Porcine epidemic diarrhea virus recombinant strain, construction method and application

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus recombinant strain, a construction method and application. The S gene sequence of the existing epidemic porcine epidemic diarrhea virus PEDV CH / SX / 2016 strain is replaced by the S2015 gene sequence shown in SEQ ID NO. 1 to construct a PEDV CH / SX / 2016 strain infectious clone. A porcine epidemic diarrhea virus recombinant strain rCH / SX / 2016-S 2015 is successfully rescued by transfecting cells with a plasmid containing the infectious cDNA shown in SEQ ID NO. 4. The problem that the original strain cannot be successfully rescued by reverse genetics engineering is solved, and the recombinant virus strain rCH / SX / 2016-S 2015 has an activity comparable to that of the original strain, and can be used for preparing a PEDV diagnostic reagent and / or vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus recombinant strain, a construction method and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family of Coronaviridae and is a member of the alpha coronavirus genus. The genome of the virus is a single-stranded positive-strand RNA virus with a capsid, and the genome size is about 28Kb, which contains 5'UTR, at least 7 open reading frames and 3'UTR. The 7 open reading frames mainly encode ORF1a, ORF1b, structural proteins S, E, M, N and auxiliary protein ORF3. According to the sequence of the S gene of PEDV, PEDV is divided into G1 and GII types. G1 is further divided into three subgroups: G1a, G1b and G1c. G1a includes CV777, DR13 and other historical strains. G1b includes OH815, USA / Ohio126 / 2014 and GER / L00719 / 2014 and some strains derived from G1a and G2 recombination. G1c is a new type, represented by the TW / Yunli550 / 2018 strain, which is a strain derived from the recombination of the Taiwan G2b strain and the wild-type G1a strain. G2 is divided into G2a and G2b. G2a is like the KNU-1302 / Korea / 2013 strain. G2b is like the USA / Illinois262 / 2014 strain.

[0003] Before the emergence of epidemic strains, attenuated live vaccines prepared from classic strains CV777 and DR13 strains can well control the epidemic of PED. Among them, inactivated vaccines mainly include TGE-PED bivalent inactivated vaccine and PED-TGEV-RoV trivalent inactivated vaccine. Live attenuated vaccines include TGE-PED-RoV (attenuated Huatuo strain + attenuated CV777 strain + NX strain) trivalent attenuated vaccine, TGE-PED (attenuated WH-1 strain + attenuated AJ1102 strain) bivalent attenuated vaccine and TGE-PED (HB08 strain + ZJ08 strain) bivalent attenuated vaccine. However, since 2010, vaccines based on CV777 and DR13 cannot produce cross protection against highly pathogenic epidemic strains in China, and the clinical application effect is not satisfactory.

[0004] Currently, the most effective method for preventing PED is immunization, and the immune protection induced by live attenuated vaccines is the most effective way to combat PEDV infection. Therefore, developing safe and effective vaccines is crucial to prevent the emergence of PED. Reverse genetics can modify and alter viruses to produce live attenuated vaccines, and this system is currently a powerful tool for producing live attenuated PEDV vaccines. In 2013, the reverse genetics system of the classic Korean PEDV vaccine strain DR13 was successfully constructed for the first time based on the targeted RNA recombination method. In 2015, an infectious clone of the classic Thai PEDV strain AVCT12 was successfully constructed based on bacterial artificial chromosomes (BAC). In 2016, researchers first constructed an infectious clone of the highly pathogenic US PEDV strain PC22A using an in vitro linking method and transcribed it in vitro to generate infectious viral RNA. Using the same method, an infectious cDNA clone of the Chinese variant PEDV strain AH2012 / 12 was also successfully constructed.

[0005] The PEDV CH / SX / 2016 strain can cause severe diarrhea and vomiting, typical symptoms of PED, in two-week-old piglets, resulting in a near 100% mortality rate in one pig farm. Evolutionary analysis indicates that it is an epidemic strain, but multiple attempts to isolate it have failed, and the PEDV CH / SX / 2016 strain obtained using a reverse genetics system has also failed to be rescued, affecting the development of diagnostic reagents and vaccines for the PEDV CH / SX / 2016 strain.

