diarrhea virus expressing the green fluorescent protein, which is constructed by the construction method of the porcine epidemic diarrhea virus expressing the green fluorescent protein based on the CRISPR/Cas9 gene editing technology, is within the protection scope of the invention.

The porcine epidemic diarrhea virus infectious cDNA clone or the porcine epidemic diarrhea virus expressing green fluorescent protein is applied to the virus rescue.

Further, a transfection reagent Lipofectamine was used^TM3000 VeroCCL-81 cells were co-transfected with the porcine epidemic diarrhea virus infectious cDNA clone of claim 3 and helper plasmid, and the viral supernatant was harvested when the typical cytopathic effect occurred to obtain rescued rPEDV.

Preferably, the helper plasmid is constructed as follows: carrying out PCR amplification by taking porcine epidemic diarrhea virus infectious cDNA clone as a template and SEQ ID NO. 17-18 as a primer to obtain an N gene fragment, and inserting the N gene fragment between SacI and XhoI sites of a pCAGGS vector to obtain an auxiliary plasmid.

The method of the present invention will be further described below by taking the HM strain of porcine epidemic diarrhea virus as an example.

The construction method of the infectious cDNA clone of the pig epidemic diarrhea virus HM strain comprises the following steps:

(1) carrying out PCR amplification by using a pYES1L vector as a template and primers pYES1L-F and pYES1L-R to obtain a linearized pYES1L vector;

(2) using a reverse transcription product of virus RNA of a porcine epidemic diarrhea virus HM strain as a template, amplifying an F1 fragment by using primers PEDV-F1 and PEDV-R1, amplifying an F2 fragment by using primers PEDV-F2 and PEDV-R2, amplifying an F3 fragment by using primers PEDV-F3 and R3, amplifying an F4 fragment by using primers PEDV-F4 and PEDV-R4, amplifying an F5 fragment by using primers V-F5 and PEDV-R5, amplifying an F6 fragment by using primers PEDV-F6 and PEDV-R6, and amplifying an F7 fragment by using primers PEDV-F7 and PEDV-R7;

(3) the linearized vector pYES1L and F1-F7 fragments are mixed and introduced into yeast for homologous recombination to obtain porcine epidemic diarrhea virus infectious cDNA clone plasmid pYES 1L-PEDV.

(7) using pEGFP-N1 as a template, and carrying out PCR amplification by using primers PEDV-EGFP-F and PEDV-EGFP-R to obtain an EGFP gene fragment;

(8) cutting pYES1L-PEDV plasmid by using Cas9 Nuclear and two guide RNAs (PEDV-sgRNA 1 and PEDV-sgRNA 2) to obtain a porcine epidemic diarrhea virus infectious cDNA cloning framework with deletion of ORF 3;

(9) EGFP and porcine epidemic diarrhea virus infectious cDNA cloning framework fragments lacking ORF3 are mixed, in vitro homologous recombination is carried out, and the mixture is transformed into DH10B shock competence to obtain recombinant plasmid pYES 1L-PEDV-EGFP.

In the invention, the application comprises the following steps: construction of expression Swine epidemicsHelper plasmid of diarrhea virus HM strain N protein, using transfection reagent Lipofectamine^TM3000 an infectious cDNA clone plasmid (pYES1L-PEDV or pYES1L-PEDV-EGFP) and an auxiliary plasmid are co-transfected into Vero CCL-81 cells, and cell cultures are harvested to obtain rescued infectious porcine epidemic diarrhea virus rPEDV or rPEDV-EGFP.

In the invention, the helper plasmid is a pCAGGS vector obtained by cloning the encoding gene of the porcine epidemic diarrhea virus N protein to obtain the pCAGGS-PEDV-N helper plasmid.

Has the advantages that:

compared with the prior art, the invention provides a rapid, efficient and stable construction method of porcine epidemic diarrhea virus infectious cDNA clone, which has the following advantages:

(1) the infectious cDNA cloning construction method provided by the invention has the characteristics of high efficiency, rapidness, stability and the like. The genome of the PEDV HM strain is divided into 7 segments, and cloning construction is completed by a one-step method based on yeast homologous recombination, so that the problem of low efficiency of traditional enzyme digestion connection is solved, and the efficiency is greatly improved.

(2) The infectious cDNA clone provided by the invention has good stability. The pYES1L vector contains yeast artificial chromosome and bacterial artificial chromosome elements, has good stability when being replicated in yeast and bacteria, and solves the problem that partial replication enzyme genes of coronavirus can not be stably stored in escherichia coli; meanwhile, the upstream of the vector is provided with a Cytomegalovirus (CMV) eukaryotic promoter sequence, and the downstream is provided with a hepatitis delta virus Ribozyme (HDV Ribozyme) sequence and a bovine growth hormone polyadenylation signal (BGH) transcription termination sequence, so that the virus rescue efficiency is greatly improved.

(3) The infectious cDNA clone provided by the invention can directly utilize CRISPR/Cas9 gene editing to modify the porcine epidemic diarrhea virus genome at the gene level, so that the efficiency of constructing recombinant viruses is improved, and a rapid and effective technical platform is established for the porcine epidemic diarrhea virus genome.

(4) The infectious cDNA clone provided by the invention directly transfects mammalian cells to rescue recombinant viruses. The CMV promoter contained in the infectious cDNA clone can start the synthesis of virus genome RNA in cells transfected by DNA, thereby avoiding the in vitro RNA transcription synthesis process, simplifying the virus rescue operation process and solving the problem of transcript heterogeneity caused by in vitro transcription.

Drawings

FIG. 1 is a schematic diagram of the construction of a porcine epidemic diarrhea virus infectious cDNA clone.

FIG. 2 is a gel electrophoresis diagram of the RT-PCR amplification of the genome fragment of HM strain of porcine epidemic diarrhea virus.

FIG. 3 is a restriction enzyme map of pYES1L-PEDV plasmid, a porcine epidemic diarrhea virus infectious cDNA clone.

FIG. 4 is a schematic diagram of the construction of pYES1L-PEDV-EGFP plasmid, a porcine epidemic diarrhea virus infectious cDNA clone expressing green fluorescent protein.

FIG. 5 is an indirect immunofluorescence assay of recombinant viruses.

FIG. 6 is a multi-step growth curve of virus rescued by infectious cDNA clones versus parental virus.

FIG. 7 is a plaque assay of virus rescued by infectious cDNA clones versus parental virus.

Detailed Description

The present invention is further described below in conjunction with specific examples, which are to be understood as being illustrative only and in no way limiting of the scope of the invention.

Example 1 construction of infectious cDNA cloning plasmid of HM Strain of porcine epidemic diarrhea Virus

The construction strategy is shown in figure 1. Seven fragments of F1, F2, F3, F4, F5, F6 and F7 were RT-PCR amplified by using Snapgene software to design 7 pairs of specific primers (see Table 1) based on the full-length genome sequence of HM strain of porcine epidemic diarrhea virus gene type II (Genbank accession number: MZ 342899).

Viral genomic RNA of PEDV HM strain was extracted according to the instructions of a viral genomic DNA/RNA extraction kit (Tiangen Biochemical technology Co., Ltd.). Using viral genomic RNA as a template according to

IV First-Strand cDNA Synthesis Reaction (Thermo Fisher Scientific) described the preparation of cDNA for HM strain of epidemic diarrhea virus. The reaction system is as follows: mu.L of 2. mu.M gene-specific reverse primer (PEDV-R7, PEDV-R4 or PEDV-R2), 1. mu.L of 10. mu.M dNTP Mix, and 11. mu.L of template RNA. The reaction procedure is as follows: 5min at 65 ℃ and 1min on ice. The reaction system is as follows: 5 XSSIV Buffer 4. mu.L, 100mM DTT 1. mu.L, recombination RNase Inhibitor 1. mu.L, Reverse Transcriptase (200U/. mu.L) 1. mu.L. The reaction procedure is as follows: cooling to 50 deg.C for 10min and 80 deg.C for 10min, and cooling to room temperature. mu.L of RNase H was added thereto and RNA was removed at 37 ℃ for 20 min.

When F1 and F2 fragments are amplified, cDNA (PEDV-R2) is used as a template, and two pairs of primers, namely PEDV-F1/PEDV-R1 and PEDV-F2/PEDV-R2, are used for amplification; when F3 and F4 fragments are amplified, cDNA (PEDV-R4) is used as a template, and two pairs of primers, namely PEDV-F3/PEDV-R3 and PEDV-F4/PEDV-R4, are used for amplification; when F3 and F4 fragments are amplified, cDNA (PEDV-R4) is used as a template, and two pairs of primers, namely PEDV-F3/PEDV-R3 and PEDV-F4/PEDV-R4, are used for amplification; when F5, F6 and F7 fragments are amplified, three pairs of primers PEDV-F5/PEDV-R5, PEDV-F6/PEDV-R6 and PEDV-F7/PEDV-R7 are used for amplifying cDNA (PEDV-R7) as a template; the reaction system is as follows: mu.L of cDNA, 0.625. mu.L each of the forward (40. mu.M) and reverse (40. mu.M) primers, 1. mu.L of 100mM dNTP Mix, 10. mu.L of 5 XQ 5 Reaction Buffer, 0.5. mu.L of Q5 High Fidelity DNA Polymerase, and 50. mu.L of ddH 2O. The amplification procedure was: 30s at 98 ℃; circulating for 30 times at 98 ℃ for 10s, 58-63 ℃ for 20s and 72 ℃ for 5 min; 72 ℃ for 2 min. The sizes of the target bands were 4050bp, 4122bp, 4010bp, 4803bp, 3417bp, 4305bp and 3612bp, respectively, and the results are shown in FIG. 2.

Using pYES1L-Vector (which was stored in this laboratory) as a template, PCR amplification was performed using primers pYES1L-F and pYES1L-R to obtain a linearized pYES1L Vector. The reaction system is as follows: pYES1L-vector 10ng, forward primer (40. mu.M) and reverse primer (40. mu.M) each 0.625. mu.L, 100mM dNTP Mix 1. mu.L, 5 XQ 5 recovery Buffer 10. mu.L, Q5 High Fidelity DNA Polymerase 0.5. mu.L, ddH₂And O is supplemented to 50 mu L. The amplification procedure was: 30s at 98 ℃; 1 at 98 DEG CCirculating for 30 times at 0s, 63 deg.C for 20s, and 72 deg.C for 5 min; 72 ℃ for 2 min. The size of the target band should be 10243 bp.