[0006] This invention utilizes a reverse genetics system to replace the S gene of the PEDV CH / SX / 2016 strain, constructing an infectious clone and successfully rescuing and obtaining a PEDV recombinant virus strain rCH / SX / 2016-S. 2015 This solved the problem that the original strain could not be successfully rescued using reverse genetics engineering, and the resulting recombinant viral strain rCH / SX / 2016-S 2015 It can be used to prepare PEDV diagnostic reagents and / or vaccines. Summary of the Invention

[0007] This invention addresses the technical problem that the PEDV CH / SX / 2016 strain cannot be isolated and cannot be rescued by directional genetics. By genetically modifying the PEDV CH / SX / 2016 strain, a recombinant porcine epidemic diarrhea virus strain, rCH / SX / 2016-S, was obtained. 2015 This solves the problem that the original strain could not be successfully rescued using reverse genetics engineering, including the following:

[0008] In a first aspect, the present invention provides a recombinant strain of porcine epidemic diarrhea virus, wherein the recombinant virus strain is obtained by replacing the S gene sequence of PEDV CH / SX / 2016 strain with the S2015 gene sequence shown in SEQ ID NO.1; the gene accession number of the PEDV CH / SX / 2016 strain is GenBank No. MT787025; the S gene sequence of the PEDV CH / SX / 2016 strain is shown in SEQ ID NO.2.

[0009] In a second aspect, the present invention provides the use of the recombinant porcine epidemic diarrhea virus strain described in the first aspect in the preparation of PEDV diagnostic reagents and / or vaccines.

[0010] Thirdly, the present invention provides an infectious clone of PEDV CH / SX / 2016 strain, the full-length cDNA sequence of which is shown in SEQ ID NO.3.

[0011] Fourthly, the present invention provides the use of the infectious clones described in the third aspect above in the preparation of PEDV diagnostic reagents, and / or PEDV recombinant virus strains, and / or PEDV vaccines.

[0012] Fifthly, the present invention provides a recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S 2015 The recombinant viral strain rCH / SX / 2016-S 2015 It was obtained by rescuing cells transfected with the infectious clone of PEDV CH / SX / 2016 strain described in the third aspect above.

[0013] Preferably, the cells are African green monkey kidney cells (Vero ccl81).

[0014] Sixthly, the present invention provides the recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S described in the fifth aspect above. 2015 Application in the preparation of PEDV diagnostic reagents and / or vaccines.

[0015] In a seventh aspect, the present invention provides a method for preparing the recombinant strain of porcine epidemic diarrhea virus as described in the fifth aspect above, characterized in that the method comprises:

[0016] pBac-CH / SX / 2016-S 2015Construction of infectious clones: Using genetic engineering techniques, the S gene in the gene sequence of PEDV CH / SX / 2016 strain was replaced with the S2015 gene sequence shown in SEQ ID NO.1 to obtain the recombinant viral strain gene sequence, which was then ligated into the pBeloBac11 plasmid to construct pBac-CH / SX / 2016-S 2015 Infectious clones;

[0017] pBac-CH / SX / 2016-S 2015 Transfection rescue: The obtained pBac-CH / SX / 2016-S 2015 Infectious clones were transfected into Vero ccl81 cells for rescue, yielding the recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S. 2015 .

[0018] Preferably, the pBac-CH / SX / 2016-S 2015 The method for constructing infectious clones is as follows:

[0019] (1) PEDV CH / SX / 2016 restriction fragments: restriction sites were introduced into the full-length sequence of CH / SX / 2016 to digest it into 9 fragments: 5', XbaI-BamHI, BamHI-pmlI, PmlI-PacI, PacI-AvrII, AvrII-kasI, KasI-Bsu36I, Bsu36I-BstBI and 3'.

[0020] (2) Construction of recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3': Construct the vector pUC17-CH / SX / 2016-5'-3' with the nucleotide sequence shown in SEQ ID NO.4; ligate the XbaI-BamHI, BamHI-pmlI, and PmlI-PacI fragments described in step (1) into the vector pUC17-CH / SX / 2016-5'-3' in sequence; ligate the digested fragments into the pBeloBac11 plasmid by digestion with RsrII and sfiI to obtain the recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3';

[0021] (3) Constructing the recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI: Amplify the Bsu36I-BstBI fragment described in step (1). During amplification, the upstream primers sequentially introduce restriction sites SfiI, PacI, AvrII, KasI, and Bsu36I, and the downstream primers sequentially introduce restriction sites BstB1 and RsrII. Using the restriction sites SfiI and RsrII, the Bsu36I-BstBI fragment is ligated into the pBeloBac11 vector to construct the vector pBAC-CH / SX / 2015-Bsu36I-BstBI. Then, the KasI-Bsu36I, AvrII-kasI, and PacI-AvrII fragments described in step (1) are sequentially ligated into the vector pBAC-CH / SX / 2015-Bsu36I-BstBI to obtain the recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI.