The linearized pYES1L vector and F1-F7 fragments are mixed and transformed into yeast by a lithium acetate transformation method. The specific operation is as follows: respectively taking 100ng of linearized vector pYES1L and uniformly mixing the linearized vector pYES1L with the fragments F1, F2, F3, F4, F5, F6 and F7; then taking out the MaV203 yeast competent cells from-80 ℃, unfreezing the MaV203 yeast competent cells at 30 ℃ for no more than 90s, adding the mixture into the MaV203 yeast competent cells, flicking the tube wall to mix the cells evenly, then adding 600 mu L of PEG/LiAc into the mixture of DNA and yeast competent cells, placing the mixture into a water bath at 30 ℃ to incubate for 30min after the mixture is mixed by soft up-down reversal, and then, re-suspending the mixture by soft up-down reversal every 10 min; adding 35.5 mu L DMSO after incubation, slightly turning upside down, mixing uniformly, placing in 42 ℃ water bath for heat shock for 20min, and slightly turning upside down and mixing uniformly every 5 min; after the heat shock is finished, centrifuging at 1800rpm (200-; 100 μ L of the suspension was spread on a tryptophan deficient yeast dish and cultured at 30 ℃ for 2 to 3 days. Since terminal homology arms exist in the linearized pYES1L vector and fragment F1, fragment F1 and fragment F2, fragment F2 and fragment F3, fragment F3 and fragment F4, fragment F4 and fragment F5, fragment F5 and fragment F6, fragment F6 and fragment F7, the linearized pYES1L vector and fragment F7, respectively, recombinant plasmids carrying CPIV3 full-length cDNA are assembled by a homologous recombination mechanism in the host cell yeast.

Picking a single colony and placing the single colony in a PCR tube containing 15 mu L of lysine Buffer, gently blowing and beating the Lysis yeast for three times, transferring 5 mu L to a new PCR tube, keeping the temperature at 4 ℃ for later use, carrying out colony PCR detection on the other 10 mu L, screening out positive clones, and then electrically transforming the positive clones into DH10B electric shock competence. Screening positive recombinants through the resistance of the Spectinomycin, and purifying a recombinant plasmid carrying the PEDV full-length cDNA through a plasmid quantitative extraction kit after proliferation, wherein the recombinant plasmid is named as pYES 1L-PEDV.

The recombinant plasmid is subjected to enzyme digestion identification by using restriction enzyme Mlu I, and the enzyme digestion map is identified through 0.8% agarose gel electrophoresis. Based on the prediction of Snapgene software, the cleavage map should contain four fragments of 4002bp, 7175bp, 10559bp and 16538bp, and the result is shown in FIG. 3.

Example 2 construction of recombinant plasmid pYES1L-PEDV-EGFP based on CRISPR/Cas9 Gene editing technology

The build strategy is shown in figure 4. Based on CRISPR/Cas9 gene editing technology, PEDV-sgRNA1 and PEDV-sgRNA2 (synthesized in Nanjing Kingsler Biotech Co., Ltd., see Table 1) two guide RNAs are designed to be used for cutting two side sequences of ORF3 in pYES1L-PEDV, and the porcine epidemic diarrhea virus infectious cDNA cloning vector skeleton with ORF3 deleted is obtained. The specific operation is as follows: cas9 nucleic Buffer 5 mu L, PEDV-sgRNA 1(300nM) and PEDV-sgRNA 2(300nM) each 3 mu L, Cas9 nucleic 3 mu L are mixed, recombinant plasmid pYES1L-PEDV 3 mu g is added after 10min at 37 ℃, and the porcine epidemic diarrhea virus infectious cDNA cloning vector backbone lacking ORF3 is recovered after 2h at 37 ℃.

Using pEGFP-N1(Clontech) as a template, primers EGFP-F and EGFP-R (see Table 1) were subjected to PCR amplification to obtain an EGFP gene fragment.

And recovering the purified vector skeleton and EGFP fragment, assembling a recombinant plasmid by using a HiFi DNA Assembly Master Mix of NEB company through an in vitro homologous recombination method (refer to the operation of a kit instruction), electrically transforming DH10B electric shock competent cells, and screening out positive clones by a colony PCR method.

Example 3: construction of helper plasmids

Specific primers PEDV-N-F and PEDV-N-R (shown in table 1) are designed by using Snapgene software according to the open reading frame region of the N gene in PEDV, and pYES1L-PEDV is used as a template for PCR amplification to obtain an N gene fragment. Through a conventional enzyme digestion connection test, the recovered and purified N gene fragment is inserted between SacI and XhoI sites of a pCAGGS vector, then a ligation product is transformed into a DH5 alpha escherichia coli competent cell, and a correct recombinant plasmid pCAGGS-PEDV-N is screened out through sequencing identification. Extracting plasmid according to the operation instruction of the endotoxin-free plasmid extraction kit, determining the concentration, subpackaging, and storing at-20 ℃.

TABLE 1 primer sequences for use in the present invention

Example 4: rescue and biological characteristic identification of porcine epidemic diarrhea virus HM strain recombinant virus and green fluorescent labeled virus

1. Rescue and amplification of recombinant viruses

Inoculating Vero CCL-81 cells into a 12-hole cell culture plate, and carrying out plasmid transfection according to the specification of a Lipofectamine 3000 transfection reagent when the cell density reaches about 80%, wherein the dosage of plasmids in each hole is as follows: infectious cDNA clone plasmids (recombinant plasmid pYES1L-PEDV or pYES1L-PEDV-EGFP) 2. mu.g and helper plasmid pCAGGS-N0.5. mu.g. 24h after transfection, the cell culture medium was changed to DMEM medium containing 10. mu.g/mL.

When typical cytopathic effect appears, cell supernatant is collected to obtain rescued virus rPEDV and rPEDV-EGFP. The rescued virus was serially passaged in Vero CCL-81 cells, and each culture was stored at-80 ℃.

2. Identification of recombinant viruses

2.1 Indirect immunofluorescence detection of viral N protein expression

Fixing the cells with 4% paraformaldehyde for 15min, drying completely, adding 0.1% Triton-X100 to cover the cells, perforating for 15min, discarding 0.1% Triton-X100, adding 1% BSA to cover the cells, sealing for 30min, adding monoclonal antibody against PEDV N protein, incubating at room temperature for 1h, washing with PBS for 5 times, and adding Alexa

488G conjugated oat Anti-Mouse IgG H&The L secondary antibody (purchased from Jackson immuneresearch Inc), washed 5 times with PBS, stained 5min with DAPI added, washed 2 times with PBS, observed with a fluorescence microscope and recorded the results of the experiment (fig. 5).

2.2RT-PCR identification

Recombinant virus (rescued virus rPEDV-EGFP) RNA was extracted separately, and RT-PCR amplification was performed using a one-step RT-PCR kit (Nanjing Novozam Biotech Co., Ltd.) using primers PEDV-EGFP-F and PEDV-EGFP-R.

The fragments were recovered after 1% agarose gel electrophoresis, and sequenced by Nanjing Kingsry Biotech Ltd to compare whether the sequence was consistent with the designed one. As a result: the amplified sequence was identical to the viral cDNA plasmid sequence.

3. Characterization of biological Properties

3.1 multistep growth Curve

Vero CCL-81 cells were inoculated into 24-well plates and cultured in a 37 ℃ incubator containing 5% CO2 until the cell density reached 100% for infection. PEDV HM, rPEDV P5 and rPEDV-EGFP P5 infected Vero CCL-81 cells at 0.01MOI, respectively, and 6 wells were infected with each virus. Viral supernatants were collected at 0h, 24h, 48h and 72h post infection, and stored at-80 ℃ for future use. And (3) diluting the collected virus supernatants by 10-fold gradient, washing Vero CCL-81 cells to be infected by PBS for 2 times to remove serum in a culture medium, adding diluted virus solution into a 96-well plate, adding 100 mu L of virus into each well, repeating the dilution for 4 times, incubating for 2 hours, discarding the culture solution, adding 100 mu L of DMEM culture medium containing 10 mu g/mL pancreatin into each well, and continuously culturing at 37 ℃. Lesions were observed and recorded day by day, cytopathic effects were observed 7 days after infection and TCID50 was calculated according to Reed-Muench method and virus growth curves were plotted (see fig. 6). The results show that the growth characteristics of the recombinant viruses (rPEDV and rPEDV-EGFP) on cells are substantially identical compared to the parental viruses. However, the viral titers of rPEDV-EGFP were 0.5log lower at 48h and 72h post-infection than the parental virus.

3.2 viral plaque assay

Vero CCL-81 cells were inoculated into 12-well plates and cultured in a 37 ℃ incubator containing 5% CO2 until the cell density reached 100% for infection. Respectively diluting PEDV HM, rPEDV P5 and rPEDV-EGFP P5 with DMEM culture medium by 10 times gradient, washing the cells to be infected with PBS for 2 times to remove serum in the culture medium, adding diluted virus solution into 12-well plate, adding 5% CO at 37 deg.C₂Under the conditions of (1). After 2h of infection, the virus fluid was discarded, and 2 XDMEM (containing 20. mu.g/mL pancreatin) and 2% UltraPure were added^TMLMP Agarose et alMixing at a certain proportion, adding into 12-hole plate, standing at 37 deg.C and 5% CO after completely solidifying₂Culturing in an incubator. After 3 days of infection, the gel was fixed with 4% paraformaldehyde overnight, the next day the 4% paraformaldehyde and gel were discarded, 300. mu.L of 1% crystal violet staining solution was added to each well, stained for 10min, the crystal violet solution was discarded, washed with water, dried and recorded by photography (see FIG. 7). The results show that there is no significant difference in plaque morphology and size for the three viruses.

Sequence listing

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aagtaaaaga tgctgaagat cagttgggac gcgtagtcta gaccagccag gacagaaatg 2520

cctcgacttc gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg 2580

cacgaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc 2640

gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg 2700

ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc 2760

agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc 2820

agcagggcag tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag 2880

tatcgactca actatcagag gtagttggcg ccatcgagcg ccatctcgaa ccgacgttgc 2940

tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 3000

atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 3060

accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 3120

ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 3180

ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 3240

ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 3300

cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 3360

ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta 3420

cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 3480

ccggctgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac 3540

aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat 3600

ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataaccc tcgagcattc 3660

aaggcgcctt gattatttga cgtggtttga tggcctccac gcacgttgtg atatgtagat 3720

gataatcatt atcactttac gggtcctttc cggtgatccg acaggttacg gggcggcgac 3780

ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg ttcttcttcg 3840

tcataactta atgtttttat ttaaaatacc tcgcgagtgg caacactgaa aatacccatg 3900

gagcggcgta accgtcgcac aggaaggaca gagaaagcgc ggatctggga agtgacggac 3960

agaacggtca ggacctggat tggggaggcg gttgccgccg ctgctgctga cggtgtgacg 4020

ttctctgttc cggtcacacc acatacgttc cgccattcct atgcgatgca catgctgtat 4080

gccggtatac cgctgaaagt tctgcaaagc ctgatgggac ataagtccat cagttcaacg 4140

gaggtctaca cgaaggtttt tgcgctggat gtggctgccc ggcaccgggt gcagtttgcg 4200

atgccggagt ctgatgcggt tgcgatgctg aaacaattat cctgagaata aatgccttgg 4260

cctttatatg gaaatgtgga actgagtgga tatgctgttt ttgtctgtta aacagagaag 4320

ctggctgtta tccactgaga agcgaacgaa acagtcggga aaatctccca ttatcgtaga 4380

gatccgcatt attaatctca ggagcctgtg tagcgtttat aggaagtagt gttctgtcat 4440

gatgcctgca agcggtaacg aaaacgattt gaatatgcct tcaggaacaa tagaaatctt 4500

cgtgcggtgt tacgttgaag tggagcggat tatgtcagca atggacagaa caacctaatg 4560

aacacagaac catgatgtgg tctgtccttt tacagccagt agtgctcgcc gcagtcgagc 4620

gacagggcga agccctcgag tgagcgagga agcaccaggg aacagcactt atatattctg 4680

cttacacacg atgcctgaaa aaacttccct tggggttatc cacttatcca cggggatatt 4740

tttataatta ttttttttat agtttttaga tcttcttttt tagagcgcct tgtaggcctt 4800

tatccatgct ggttctagag aaggtgttgt gacaaattgc cctttcagtg tgacaaatca 4860

ccctcaaatg acagtcctgt ctgtgacaaa ttgcccttaa ccctgtgaca aattgccctc 4920

agaagaagct gttttttcac aaagttatcc ctgcttattg actctttttt atttagtgtg 4980

acaatctaaa aacttggcac acttcacatg gatctgtcat ggcggaaaca gcggttatca 5040

atcacaagaa acgtaaaaat agcccgcgaa tcgtccagtc aaacgacctc actgaggcgg 5100

catatagtct ctcccgggat caaaaacgta tgctgtatct gttcgttgac cagatcagaa 5160

aatctgatgg caccctacag gaacatgacg gtatctgcga gatccatgtt gctaaatatg 5220

ctgaaatatt cggattgacc tctgcggaag ccagtaagga tatacggcag gcattgaaga 5280

gtttcgcggg gaaggaagtg gttttttatc gccctgaaga ggatgccggc gatgaaaaag 5340

gctatgaatc ttttccttgg tttatcaaac gtgcgcacag tccatccaga gggctttaca 5400

gtgtacatat caacccatat ctcattccct tctttatcgg gttacagaac cggtttacgc 5460

agtttcggct tagtgaaaca aaagaaatca ccaatccgta tgccatgcgt ttatacgaat 5520

ccctgtgtca gtatcgtaag ccggatggct caggcatcgt ctctctgaaa atcgactgga 5580

tcatagagcg ttaccagctg cctcaaagtt accagcgtat gcctgacttc cgccgccgct 5640

tcctgcaggt ctgtgttaat gagatcaaca gcagaactcc aatgcgcctc tcatacattg 5700

agaaaaagaa aggccgccag acgactcata tcgtattttc cttccgcgat atcacttcca 5760

tgacgacagg atagtctgag ggttatctgt cacagatttg ggggtggttc gtcacatttg 5820

ttctgaccta ctgagggtaa tttgtcacag ttttgctgtt tccttcagcc tgcatggatt 5880

ttctcatact ttttgaactg taatttttaa ggaagccaaa tttgagggca gtttgtcaca 5940

gttgatttcc ttctctttcc cttcgtcatg tgacctgata tcgggggtta gtttgtcatc 6000

attgatgagg gttgattatc acagtttatt actctgaatt ggctatccgc gtgtgtacct 6060

ctacctggag tttttcccac ggtggatatt tcttcttgcg ctgagcgtaa gagctatctg 6120

acagaacagt tcttctttgc ttcctcgcca gttcgctcgc tatgctcggt tacacggctg 6180

cggcgagcgc tagtgataat aagtgactga ggtatgtgct cttcttatct ccttttgtag 6240

tgttgctctt attttaaaca actttgcggt tttttgatga ctttgcgatt ttgttgttgc 6300

tttgcagtaa attgcaagat ttaataaaaa aacgcaaagc aatgattaaa ggatgttcag 6360

aatgaaactc atggaaacac ttaaccagtg cataaacgct ggtcatgaaa tgacgaaggc 6420

tatcgccatt gcacagttta atgatgacag cccggaggcg aggaaaataa cccggcgctg 6480

gagaataggt gaagcagcgg atttagttgg ggtttcttct caggctatca gagatgccga 6540

gaaagcaggg cgactaccgc acccggatat ggaaattcga ggacgggttg agcaacgtgt 6600

tggttataca attgaacaaa ttaatcatat gcgtgatgtg tttggtacgc gattgcgacg 6660

tgctgaagac gtatttccac cggtgatcgg ggttgctgcc cataaaggtg gcgtttacaa 6720

aacctcagtt tctgttcatc ttgctcagga tctggctctg aaggggctac gtgttttgct 6780

cgtggaaggt aacgaccccc agggaacagc ctcaatgtat cacggatggg taccagatct 6840

tcatattcat gcagaagaca ctctcctgcc tttctatctt ggggaaaagg acgatgtcac 6900

ttatgcaata aagcccactt gctggccggg gcttgacatt attccttcct gtctggctct 6960

gcaccgtatt gaaactgagt taatgggcaa atttgatgaa ggtaaactgc ccaccgatcc 7020

acacctgatg ctccgactgg ccattgaaac tgttgctcat gactatgatg tcatagttat 7080

tgacagcgcg cctaacctgg gtatcggcac gattaatgtc gtatgtgctg ctgatgtgct 7140

gattgttccc acgcctgctg agttgtttga ctacacctcc gcactgcagt ttttcgatat 7200

gcttcgtgat ctgctcaaga acgttgatct taaagggttc gagcctgatg tacgtatttt 7260

gcttaccaaa tacagcaata gtaatggctc tcagtccccg tggatggagg agcaaattcg 7320

ggatgcctgg ggaagcatgg ttctaaaaaa tgttgtacgt gaaacggatg aagttggtaa 7380

aggtcagatc cggatgagaa ctgtttttga acaggccatt gatcaacgct cctcaactgg 7440

tgcctggaga aatgctcttt ctatttggga acctgtctgc aatgaaattt tcgatcgcct 7500

gattaaacca cgctgggaga ttagataatg aagcgtgcgc ctgttattcc aaaacatacg 7560

ctcaatactc aaccggttga agatacttcg ttatcgacac cagctgcccc gatggtggat 7620

tcgttaattg cgcgcgtagg agtaatggct cgcggtaatg ccattacttt gcctgtatgt 7680

ggtcgggatg tgaagtttac tcttgaagtg ctccggggtg atagtgttga gaagacctct 7740

cgggtatggc caggtaatga acgtgaccag gagctgctta ctgaggacgc actggatgat 7800

ctcatccctt cttttctact gactggtcaa cagacaccgg cgttcggtcg aagagtatct 7860

ggtgtcatag aaattgccga tgggagtcgc cgtcgtaaag ctgctgcact taccgaaagt 7920

gattatcgtg ttctggttgg cgagctggat gatgagcaga tggctgcatt atccagattg 7980

ggtaacgatt atcgcccaac aagtgcttat gaacgtggtc agcgttatgc aagccgattg 8040

cagaatgaat ttgctggaaa tatttctgcg ctggctgatg cggaaaatat ttcacgtaag 8100

attattaccc gctgtatcaa caccgccaaa ttgcctaaat cagttgttgc tcttttttct 8160

caccccggtg aactatctgc ccggtcaggt gatgcacttc aaaaagcctt tacagataaa 8220

gaggaattac ttaagcagca ggcatctaac cttcatgagc agaaaaaagc tggggtgata 8280

tttgaagctg aagaagttat cactctttta acttctgtgc ttaaaacgtc atctgcatca 8340

agaactagtt taagctcacg acatcagttt gctcctggag cgacagtatt gtataagggc 8400

gataaaatgg tgcttaacct ggacaggtct cgtgttccaa ctgagtgtat agagaaaatt 8460

gaggccattc ttaaggaact tgaaaagcca gcaccctgat gcgaccacgt tttagtctac 8520

gtttatctgt ctttacttaa tgtcctttgc tacaggccag aaagcataac tggcctgaat 8580

attctctctg ggcccactgt tccacttgta tcgtcggact gataatcaga ctgggaccac 8640

ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacggtccca ctcgtatcgt 8700

cggtctgatt attagtctgg gaccacggtc ccactcgtat cgtcggtctg ataatcagac 8760

tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc atggtcccac 8820

tcgtatcgtc ggtctgatta ttagtctggg accacggtcc cactcgtatc gtcggtctga 8880

ttattagtct ggaaccacgg tcccactcgt atcgtcagtc tgattattag tctgggacca 8940

cggtcccact cgtatcgtcg gtctgattat tagtctggga ccacgatccc actcgtgttg 9000

tcggtctgat tatcggtctg ggaccacggt cccacttgta ttgtcgatca gactatcagc 9060

gtgagactac gattccatca atgcctgtca agggcaagta ttgacatgtc gtcgtaacct 9120

gtagaacgga gtaacctcgg tgtgcggttg tatgcctgct gtggattgct gctgtgtcct 9180

gcttatccac aacattttgc gcacggttat gtggacaaaa tacctggtta cccaggccgt 9240

gccggcacgt taaccgggct gcatccgatg caagtgtgtc gctgtcgacg gcctcctcac 9300

ccggtcacgt gagctcattt aacccactcc acaaaaaggc tcaacaggtt ggtggttctc 9360

accaccaaaa gcaccacacc ccacgcaaaa acaagttttt gctgattttt ctttataaat 9420

agagtgttat gaaaaattag tttctcttac tctctttatg atatttaaaa aagcggtgtc 9480

ggcgcggcta caacaacgcg ccgacaccgt tttgtagggg tggtactgac tatttttata 9540

aaaaacatta ttttatatta ggggtgctgc tagcggcgcg gtgtgttttt ttataggata 9600

ccgctagggg cgctgctagc ggtgcgtccc tgtttgcatt atgaattagt tacgctaggg 9660

ataacagggt aatatagaac ccgaacgacc gagcgcagcg gcggccgcgc tgataccgcc 9720

gccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 9780

ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 9840

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 9900

ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 9960

tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 10020

accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 10080

ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 10140

cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 10200

gtacggtggg aggtctatat aagcagagct tgagctctaa tag 10243

<210> 2

<211> 28064

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acttaaaaga ttttctatct acggatagtt agctctttct ctagactctt gtctactcaa 60