[0022] (4) Construct pBac-CH / SX / 2016-S 2015 Infectious clones: The Bsu36I-BstBI fragment and AvrII-Bsu36I fragment described in step (1) were fused with the S2015 gene by overlap PCR to obtain the fused Bsu36I-BstBI fragment and AvrII-Bsu36I fragment; the fused Bsu36I-BstBI fragment, AvrII-Bsu36I fragment, and the PacI-AvrII fragment described in step (1) were sequentially ligated into the pBeloBac11 vector to construct the recombinant plasmid pBAC-CH / SX / 2016-S 2015 -PacI-BstBI; the PacI-BstBI fragment was obtained by enzyme digestion and ligated into the plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3' described in step (2) to obtain the full-length plasmid pBAC-CH / SX / 2016-S, which is the infectious clone of the CH / SX / 2016 genome-wide cDNA. 2015 .

[0023] Preferably, the pBac-CH / SX / 2016-S 2015 The transfection rescue method is as follows: pBac-CH / SX / 2016-S 2015 Infectious clones were transfected into Vero ccl81 cells and cultured at 37°C in a 5% CO2 cell incubator until the virus stably induced cytopathic effects, thus obtaining the recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S. 2015 .

[0024] The beneficial effects of this invention are: ① This invention first discovered the problem that the existing prevalent PEDV CH / SX / 2016 strain cannot be naturally isolated and cannot be rescued by conventional reverse genetics; ② Based on the above problems, this invention unexpectedly discovered that after replacing the S gene of the PEDV CH / SX / 2016 strain with the S2015 gene described in this invention, the constructed infectious clone can be successfully rescued to obtain the recombinant strain rCH / SX / 2016-S. 2015 ③ Obtain the recombinant strain rCH / SX / 2016-S 2015 It has the same activity as the parent strain PEDV CH / SX / 2016, but has a lower viral titer. It can be used to prepare PEDV diagnostic reagents and / or vaccines, and can also provide effective assistance for basic research on the pathogenesis mechanism, immune evasion mechanism, cell tropism alteration and tissue tropism mechanism of PEDV strains. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 Identification diagram of cytopathic effect (CPE) caused by the CH / SX / 2016 strain;

[0027] Figure 2 Image of CH / SX / 2016 strain identified by indirect immunofluorescence assay (IFA);

[0028] Figure 3 Schematic diagram of the construction of an infectious clone of the CH / SX / 2016 strain;

[0029] Figure 4 IFA identification image of the rescued strain rCH / SX / 2016;

[0030] Figure 5 Sequencing results of genome markers for the rescue strain rCH / SX / 2016;

[0031] Figure 6 Recombinant strain rCH / SX / 2016-S 2015 Schematic diagram of infectious clone construction;

[0032] Figure 7 Saving the recombinant strain rCH / SX / 2016-S 2015 IFA identification diagram;

[0033] Figure 8 Saving the recombinant strain rCH / SX / 2016-S 2015 The growth curve diagram. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The restriction endonucleases and T4 DNA ligases used in this invention were purchased from NEB; the high-fidelity amplification enzymes used were also purchased from NEB. GXL HS DNA polymerase, cloning vector pMD19-T, and competent JM109 cells were all purchased from TAKARA. The cloning vector pEASY-Blunt (Simple), competent Trans5α cells, and fluorescent secondary antibody were all purchased from Beijing TransGen Biotech Co., Ltd. Fetal bovine serum used in cell culture was from BI, and DMEM culture medium / Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher.

[0036] PEDV IFA was identified using a polyclonal antibody against the PEDV N protein prepared in our laboratory. The Vero cells used for virus isolation were all preserved in our laboratory.

[0037] Example 1: Isolation of PEDV CH / SX / 2016 strain

[0038] (1) Samples of small intestine, intestinal contents, and feces were collected from diarrheal piglets. After soaking in 1×PBS, the samples were ground and the liquid was collected in 1.5 ml EP tubes. The tubes were centrifuged at 6000 g and 4℃ for 10 min. The supernatant was collected, filtered through a 0.22 μm filter membrane, and then frozen for later use. Vero cells were revived and passaged into DMEM medium containing 10% FBS. When the cells reached a suitable density, they were inoculated with PEDV CH / SX / 2016 strain and cultured in a constant temperature incubator at 37℃ and 5% CO2 for 3-4 days. When 80% of the cells showed cytopathic effect (CPE), the cell supernatant was collected to obtain the virus solution.

[0039] (2) CPE Identification: Vero cells were seeded at an appropriate density in 6-well plates for CPE verification of the isolated strain. Virus solution was inoculated into the cell plates and cultured in a suitable environment for 3–4 days, observing changes in cell morphology. When Vero cells exhibited CPE morphologies such as syncytia, shrinkage, or detachment, photographs were taken under a microscope. The results are as follows: Figure 1 As shown: A represents the normal cell morphology, and B represents the morphology of the Vero ccl81 cell inoculated with the CH / SX / 2016 strain.

[0040] (3) IFA identification: Vero cells were seeded onto 24-well plates with pre-placed cell spreaders for indirect immunofluorescence identification. Cells were inoculated according to the method described in step (2), with the uninoculated group serving as a negative control. When significant CPE appeared in the inoculated group, the cell plates were collected, the culture medium was discarded, and the cells were washed once with 1×PBS. 300 μl of 4% paraformaldehyde was added, and the cells were fixed at 37°C for 15 min. The fixative was discarded, and 300 μl of 0.25% Triton X-100 solution was added, and the cells were incubated at 37°C for 15 min to break the membrane. The liquid was carefully discarded, and the cells were washed twice with 1×PBS. 1% BSA solution was added, and the cells were blocked at 37°C for 30 min. The cells were washed three times with 200 μl of 1×PBS, and 300 μl of PEDV diluted 1:2000 was added to each well. Polyclonal antibody against N protein was incubated at 37°C for 1 hour; the cells were washed three times with PBS, and 300 μl of 1:300 diluted fluorescent secondary antibody was added under light-protected conditions, followed by incubation at 37°C for 1 hour; the cells were washed three times with PBS, and the nuclear dye DAPI was added, followed by incubation at 37°C for 5-10 minutes; after washing three times with 1×PBS, the cell slides were mounted onto glass slides and photographed using a fluorescence microscope. Results are as follows: Figure 2 As shown: all cells exhibited blue fluorescence under DAPI excitation light, indicating normal nuclear staining; meanwhile, the normal control group cells and the CH / SX / 2016 infection group showed no fluorescence under FITC excitation light, indicating that the experimental control group met expectations, the experiment was successful, and the results were reliable; this also shows that CH / SX / 2016 did not infect Vero cells, indicating that the PEDV CH / SX / 2016 strain could not be directly isolated.

[0041] Example 2: Construction of a full-length infectious cDNA clone of PEDV CH / SX / 2016 and virus rescue

[0042] (1) Construction of the infectious clone pBac-CH / SX / 2016:

[0043] A schematic diagram of the construction of a full-length cDNA infectious clone of PEDV CH / SX / 2016 is shown below. Figure 3 As shown, the specific steps are as follows:

[0044] Construction of recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3': The full-length genome sequence of PEDV CH / SX / 2016 strain was synthesized (GenBank accession number: MT787025); the full-length PEDV CH / SX / 2016 genome was divided into 9 fragments: 5', XbaI-BamHI, BamHI-pmlI, PmlI-PacI, PacI-AvrII, AvrII-kasI, KasI-Bsu36I, Bsu36I-BstBI, and 3'; a partial sequence of pBeloBac11, the 5' terminal sequence of the virus, and the 3' terminal sequence were synthesized into the vector pUC17, and restriction enzyme sites XbaI, BamHI, PmlI, PacI, and BstBI were inserted to obtain the modified vector pUC17-CH / SX / 2016-5'-3' (gene sequence as shown in SEQ ID). (As shown in NO.4); the XbaI-BamHI and BamHI-PmlI fragment sequences were amplified respectively and sequentially ligated into the modified vector pUC17-CH / SX / 2016-5'-3' to obtain the plasmid pUC17-CH / SX / 2016-5'-XbaI-PmlI-3'; among them, when inserting the BamHI-PmlI fragment, Overlap was used PCR was used to mutate the A at position 3493 to C as a marker to distinguish between the parental CH / SX / 2016 strain and the rescue strain. After correct sequencing, the target fragment of the pUC17-CH / SX / 2016-5'-XbaI-PmlI-3' vector was excised using RsrII and sfiI restriction enzymes and ligated into the pBeloBac11 plasmid to obtain the pBAC-CH / SX / 2016-5'-XbaI-PmlI-3' plasmid. The amplified fragment PmlI-PacI was then ligated into the pBAC-CH / SX / 2016-5'-XbaI-PmlI-3' plasmid using the restriction enzyme sites PmlI and PacI. When inserting the PmlI-PacI fragment, the T at position 7615 was mutated to A using OverlapPCR as a second marker to distinguish between the parental CH / SX / 2016 strain and the rescue strain. The entire first half of the infectious whole-genome cDNA clone of CH / SX / 2016 was obtained from the recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3'; the sequencing results of the rCH / SX / 2016 genome marker are as follows. Figure 5 As shown.