ttcaactaaa cgaaattttg tccttccagt cgcatgtcta tgctgctgga agctgacgtg 120

gaatttcatt aggtttgctt aagtagccat cgcaagtgct gtgctgtcct ctagttcctg 180

gttggcgttc cgtcgccttc tacatactag acaaacagcc ttcctccggt tccgtctggg 240

ggttgtgtgg ataactagtt ccgtctagtt tgaaaccagt aactgtcggc tatggctagc 300

aaccatgtta cattggctgt tgccaatgat gcagaaattt cagcctttgg cttttgcact 360

gctagtgaag ccgtctcata ctattctgag gccgccgcta gtggatttat gcaatgccgt 420

ttcgtgtcct tcgatctcgt tgacactgtt gagggattgc ttcccgaaga ctatgtcatg 480

gttgtggtcg gcactaccaa gcttagtgcg tatgtggaca cttttggtag ccgccccaga 540

aacatctgtg gttggctatt attttctaac tgtaattact tcctcgaaga gttagagctc 600

acttttggtc gtcgtggtgg taacatcgtg ccagttgatc aatacatgtg tggtgctgac 660

gggaaacctg ttcttcagga atccgagtgg gagtatacag acttctttgc tgactccgag 720

gacggtcaac tcaacattgc tgggatcact tatgtgaagg cctggattgt agagagatcg 780

gatgtctctt atgcgagtca gaatttaaca tctattaaat ctattactta ttgttcaacc 840

tatgagcata cttttcctga tggtaccgcc atgaaggttg cacgtaatcc aaagatcaag 900

aagaatgttg tcttgtctga gtcacttgct actatctaca gggaaattgg ttctcctttt 960

gtggataatg ggagcgatgc tcgttctatc attaagagac cagtgttcct ccacgctttt 1020

gttaagtgta agtgtggtag ttatcattgg actgttggtg attggacctc ctatgtctcc 1080

acctgctgtg gctttaagtg caagccagtc cttgtggctt catgctctgc tacgcctggt 1140

tctgttgtgg ttacgcgcgc tggtgctggc actggtgtta agtattacaa caacatgttc 1200

ctgcgccatg tggcagacat tgatgggttg gcattctggc gaattcttaa ggtgcagtcc 1260

aaagacgacc tcgcttgctc tggtaaattc cttgaacacc atgaggaagg tttcacagat 1320

ccttgctact ttttgaatga ctcgagcatt gctactaagc tcaagtttga catccttagt 1380

ggcaagtttt ctgatgaagt caaacaagct atctttgctg gtcatgttgt tgttggcagt 1440

gcgctcgttg acattgttga cgatgcactg ggacaacctt ggtttatacg taagcttggt 1500

gaccttgcaa gtgcagcctg ggagcagctt aaggctgtcg ttagaggcct taacctcctg 1560

tctgatgaag tcgtgctctt tggcaaaaga cttagctgtg ccactcttag tatcgttaac 1620

ggtgtttttg agtttgtcgc cgaagtgcca gagaagttgg ctgcggccgt tacagttttt 1680

gtcaacttct tgaatgagct ttttgagtct gcctgtgact gtttaaaggt cggaggtaaa 1740

acctttaaca gggttggctc ttatgttctt tttgacaatg cattggttaa gcttgtcaag 1800

gcaaaagttc gcggcccacg acaggcaggt gtttgtgaag ttcgttacac aagccttgtt 1860

attgggagta ctaccaaggt ggtttccaag cgcgttgaaa atgccaatgt gaatctcatc 1920

gtcgttgacg aggatgtgac cctcaacacc actggtcgta cagttgttgt tgatggactt 1980

gcattcttcg agagtgacgg gttttacaga catcttgctg atgctgacgt tgtcattgaa 2040

catcctgttt ataagtctgc ttgtgagctc aagccagtct ttgagtgtga cccaatacct 2100

gattttccca tgcctgtggc cgctagtgtt gcagagcttt gtgtgcaaac tgatctgttg 2160

cttaaaaatt acaacactcc ttataaaact tacagctgcg tggtgagagg tgataagtgt 2220

tgcattactt gcaccttaca tttcacagca ccaagttata tggaggatgc tgctaatttt 2280

gtagacctct gtaccaagaa cattggtact gctggttttc atgagtttta cattacggcc 2340

catgaacaac agaatctgca agggttcgta accacttgtt gcacgatgtc aggttttgag 2400

tgttttatgc ctataatccc acagtgtcca gcagtgcttg aagagattga tggtggtagc 2460

atctggcggt cttttatcac tggtcttaat acaatgtggg atttttgcaa gcatcttaaa 2520

gtcagctttg gactagatgg cattgttgtc actgtagcac gcaaatttaa acgacttggt 2580

gctctcttgg cagaaatgta taacacttac ctttcaactg tggtggaaaa cttggtactg 2640

gccggtgtta gcttcaagta ttatgccacc agtgtcccaa aaattgtttt gggctgttgt 2700

ttttacagtg ttaaaagtgt tcttgcaagt gccttccaga ttcctgtcca ggcaggcatt 2760

gagaatttta aagtctttct taactgtgtt caccctgttg taccacgcgt cattgaaact 2820

tcttttgtgg aattagaaga gacgacattt aaaccaccag cactcaatgg tagtattgct 2880

attgttgatg gctttgcttt ctattatgat ggaacactat actatcccac cgatggtaat 2940

agcgttgttc ctatctgctt taagaagaaa ggtggtggtg atgtcaaatt ctctgatgaa 3000

gtttctgtta aaaccattga cccagtttat aaggtctccc ttgaatttga gttcgagtct 3060

gagactatta tggctgtgct taataaggct gttggtaacc gtatcaaggt tacaggtggt 3120

tgggacgatg ttgttgagta tatcaacgtt gccattgagg ttcttaaaga tcacatcgat 3180

gtgcctaagt actacattta tgatgaggaa ggtggcaccg atcctaatct tcccgtaatg 3240

gtttctcagt ggccgctgaa tgatgacacg acctcacagg atctgcttga tgtggaagtt 3300

gttacggatg caccaagtga ctacgagggt gatgaagtgg actcctctga ccctgataag 3360

gtggcagatg tggctaactc tgagcctgag gatggtgttc ttaatgtagc tcctgaaaca 3420

aatgtagagt ctgaagttga ggaagttgcc gcaaccttgt cctttattaa agatacacct 3480

tccacagtta ctaaggatcc ttttgctttt gactttgcaa gctatggagg acttaaggtt 3540

ttaagacaat ctcataacaa ctgttgggtt acttctacct tggtgcagct acaattgctt 3600

ggcatcgttg atgaccctgc aatggagctc tttagtgctg gtagagtcgg tccaatggtt 3660

cgcaaatgct atgagtcaca aaaggctatt ttgggatctt tgggtgatgt atcggcttgt 3720

ctagagtctc ttactaagaa cctacacaca cttaagatta cctgttctgt agtctgtggt 3780

tgtggtactg gtgaacgcat ttatgagggt tgtgcttttc gtatgacgcc aactttggaa 3840

ccgttcccat atggtgcttg tgctcagtgt gctcaagttt tgatgcacac ttttaaaagt 3900

attgttggca ccggcatctt ttgtcgagat actactgctc tctccttgga ttctttggtt 3960

gtaaaacctc tttgtgcggc tgcttttata ggcaaggata gtggtcatta tgtcaccaac 4020

ttttatgatg ctgctatggc tattgatggt tatggtcgtc atcagataaa gtatgacaca 4080

ctgaacacca tttgtgttaa agacgtgaat tggacagcac cttctgtcct tgacgttgcg 4140

cctgtattga agcctgttgt caaacctttc tattcttata agaatgttga tttttaccaa 4200

ggagatttta gtgaccttgt taaacttcca tgtgactttg ttgttaatgc tgcaaatgag 4260

aatttgtctc acggtggcgg catagcaaag gccattgatg tttataccaa gggcatgttg 4320

cagaagtgct cgaatgatta cattaaagca cacggtccca ttaaagttgg acgtggtgtc 4380

atgttggagg cattaggtct taaggtcttt aatgttgttg gtccacgtaa gggtaagcat 4440

gcacctgagc ttcttgttaa ggcttataag tccgtttttg ctaattcagg tgttgctctt 4500

acacctttga ttagtgttgg aatttttagt gttcctttgg aagaatcttt atctgctttt 4560

cttgcatgtg ttggtgatcg ccactgtaag tgcttttgtt atagtgacaa agagcgcgag 4620

gcgatcatta attacatgga tggcttggta gatgctattt tcaaagatgc gcttgttgat 4680

actactcctg tccaggaaga tgttcaacaa gtttcacaaa aaccagtttt gcctaatttt 4740

gaacctttca ggattgaagg tgctcatgct ttctatgagt gtaaccctga aggtttgatg 4800

tccttaggtg ctgacaagct ggtgttgttt acaaattcca ctttggattt ttgtagcgtt 4860

ggtaagtgtc ttaacaatgt gaccgccggt gcattgcttg aagccataaa tgtatttaaa 4920

aagagtaaca aaacagtgcc tgctggcaac tgtgttactt ttgagtgtgc agatatgatt 4980

tctattacta tggtagtatt gccagctgat ggtgatgcta attatgacaa aaattatgca 5040

cgcgccgttg ttaaggtatc taagcttaaa ggcaagttat tgcttgctgt tggtgatgcc 5100

acgttgtatt ccaagttgtc ccaccttagc gtggtaggtt tcgtatccac acctgatgat 5160

gtggagcgtt tctacgcaaa taagagtgtg gttattaaag tcactgagga tacacgtagt 5220

gttaaggctg ttaaagtaga atctactgtt acttatggac aacaaatcgg accttgtctt 5280

gttaatgaca ccgttgttac agacaacaaa cctgttgttg ctgatgttgt agctaaggtt 5340

gtaccaagtg ctaattggga ttcacattgt ggttttgata aggcaggtga gttccatatg 5400

ctagaccata ctgggtttgc ctttcctagt gaagttgtta acggtaggcg tgtgcttaaa 5460

accacagata ataactgttg ggttaatgtt acatgtttac aattacagtt tgctagattt 5520

aggttcaagt cagcaggtct acaggctatg tgggagtcct attgtactgg tgatgttgct 5580

atgtttgtgc attggttgta ctggcttact ggtgttgaca aaggtcagcc tagtgattca 5640

gaaaatgcac ttaacatgtt gtccaagtac attgttcctg ctggttctgt cactattgaa 5700

cgtgtcacgc atgacggctg ttgttgtagt aaacgtgttg tcactgcacc agttgttaat 5760

gctagcgttt tgaagcttgg cgtcgaggat ggtctttgtc cacatggtct taactacatt 5820

gacaaagttg ttgtagttaa aggtactaca attgttgtca atgttggaaa acctgtggtg 5880

gcaccttcac acctctttct caagggtgtt tcctacacaa cattcctaga taatggtaac 5940

ggtgttgtcg gccattatac tgtttttgat catgacactg gtatggtgta tgatggagat 6000

gcttttgtac cgggtgacct caatgtatcc cctgttacaa atattgttgt ctcagagcag 6060

acggctgttg tgattaaaga ccccgtgaag aaagtagagt tagacgctac aaagctgtta 6120

gacactatga attatgcatc ggaaagattc ttttcctttg gtgatttcat gtcacgtaat 6180

ttaattacag tgtttttgta catccttagc attttgggtc tctgttttag ggcctttcgt 6240

aagagagatg ttaaagttct agctggtgta ccccaacgta ctggtattat attgcgtaaa 6300

agtgtgcgct ataatgcaaa ggcgttgggt gtctttttca agctaaagct ttattggttc 6360

aaagttcttg gtaagtttag tttgggtgtt tatgcattgt atgcactact attcatgaca 6420

atacgcttta cacctatagg tagccctgtt tgtgatgatg ttgttgctgg ttatgctaat 6480

tctagttttg ataagaatga gtattgcaac agtgttattt gtaaggtctg tctatatggg 6540

taccaggaac tctcggactt ctcccacaca caggtagtat ggcaacacct tagagaccca 6600

ttaattggta atgtgatgcc tttctcttat ttggcatttt tggcaatttt tgggggtgtt 6660

tatgtaaagg ctattactct ctattttatt tttcagtatc ttaacattct tggtgtgttt 6720

ttgggcttac aacagtccat ttggtttttg cagcttgtgc cttttgatgt ttttggcgac 6780

gagatcgtcg tctttttcat cgttacacgc gtattgatgt tccttaagca tgttttcctt 6840

ggctgtgata aggcatcttg tgtggcttgc tctaagagtg ctcgtcttaa gcgcgttcct 6900

atccagacta tctttcaggg tactagcaaa tccttctacg tacatgccaa tggtggttct 6960

aagttctgta agaagcacaa tttcttttgt ttaaattgtg attcttatgg tccaggctgc 7020

acttttatta atgacgtcat tgcaactgaa gttggtaatg tcgtcaaact taatgtgcaa 7080

ccgacaggtc ctgccactat tcttattgac aaggttgaat tcagtaatgg tttttactat 7140

ctttatagtg gtgacacatt ttggaagtac aactttgaca taacagatag caaatacact 7200

tgcaaagagg cacttaaaaa ttgtggcata atcacagact ttattgtttt taacaataat 7260

ggttccaatg taaatcaggt taagaatgca tgtgtgtatt tttcacagat gctttgtaaa 7320

cctgttaaat tagtggactc agcgttgttg gccagtttgt ctgttgattt tggtgcaagt 7380

ttacatagtg cttttgttag tgtgttgtcg aatagtttcg gcaaagacct gtcaagttgt 7440

aatgacatgc aggattgcaa gagcacattg ggttttgatg atgtaccatt ggataccttt 7500

aatgctgctg ttgctgaggc tcatcgttat gatgtcctct tgactgacat gtcattcaac 7560

aattttacca ccagttatgc aaaaccagag gaaaaatttc ccgtccatga cattgccacg 7620

tgtatgcgtg taggtgccaa gattgttaat cataacgttc ttgtcaagga tagtatacct 7680

gtggtgtggc tcgtacgtga tttcattgcc ctttcggaag aaactaggaa gtacattatt 7740

cgtacgacta aagttaaggg tataacattt atgctgacct ttaatgattg tcgtatgcat 7800

actaccatac ctactgtttg cattgcaaat aagaagggtg caggtcttcc tagtttttca 7860

aaggttaaga aattcttttg gtttttgtgt ctattcatag ttgctgtttt ctttgcacta 7920

agctttcttg attttagtat tcaggttagc agtgatagcg attatgattt caagtatatt 7980

gagagtggcc agttgaagac ttttgacaat ccacttagtt gtgtgcataa tgtctttagt 8040

aacttcgacc agtggcatga tgccaagttt ggtttcaccc ccgtcaacaa tcctagttgt 8100

cctatagttg ttggtgtatc agacgaagct cgcactgttc caggtatccc agcaggtgtt 8160

tatttagctg gtaaaacact tgtgtttgct attaacacca tttttggtac atctggtttg 8220

tgctttgatg ctagtggcgt tgctgataag ggcgcttgca tttttaattc ggcttgcacc 8280

acattatctg gtttgggtgg aactgctgtc tactgttata agaatggtct agttgaaggt 8340

gctaaacttt atagtgagtt ggcacctcat agctactata aaatggtaga tggtaatgct 8400

gtgtctttac ctgaaattat ctcacgcggc tttggcatcc gtactatccg tacaaaggct 8460

atgacctact gtcgcgttgg ccagtgtgtg caatctgcag aaggtgtttg ttttggcgcc 8520

gatagattct ttgtctataa tgcagactct ggttctgact ttgtttgtgg cacggggctc 8580

ttcacattgt tgatgaacgt tattagtgtt ttttccaaga cagtaccagt aactgtgttg 8640

tctggtcaaa tactttttaa ttgcattatt gcttttgctg ctgttgcggt gtgtttctta 8700

tttacaaagt ttaagcgcat gttcggtgat atgtctgttg gcgttttcac tgtcggtgct 8760

tgtactttgt tgaacaatgt ttcttacatt gtaacacaga atacacttgg catgttgggc 8820

tatgcaactt tgtacttttt gtgcactaaa ggtgttagat atatgtggat ttggcatttg 8880

ggatttttga tctcatatat acttattgca ccatggtggg ttttgatggt ttatgccttt 8940

tcagccattt ttgagtttat gcctaacctt tttaagctta aggtttcaac acaacttttt 9000

gagggtgaca agttcgtagg ctcttttgaa aatgctgcag caggtacatt tgtgcttgat 9060

atgcatgcct atgagagact tgccaattct atctcaactg aaaaactgcg tcagtatgct 9120

agtacttaca ataagtacaa gtattattca ggcagtgctt cagaggctga ttataggctt 9180

gcttgttttg cccatttggc caaggctatg atggattatg cttctaatca caacgacacg 9240

ttatacacac cacccactgt gagttataat tcaactctac aggctggcct gcgtaagatg 9300

gcacaaccat ctggtgttgt tgagaagtgc atagttcgtg tttgctatgg taatatggct 9360

cttaatggcc tatggcttgg tgatactgtt atgtgcccac gccatgttat agcgtctagt 9420

actactagca ctatagatta tgactatgcc ctttctgttt tacgcctcca caacttctcc 9480

atttcatctg gtaatgtttt cctaggtgtt gtgggtgtaa ccatgcgagg ttctttgttg 9540

cagataaagg ttaatcaaaa caatgtccac acgcctaagt acacctatcg cacagttaga 9600

ccgggtgaat cttttaatat cttggcgtgc tatgatggtt ctgcagctgg tgtttatggc 9660

gttaacatgc gctctaatta cactattaga ggctcgttca ttaatggcgc ttgtggttca 9720

cctggttata atattaacaa tggtaccgtt gagttttgct atttacacca gctcgaactt 9780

ggttcgggct gtcatgttgg tagcgactta gatggtgtta tgtatggtgg ttatgaggac 9840

caacctacgt tgcaagttgg aggcgctagt agtctgttta cagagaatgt gttggcattt 9900

ctttatgcag cactcattaa tggttctatc tggtggctta gttcttctag gaccgctgta 9960

gacaggttta atgagtgggc tgttcataat ggtatgacaa cagtgggtaa tactgattgc 10020

ttttctattc ttgctgctaa gactggcgtt gatgtacaac gtttgttggc ctcaatccag 10080

tctctacata agaattttgg tggaaagcaa attcttggct atacctcttt gacagatgag 10140

tttactacag gtgaggttat acgtcaaatg tatggcgtta atcttcagag tggttatgtt 10200

tcacgcgcct gcagaaatgt cttgctggtt ggttcctttc tgactttctt ttggtcagaa 10260

ttagtctcct acactaagtt cttttgggta aatcctggtt atgtcacacc tatgtttgcg 10320

tgtttgtcat tgttgtcctc acttttgatg ttcacactca agcataagac attgtttttc 10380

caggtcttct taatacctgc tctgattgtt acatcttgca ttaatttggc atttgatgtt 10440

gaagtctaca actatttggc agagcatttt gattaccatg tttctcttat gggttttaat 10500

gcacaaggtc ttgttaatat ttttgtctgc tttgttgtta ccattttaca cggcacatac 10560

acatggcgct ttttttatac acccgtgagt tctgtcactt atgtggtagc tttgctgact 10620

gcggcatata actatttcta cgctagtgac actcttagtt gtgctatgac actattcgct 10680

agtgtgactg gcaactggtt tgttggtgct gtttgttata aagctgctgt ttatatggcc 10740

ttgaggtttc ctacttttgt ggctattttt ggtgatatta agagtgttat gttttgttac 10800

cttgtgttgg gttattttac ctgttgcgtc tatggtattc tctactggtt caacaggttc 10860

tttaaggtta gtgtaggtgt ctatgactat actgttagtg ctgctgagtt taagtatatg 10920

gttgctaacg gcttacgtgc accaactgga acacttgatt cactacttct gtctgccaaa 10980

ttgattggta ttggtggtga gcggaatatt aagatctctt ccgttcagtc taaattgact 11040

gatattaagt gtagtaacgt tgtgctttta ggctgtctct ctagcatgaa tgtctcagca 11100

aattcaacag aatgggccta ttgtgttgac ttgcataaca agatcaactt gtgtaatgac 11160

ccagaaaaag cgcaggaaat gctacttgct ttgttggcat ttttccttag taagaatagt 11220

gcttttggtt tagatgactt attggaatcc tattttaatg acaatagtat gttgcagagt 11280

gttgcatcta cttatgtcag tttgccttct tatgtcattt atgaaagtgc acgccaacag 11340

tatgaagatg ctgttaataa tggttctcca cctcagttgg ttaagcaatt gcgtcatgct 11400

atgaatgtag caaagagcga atttgaccgt gaggcttcta ctcagcgtaa gcttgataga 11460

atggcggaac aggctgcagc acagatgtac aaagaggcac gagcagtcaa taggaagtcc 11520

aaagttgtaa gtgctatgca ctcactgctt tttggtatgt tgagacgttt ggatatgtct 11580

tctgtagaca ccattctcaa cttggcaaag gatggggttg tacctctgtc tgtcataccg 11640

gcagtcagtg ctactaagct taacattgtt acttctgata tcgattctta taatcgtatc 11700

cagcgtgagg gatgtgtcca ttacgctggt accatttgga atataattga tatcaaggac 11760

aatgatggca aggtggtaca cgttaaggag gtaaccgcac agaatgctga gtccctgtca 11820

tggcccctgg tcctagggtg tgagcgtatt gtcaagctcc agaataatga aattattcct 11880

ggtaagctga agcagcgctc cattaaggca gaaggagatg gcatagttgg agaaggtaag 11940

gcactttaca ataatgaggg tggacgtact tttatgtatg ctttcatctc ggataaaccg 12000

gacctgcgtg tagttaagtg ggagttcgat ggtggttgta acactattga gctagaacca 12060

ccacgtaagt ttttggtgga ttctcctaat ggtgcacaga tcaagtatct ctactttgtt 12120

cgtaacctta acacgttacg taggggtgct gttcttggct acataggtgc cactgtacgc 12180

ttgcaggctg gtaaacaaac agaacaggct attaactctt cattgttgac actttgcgct 12240

ttcgctgtgg atcctgctaa gacctacatc gatgctgtca aaagtggtca caaaccagta 12300

ggtaactgtg ttaagatgtt ggccaatggt tctggtaatg gacaagctgt tactaatggt 12360

gtggaggcta gtactaacca ggattcatac ggtggtgctt ccgtgtgtct atattgtaga 12420

gcacatgttg agcatccatc tatggatggt ttttgcagac tgaaaggcaa gtacgtacag 12480

gtgccactag gtacagtgga tcctatacgt tttgtacttg agaatgacgt ttgcaaggtt 12540

tgtggttgtt ggctggctaa tggctgcact tgtgacagat ccattatgca aagcactgat 12600

atggcttatt taaacgagta cggggctcta gtgcagctcg actagagccc tgtaacggta 12660

ctgatacaca acatgtgtat cgtgcttttg acatctacaa caaggatgtt gcttgtctag 12720

gtaaattcct caaggtgaac tgtgttcgcc tgaagaattt ggataagcat gatgcattct 12780

atgttgtcaa aagatgtacc aagtctgcga tggaacacga gcaatccatt tatagcagac 12840

ttgaaaagtg tggagccgta gccgaacacg atttcttcac ttggaaggat ggtcgtgcaa 12900

tctatggtaa cgtttgtaga aaggatctta ccgagtatac tatgatggat ttgtgttacg 12960

ctttacgtaa ctttgatgaa aacaattgcg atgttcttaa gagcatttta attaaggtag 13020

gtgcttgtga ggagtcctac ttcaataata aagtctggtt tgaccctgtt gaaaatgaag 13080

acattcatcg tgtctatgca ttgttaggta ccattgtttc acgtgctatg cttaaatgcg 13140

ttaagttctg tgatgcaatg gttgaacaag gtatagttgg tgttgtcaca ttagataatc 13200

aggatcttaa tggtgatttt tatgactttg gtgattttac ttgtagcatc aagggaatgg 13260

gtatacccat ttgcacatca tattactctt atatgatgcc tgttatgggt atgactaatt 13320

gccttgctag tgagtgtttt gttaagagtg atatatttgg tgaggatttc aagtcatatg 13380

accttctgga atatgatttc acggagcata agacatcact ctttaacaag tatttcaagt 13440

attggggact gcaataccac cctaattgtg tggactgcag tgatgagcag tgcatagttc 13500

actgtgctaa cttcaatacg ttgttttcca ctactatacc tattacggca tttggacctt 13560

tgtgtcgcaa gtgctggatt gatggtgttc cactggtaac tacagctggt taccatttta 13620

aacagttagg tatagtttgg aacaatgacc tcaacttaca ttctagcagg ctctctatta 13680

atgaactact ccagttttgt agtgatcctg cattgcttat agcatcatca ccagcccttg 13740

ttgatcagcg tactgtttgc ttttcagttg cagcgctagg tacaggtatg actaaccaga 13800

ctgtgaaacc tggccatttc aataaggagt tttatgactt cttacttgag caaggtttct 13860

tctctgaggg ctctgagctt actttaaagc acttcttctt tgcacagaag ggtgatgcag 13920

ctgttaagga ttttgactac tataggtata atagacccac tgttctggac atttgccaag 13980

cacgcgtcgt gtatcaaata gtgcaacgct attttgatat ctacgaaggt ggttgtatca 14040

ctgctaaaga agtggttgtt acaaacctta acaagagcgc aggctatcct ttgaacaagt 14100

ttggtaaagc tggtctttac tatgagtctt tatcctatga ggaacaggat gaactttatg 14160

cttatactaa gcgtaacatc ctgcccacta tgacacagct caaccttaaa tatgctataa 14220

gtggcaaaga acgtgcacgc acagtgggtg gtgtttcgct tttgacaacc atgactactc 14280

ggcagtatca tcagaaacac cttaagtcca tagttaatac taggggcgct tcggttgtta 14340

ttggtactac taagttttat ggtggttggg acaatatgct taagaacctt attgatggtg 14400

ttgaaaatcc gtgtcttatg ggttgggatt acccaaagtg tgacagagca ctgcccaata 14460

tgatacgcat gatttcagcc atgattttag gctctaagca caccacatgc tgcagttcca 14520

ctgaccgctt tttcaggttg tgcaatgaat tggctcaagt ccttaccgag gttgtttatt 14580

ctaatggagg tttttatttg aagccaggtg gtactacctc tggtgatgca accaccgcat 14640

atgcaaactc agttttcaat atcttccaag cagtaagtgc caatgttaac aaacttctta 14700

gtgttgacag caatgtctgt cataatttag aagttaagca attgcagcgt aagctttatg 14760

agtgctgtta tagatcaacc accgtcgatg accagttcgt cgttgagtat tatggttact 14820

tgcgtaaaca tttctcaatg atgattcttt ctgatgatgg cgttgtttgt tataacaatg 14880

actatgcatc acttggttat gtcgctgatc ttaacgcatt caaggctgtt ttgtattacc 14940

agaacaatgt tttcatgagc gcctctaaat gttggatcga gcctgacatt aataaaggtc 15000

ctcatgaatt ttgctcgcag catactatgc agattgttga taaggatggt acttattacc 15060

ttccttaccc tgatccttca agaatcctct ctgcaggtgt gtttgttgac gacgttgtta 15120

aaactgatgc agttgtattg cttgaacgtt atgtgtcatt ggctatagat gcctacccgt 15180

tatctaagca tgaaaaccct gaatataaga aggtgtttta tgtgcttttg gattgggtta 15240

agcatctgta caaaactttg aatgctggtg tgttagagtc tttttctgtc acacttctgg 15300

aagattctac tgctaaattc tgggatgaga gcttttatgc caacatgtat gagaaatctg 15360

cagttttaca atctgcaggg ctttgtgttg tttgtggctc tcaaactgtt ttacgttgtg 15420

gtgattgtct acggcgtcct atgctttgta ctaagtgtgc ttatgatcat gtcattggaa 15480

caactcacaa gttcattttg gccatcactc catatgtgtg ttgtgcttca gattgtggtg 15540

tcaatgatgt aactaagctc tacttaggtg gtcttagtta ttggtgtcat gaacacaagc 15600

cacgtcttgc attcccgttg tgctctgctg gtaatgtttt tggcttgtac aaaaattctg 15660

ctaccggctc acccgatgtt gaagacttta atcgcattgc tacatccgat tggactgatg 15720

tttctgacta caggttggca aatgatgtca aggactcatt gcgtctgttt gcagcggaaa 15780

ccatcaaggc caaggaggag agcgttaagt catcctacgc ttgtgcaaca ctacatgagg 15840

ttgtaggacc taaagagttg ttgctcaaat gggaagtcgg cagacccaaa ccacccctta 15900

atagaaattc ggttttcact tgttatcata taacgaagaa caccaaattt caaattggtg 15960

agtttgtgtt tgagaaggca gaatatgata atgatgctgt aacatataaa actaccgcca 16020

caacaaaact tgttcctggc atggtttttg tgcttacctc acataatgtt cagccattgc 16080

gtgcaccaac cattgctaat caagaacgtt attccactat acataagttg catcctgctt 16140

ttaacatacc tgaagcttat tctagcttag tgccctatta ccaattgatc ggtaagcaga 16200

agattacaac tatccaggga cctcccggta gtggtaaatc tcactgcgtc atagggctag 16260

gtttgtacta tccaggtgca cgtatagtgt ttacagcttg ttctcatgca gcggtcgatt 16320

cactttgtgt gaaagcctcc actgcttata gcaatgacaa atgttcacgc atcataccac 16380

agcgcgctcg tgttgagtgt tatgacggtt tcaagtctaa taatactagt gctcagtacc 16440

ttttttccac tgtaaacgct ttgccagagt gcaatgcgga cattgttgtg gtggatgagg 16500

tctctatgtg cactaattac gacttgtctg tcataaatca gcgcatcagc tataggcatg 16560

tagtctatgt tggtgaccct caacagctgc ccgcaccacg tgttatgatt tcccgtggca 16620

ctttggaacc aaaggactac aacgttgtca ctcaacgcat gtgtgccctt aagcctgatg 16680

ttttcttgca taagtgttat cgctgtcctg ctgagatagt gcgtactgtg tctgagatgg 16740

tttacgaaaa ccaattcatt cctgttcacc tagatagcaa gcagtgtttt aaaatctttt 16800

gcaagggtaa tgttcaggtg gagaatggtt caagcattaa tcgcaggcaa ttggatgttg 16860

tgcgtatgtt tttggctaaa aaccctaggt ggtcaaaggc tgtttttatt tctccttata 16920

acagccagaa ttatgttgcc agccgcatgc taggtttaca aattcagaca gttgactcat 16980

cccagggtag tgagtatgac tatgtcattt atacacaaac ttcagatact gcccatgcct 17040

gtaatgttaa caggtttaat gttgccatca caagggctaa gaaaggcata ttatgtataa 17100

tgtgcgatag gtcccttttt gatgtgctta aattttttga gcttaaattg tctgatttgc 17160

aggctaatga gggttgtggt ctttttaaag actgtagcag aggtgatgat ctgttgccac 17220

catctcacgc taataccttc atgtctttag cggacaattt taagactgat caagatcttg 17280

ctgttcaaat aggtgttaat ggacccatta agtatgagca tgttatctcg tttatgggct 17340

tccgttttga tatcaacata cccaaccatc acactctctt ttgcacacgc gattttgcca 17400

tgcgcaatgt tagaggttgg ttgggttttg acgttgaagg agcacatgtt gttggctcta 17460

acgtcggtac aaatgtccca ttgcaattag ggttttctaa cggtgttgat tttgttgtca 17520

gacctgaagg ttgcgttgta actgagtctg gtgactacat taaacccgtc agagctcgtg 17580

ctccaccagg ggaacaattt gcacaccttt tgcctctact taaacgcggc caaccatggg 17640

atgtggttcg taagcgtata gtgcaaatgt gtagtgacta cctggccaac ctatcagaca 17700

tactaatttt tgtgttgtgg gctggtggtt tggagttgac aactatgcgt tattttgtca 17760

agattggacc aagtaagagt tgtgattgtg gtaaggttgc tacttgttac aatagtgcgc 17820

tgcatacgta ctgttgtttc aaacatgccc ttggttgtga ttacctgtac aatccatact 17880

gtattgatat acagcagtgg ggatacaagg gatcacttag ccttaaccac catgagcatt 17940

gtaatgtaca tagaaacgag catgtggctt ctggtgatgc cataatgact cgctgtctgg 18000

ccatacatga ttgctttgtc aagaacgttg actggtccat cacataccca tttattggta 18060

atgaggctgt tattaataag agcggccgaa ttgtgcaatc acacaccatg cggtcagttc 18120

ttaagttata caatccgaaa gccatatatg atattggcaa tcctaagggc attagatgtg 18180

ccgtaacgga tgctaagtgg ttttgctttg acaagaatcc tactaattct aatgtcaaga 18240

cattggagta tgactatata acacatggcc aatttgatgg gttgtgcttg ttttggaatt 18300

gcaatgtaga catgtatcca gaattttctg tggtctgtcg ttttgacact cgctgtaggt 18360

caccactcaa cttggagggt tgtaatggtg gttcactgta tgttaataat catgcattcc 18420

atacaccggc ttttgacaag cgtgcttttg ctaagttgaa gccaatgcca tttttctttt 18480

atgatgatac tgagtgtgac aagttacagg actccataaa ctatgttcct cttagggcta 18540

gtaactgcat tactaaatgt aatgttggtg gagctgtctg tagtaagcat tgtgctatgt 18600

atcatagcta tgttaatgct tacaacactt ttacgtcggc gggctttact atttgggtgc 18660

ctacttcgtt tgacacctat aatctgtggc agacatttag taacaatttg caaggtcttg 18720

agaacattgc tttcaatgtc gtaaagaaag gatcttttgt tggtgccgaa ggtgaacttc 18780

ctgtagctgt ggttaatgac aaagtgctcg ttagagatgg tactgttgat actcttgttt 18840

ttacaaacaa gacatcacta cccactaacg tagcttttga gttgtatgcc aagcgtaagg 18900

taggactcac cccacccatt acgatcctac gtaacttggg tgtagtttgt acatctaagt 18960

gtgtcatttg ggactatgaa gccgaacgtc cacttactac ttttacaaag gatgtttgta 19020

aatataccga ctttgagggt gacgtctgta cactctttga taacagcatt gttggttcat 19080

tagagcgatt ctctatgacc caaaatgctg tgcttatgtc acttacagcc gttaaaaagc 19140

ttactggcat aaagttaact tatggttatc ttaatggtgt cccagttaac acacatgaag 19200

ataaaccttt tacttggtat atttacacta ggaagaacgg caaattcgag gactatcctg 19260

atggctattt tacccaaggt agaacaaccg ctgattttag ccctcgtagc gatatggaaa 19320

aggacttcct aagtatggat atgggtctgt ttattaacaa gtacggactt gaagattacg 19380

gctttgagca cgttgtgtat ggtgatgttt caaaaaccac ccttggtggt ttacatctac 19440

taatttcgca ggtgcgtctg tcctgtatgg gtgtgcttaa aatagacgag tttgtgtcta 19500

gtaatgatag cacgttaaag tcctgtactg ttacatatgc tgataaccct agtagtaaga 19560

tggtttgcac gtatatggat ctcctgcttg acgattttgt cagcattctt aaatcgttgg 19620

atttgagcgt tgtatctaaa gttcatgaag ttatggtcga ttgtaaaatg tggaggtgga 19680

tgttgtggtg taaggatcat aaactccaga cattttatcc gcaacttcag gccagtgaat 19740

ggaagtgtgg ttattccatg ccttctattt acaagataca acgtatgtgt ttagaacctt 19800

gcaatctcta taactatggc gctggtatta agttacctga tggcattatg tttaacgtag 19860

ttaaatacac acagctttgt caatatctca atagcaccac aatgtgtgta ccccatcaca 19920

tgcgtgtgct acatcttggt gctggctccg ataagggtgt tgcacctggc acggctgtct 19980

tacgacgttg gttgccactg gatgccatta tagttgacaa tgatagtgtg gattacgtta 20040

gcgatgctga ttatagtgtt acaggagatt gctctacctt atacctgtca gataagtttg 20100

acttagttat atctgatatg tatgatggta agattaaaag ttgtgatggg gagaacgtgt 20160

ctaaagaagg cttctttccc tatattaatg gtgtcatcac tgaaaagttg gcacttggtg 20220

gtactgtggc tattaaggtg acggagttta gttggaataa gaagttgtat gaactcattc 20280

agaagtttga gtattggaca atgttctgta ccagtgttaa cacgtcatcg tcagaggcat 20340

ttttaattgg tgttcactat ttaggtgatt ttgcaagtag cgctgttatt gatggcaaca 20400

ctatgcatgc caattatatc ttctggcgta attccacaat tatgactatg tcttacaata 20460

gtgtacttga tttaagcaag ttcaattgta agcataaggc tacagttgtt attaatttaa 20520

aggattcatc cattagtgat gttgtgttag gtttgttgaa gaatggtaag ttgctagtgc 20580

gtaataatga cgccatttgt ggtttttcta atcatttggt caacgtaaac aaatgaagtc 20640

tttaacttac ttctggttgt tcttaccagt actttcaaca cttagcctac cacaagatgt 20700

caccaggtgc tcagctaaca ctaattttag gcggttcttt tcaaaattta atgttcaggc 20760

gcctgcagtt gttgtactgg gcggttatct acctattggt gaaaaccagg gtgttaattc 20820

aacttggtac tgtgctggcc aacatccaac tgctagtggc gttcatggta tctttcttag 20880

ccatattaga ggtggtcatg gctttgagat tggcatttcg caagagcctt ttgaccctag 20940

tggttaccag ctttatttac ataaggctac taatggtaac actaatgcta ctgcgcgatt 21000

gcgcatttgc cagtttccca gcattaaaac attgggcccc actgctgata acgatgttac 21060

aacaggtcgt aactgcctat ttaacaaagc catcccagct catatgagtg aacatagtgt 21120

tgtcggcata acatgggata atgatcgtgt cactgtcttt tctgacaaga tctatcattt 21180

ttattttaaa aatgattggt cccgtgttgc gacaaagtgt tacaacagtg gaggttgtgc 21240

tatgcaatat gtttacgaac ccacttacta catgcttaat gttactagtg ctggtgagga 21300

tggtatttct tatcaacact gtacagctaa ttgcattggt tatgctgcca atgtatttgc 21360

tactgagccc aatggccaca taccagaagg ttttagtttt aataattggt ttcttttgtc 21420

caatgattct actttggtgc atggtaaggt ggtttccaac caaccattgt tggtcaattg 21480

tcttttggcc atgcctaaga tttatggact aggccaattt ttctccttca atcaaacgat 21540

cgatggtgtt tgtaatggag cttctgcgca gcgtgcacca gaggctctga ggtttaatat 21600

taatgatacc tctgtcattc ttgctgaagg ctcaattgta cttcatactg ctttaggaac 21660

aaatttttct tttgtttgca gtaattcttc agatcctcat ttagctacct tcaccatacc 21720

tctgggtgcc acccaagtac cctattattg ttttcttaaa gtggatactt acaactccac 21780

tgtttataaa ttcttggctg ttttacctcc taccgtcagg gaaattgtca tcaccaagta 21840

cggtgatgtt tatgtcaatg ggtttggcta cttgcatctc ggtttgttgg atgctgttac 21900

aattaatttc actggtcatg gcactgacga tgacgtttct ggtttttgga ccatagcatc 21960

gactaatttt gttgatgcac ttattgaagt tcaaggaact gccattcagc gtattcttta 22020

ttgtgatgat cctgttagcc aacttaagtg ttctcaggtt gcttttgacc ttgacgatgg 22080

tttttaccct atttcctcta caaaccttct gagtcatgaa cagccaactt cttttgttac 22140

tttgccatca tttaatgatc attcttttgt taatattact gtctctgctg cttttggcgg 22200

tcatagtggt gccaacctta ttgcatctga cactactatc aatgggttta gttctttctg 22260

tgttgatact agacaattta ccatttcact gttttataac gttacaaaca gttatggtta 22320

cgtgtctaaa tcacaggaca gtaattgccc ttttaccttg caatctgtta atgattacct 22380

gtcttttagc aaattttgtg tttctaccag ccttttggct agtgcttgta ccatagatct 22440

ttttggttac cctgagtttg gtagtggtgt taagttcacg tccctttact ttcaattcac 22500

aaagggtgag ttgattactg gcacgcctaa accacttgaa ggtgttacgg acgtttcttc 22560

tatgactctg gatgtgtgta ccaagtatac tatctatggc tttaaaggtg agggtattat 22620

tacccttaca aattctagtt ttttggcagg tgtttattac acatctgatt ctggacagtt 22680

gttagctttt aagaatgtca ctagtgatgc tgtttattct gttacgccat gttctttttc 22740

agagcaggct gcatatgttg atgatgatat agtgggtgtt atttctagtt tgtctagctc 22800

cacttttaac agtactaggg agttgcctgg tttcttctac cattctaatg atggctctaa 22860

ttgtacagag cctgtgttgg tgtatagtaa cataggtgtt tgtaaatctg gcagtattgg 22920

ctatgtccca tctcagtctg gtcaagtcaa gattgcaccc acggttactg ggaatattag 22980

tattcccacc aactttagta tgagtattag gacagaatat ttacagcttt acaacacgcc 23040

tgttagtgtt gattgtgcta catatgtttg taatggtaac tctcgttgta aacaattact 23100

cacccagtac actgcagcat gtaagaccat agagtcagca ttacaactca gcgctaggct 23160

tgagtctgct gaagtcaact ctatgcttac tatttctgaa gaggctctac agttagctac 23220

catcagttcg tttaatggtg atgggtataa ttttactaat gtgctgggtg tttccgtgta 23280

tgaccctgca agtggcaggg tggtacaaaa aaggtctttt attgaagacc tgctttttaa 23340

taaagtggtt actaatggcc ttggtactgt tgatgaagac tataagcgct gttctaatgg 23400

tcgctctgtg gcagatctag tctgtgcaca gtattactct ggtgtcatgg tactacctgg 23460

tgttgttgac gctgagaagc ttcacatgta tagtgcgtct ctcatcggtg gtatggcgct 23520

aggaggtttt acttctgcag cggcattgcc ttttagctat gctgttcaag ctagactcaa 23580

ttatcttgct ctacagacgg atgttctaca gcggaaccag caaatgcttg ctgagtcttt 23640

taactctgct attggtaata taacttcagc ctttgagagt gttaaagagg ctattagtca 23700

aacttccaag ggtttgaaca ctgtggctca tgcgcttact aaggttcaag aggttgttaa 23760

ctcgcagggt gcagctttga ctcaacttac cgtacagctg caacacaact tccaagccat 23820

ttctagttct attgatgaca tttactctcg actggacatt ctttcagccg atgttcaggt 23880

tgaccgtctc atcaccggca gattatcagc acttaatgct tttgttgctc aaaccctcac 23940

taagtatact gaggttcagg ctagcaggaa gctagcacag caaaaggtta atgagtgcgt 24000

taaatcgcaa tctcagcgtt atggtttttg tggtggtgat ggcgagcaca ttttctctct 24060

ggtacaggcc gcacctcaag gcctgctgtt tttacacaca gtacttgtac cgggtgactt 24120

tgtaaatgtt attgccatcg ctggcttatg tgttaacgat gaaatttcct tgactctacg 24180

tgagcctggc ttagtcttgt ttacgcatga acttcaagat actgcgacgg aatattttgt 24240

ttcatcgcga cgtatgtatg aacctagaaa acctaccgtt ggtgattttg ttcaaattga 24300

gagttgtgtg gtcacctatg tcaatttgac tagagaccaa ctaccagaag taatcccaga 24360

ttacatcgat gttaacaaaa cacttgatga gattttagct tctctgccca atagaactgg 24420

tccaagtctt tctctagatg tttttaatgc cacttatctt aatctcactg gtgaaattgc 24480

agatttagag cagcgttcag agtctctccg taatactaca gaagagctcc aaagtcttat 24540

atataatatc aacaacacac tagttgacct tgagtggctc aaccgagttg agacatatat 24600

caagtggccg tggtgggttt ggttgattat ttttattgtt ctcatttttg ttgtgtcatt 24660

actagtgttc tgctgcattt ccacgggttg ttgtggatgc tgcggctgct gtggtgcttg 24720

tttttcaggt tgttgtaggg gtcctagact tcaaccttac gaagcttttg aaaaggtcca 24780

cgtgcagtga tgtttcttgg actttttcaa tacacgattg acacagtcgt caaagatgtc 24840

tctaagtctg ccaacttgtc ttcggacgct gtccaagagt tggagcttaa tgtagttcca 24900

attagacaag cttcaaatgt gactggtttt cttttcacca gtgtttttat ttacttcttt 24960

gcactgttta aagcttcttc tttgaggcgc aattatgtta tgttggcagc gcgttttgct 25020

gtcatctttc tttattgccc acttttatat tactgtggtg catttttaga tgcaactatt 25080

atctgttgca cacttattgg caggctcttt ttagtctgct tttattcctg gcgctataaa 25140

aatgcgctct ttattatctt taatactact acactttctt ttctcaatgg taaagcagct 25200

tattacggca aatccattgt gattctagaa ggtggtgatc attacatcac ttttggcaac 25260

tctttcgttg ctttcgttag tagcattaac ttgtatctag ctatacgtgg gcggcaagaa 25320

gctgacctac atctgttgcg aactgttgag cttcttgatg gcaagaagct ttatgtcttt 25380

tcgcaacatc aaattgttgg cattactaat gctgcatttg actcaattca actagacgag 25440

tatgctacaa ttagtgaatg ataatggtct agtagttaat gttatacttt ggcttttcgt 25500

actctttttc ctgcttatta taagcattac tttcgtccaa ttggttaatc tgtgcttcac 25560

ttgtcaccgg ttgtgtaata gcgcagttta cacacctata gggcgtttgt atagagttta 25620

taagtcttac atgcaaattg accccctccc cagtactgtt attgacgtat aaacgaaata 25680

tgtctaacgg ttctattccc gttgatgagg tgattgaaca ccttagaaac tggaatttca 25740

catggaatat catactgacg atactacttg tagtgcttca gtatggccat tacaagtact 25800

ctgcgttctt gtatggtgtc aagatggcta ttctatggat actttggcct cttgtgttgg 25860

cactttcact ttttgatgca tgggctagct ttcaggtcaa ctgggtcttt tttgctttca 25920

gcatccttat ggcttgcatc actcttatgc tgtggataat gtactttgtc aatagcattc 25980

ggttgtggcg caggacacat tcttggtggt ctttcaatcc tgaaacagac gcgcttctca 26040

ctacttctgt gatgggccga caggtttgca ttccagtgct tggagcacca actggtgtaa 26100

cgctaacact ccttagtggt acattgcttg tagagggcta taaggttgct actggcgtac 26160

aggtaagtca attacctaat ttcgtcacag tcgccaaggc cactacaaca attgtctatg 26220

gacgtgttgg tcgttcagtc aatgcttcat ctagcactgg ttgggctttc tatgtccggt 26280

caaaacacgg cgactactca gctgtgagta atccgagtgc ggttctcaca gatagtgaga 26340

aagtgcttca tttagtctaa acagaaactt tatggcttct gtcagcttcc aggatcgtgg 26400

ccgcaaacgg gtgccattat ccctctatgc ccctcttagg gttactaatg acaaacccct 26460

ttctaaggta cttgcaaaca atgctgtacc cactaataaa ggaaataagg accagcaaat 26520

tggatactgg aatgagcaaa ttcgctggcg catgcgccgt ggtgagcgaa ttgagcaacc 26580

ttccaattgg catttctact acctcggaac aggacctcac gccgacctcc gctataggac 26640

tcgtactgag ggtgttttct gggttgctaa agaaggcgca aagactgaac ccactaacct 26700

gggtgtcaga aaggcgtctg aaaagccaat cattccaaat ttctctcaac agcttcccag 26760

cgtagttgag attgttgaac ctaacacacc tcctacttca cgtgcaaatt cacgtagcag 26820

gagtcgtggt aatggcaaca acaggtccag atctccaagt aacaacagag gcaataacca 26880

gtcccgcggt aattcacaga atcgtggaaa taaccagggt cgtggagctt ctcagaacag 26940

aggaggcaat aataataaca ataacaagtc tcgtaaccag tccaagaaca gaaaccagtc 27000

aaatgaccgt ggtggaatga catcacgcga tgatctggtg gctgctgtca aggatgccct 27060

taaatctctg ggtattggag aaaatcctga taggcttaag caacaacaga agcctaagca 27120

ggaaaagtct gacaacagcg gcaaaaatac acctaagaag aacaaatcca gggccacttc 27180

gaaggaacgt gacctcaagg acatcccaga gtggaggcga attcccaagg gcgaaaatag 27240

cgtagcagct tgcttcggac ccaggggggg cttcaaaaat tttggagatg cggagtttgt 27300

cgaaaaaggt gttgatgcct caggctatgc tcagatcgcc agtttggcac caaatgttgc 27360

agcattgctc tttggtggta atgtggctgt tcgcgagcta gcggactctt acgagattac 27420

atacaattat aaaatgactg tgccaaagtc tgatccaaat gttgagcttc ttgtttcaca 27480

ggttgatgca tttaaaactg gtaatgcaaa accccagaga aagaaggaaa agaagaacaa 27540

gcgtgaaacc acgcagcagc ttaatgaaga tgccatctac gatgacgtgg gtgtgccatc 27600

tgatgtgact catgccaatt tggaatggga cacagctgtt gatggtggtg acacggccgt 27660

tgaaattatc aacgagatct tcgatacagg aaattaaata atgtttgact ggcttatcct 27720

ggctatgtcc cagggtagtg ccattacact gttattactt agtgtttttc tagcgacttg 27780

gctgctgggc tatggctttg ccctctaact agcggtcttg gtcttgcaca caacggtaag 27840

ccagtggtaa tgtcagtgca agaaggatat taccatagca ctgtcacgag gggaacgcag 27900

taccttttca tctaaacctt tgcacgagta atcaaagatc cgcttgacga gcctatatgg 27960

aagagcgtgc caggtatttg actcaaggac tgttagtaac tgaagacctg acggtgttga 28020

tatggataca caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 28064

Claims

1. A method for constructing porcine epidemic diarrhea virus infectious cDNA clone is characterized by comprising the following steps:

(2) pYES1L-vector as template, SEQ ID No.: 15-16 is used as a primer, and a linearized vector pYES1L is obtained by amplification, wherein the nucleotide sequence of pYES1L-vector is shown in SEQ ID No.: 23 is shown;

2. The method for constructing the porcine epidemic diarrhea virus infectious cDNA clone according to claim 1, wherein the genome sequence of the porcine epidemic diarrhea virus is as set forth in SEQ ID No.: as shown at 24.

3. The porcine epidemic diarrhea virus infectious cDNA clone constructed by the method for constructing porcine epidemic diarrhea virus infectious cDNA clone of any one of claims 1-2.

4. The method for constructing the porcine epidemic diarrhea virus expressing green fluorescent protein based on CRISPR/Cas9 gene editing technology comprises the following steps:

(1) EGFP gene as template, SEQ ID No.: performing PCR amplification by taking the sequence shown by 19-20 as a primer to obtain an EGFP gene segment;

(2) cleaving the porcine epidemic diarrhea virus infectious cDNA clone obtained in claim 1 with Cas9 Nuclear and two guide RNAs, the sequence of which is as set forth in SEQ ID No.: 21-22, obtaining a porcine epidemic diarrhea virus infectious cDNA cloning framework lacking ORF 3;

(3) EGFP and the porcine epidemic diarrhea virus infectious cDNA clone framework segment lacking ORF3 are mixed, in vitro homologous recombination is carried out, and the mixture is transformed into DH10B electric shock competence to obtain recombinant plasmid, namely the porcine epidemic diarrhea virus clone expressing green fluorescent protein.

5. The porcine epidemic diarrhea virus clone expressing green fluorescent protein, which is constructed by the porcine epidemic diarrhea virus construction method expressing green fluorescent protein based on CRISPR/Cas9 gene editing technology of claim 4.

6. Use of the porcine epidemic diarrhea virus infectious cDNA clone of claim 3, the porcine epidemic diarrhea virus clone expressing green fluorescent protein of claim 5 for rescuing viruses.

7. Use according to claim 6, characterized in that the transfection reagent Lipofectamine is used^TM3000 cloning of the porcine epidemic diarrhea virus infectious cDNA according to claim 3 or expressing green fluorescence according to claim 5The porcine epidemic diarrhea virus clone of the protein and the helper plasmid are cotransfected with VeroCCL-81 cells, and virus supernatant is collected when typical cytopathic effect occurs, so that rescued rPEDV is obtained.

8. The use of claim 6, wherein the mass ratio of the porcine epidemic diarrhea virus infectious cDNA clone or porcine epidemic diarrhea virus clone expressing green fluorescent protein to helper plasmid is 4: 1.

9. The use according to claim 7, wherein the helper plasmid is constructed as follows: carrying out PCR amplification by taking porcine epidemic diarrhea virus infectious cDNA clone as a template and SEQ ID NO. 17-18 as a primer to obtain an N gene fragment, and inserting the N gene fragment between SacI and XhoI sites of a pCAGGS vector to obtain an auxiliary plasmid.