[0045] Construction of recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI: First, the latter half of the Bsu36I-BstBI fragment was amplified. During amplification, the upstream primers introduced SfiI, PacI, AvrII, KasI, and Bsu36I, and the downstream primers introduced BstB1 and RsrII restriction sites. Using the SfiI and RsrII restriction sites, the Bsu36I-BstBI fragment was ligated into the pBeloBac11 vector to construct pB... AC-CH / SX / 2015-Bsu36I-BstBI was then used to ligate the KasI-Bsu36I, AvrII-kasI, and PacI-AvrII fragments into pBAC-CH / SX / 2015-Bsu36I-BstBI according to their respective restriction sites, thus obtaining the entire latter half of the infectious clone of the CH / SX / 2016 genome cDNA, recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI.

[0046] Construction of pBac-CH / SX / 2016: The entire PacI-BstBI fragment was digested from the recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI using the PacI and BstBI restriction sites, and then ligated into the recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3' to obtain the full-length plasmid pBac-CH / SX / 2016, which is the infectious clone of the CH / SX / 2016 genome cDNA. A schematic diagram of the construction is shown below. Figure 3 As shown.

[0047] (2) pBAC-CH / SX / 2016 transfection rescue

[0048] Vero ccl81 cells were stored at a density of 1×10⁻⁶. 6Cells were seeded per well in 6-well cell culture plates. When the cells reached approximately 80% confluence, the full-length clone pBAC-CH / SX / 2016 was transfected into Vero ccl81 cells for rescue, following the instructions of the Lipofectamine 3000 transfection reagent. The specific procedure for transfecting the plasmid was as follows: The culture medium in the 6-well cell culture plate was replaced with fresh 10% FBS DMEM, 2 mL / well; two sterile EP tubes were taken, and 125 μL of opti-MEM medium was added to one EP tube, followed by 3.75 μL of Lipofectamine 3000 transfection reagent, and the mixture was gently pipetted to mix; 125 μL of opti-MEM was also added to the other EP tube, followed by 2.5 μg of plasmid and 5 μL of P3000 reagent, and the mixture was gently pipetted to mix; the opti-MEM mixture containing the plasmid was then added dropwise to the EP tube containing the transfection reagent, and the mixture was incubated at room temperature for 5 min. The static mixture was added dropwise to a 6-well cell culture plate, and then the cell culture plate was placed in a 37°C, 5% CO2 cell culture incubator until CPE appeared.

[0049] (3) IFA identification of the rescue strain rCH / SX / 2016

[0050] healthy Vero ccl81 cells were loaded with 2 × 10 5Cells were seeded per well in a cell culture plate containing 24-well cell spreaders. When the confluence was approximately 80%, rCH / SX / 2016 cells were inoculated and cultured at 37°C in a 5% CO2 incubator. After 36 hours, IFA (Intracytoplasmic Saturation) was performed: the cell culture medium was discarded, the cells were washed three times with PBS, and 200 μL / well was added to 4% paraformaldehyde and incubated at 37°C for 10 min. 200 μL / well was added to 0.25% Triton X-100 to permeate the cell membrane and incubated at 37°C for 10 min. The cells were washed three times with PBS, and 300 μL / well was added to 1% BSA for blocking and incubated at 37°C for 30 min. 200 μL / well was added to a polyclonal antibody (mouse-derived, 1:2000 dilution) against PEDV N protein and incubated at 37°C for 1 hour. The cells were washed three times with PBS, and 200 μL / well was added to a 1:500 dilution of the fluorescent secondary antibody Rhodamine (TRITC) Affini Pure Goat Anti-Mouse. IgG (H+L) was incubated at 37°C for 1 hour, washed three times, and 200 μL / well of DAPI was added for nuclear staining. After incubation at room temperature for 5 minutes and washing with PBS, images were taken using a Leica microscope. The results, shown in Figure 4, indicated that the specific polyclonal antibody against PEDV N protein failed to detect viral proteins in Vero ccl81 cells infected by rCH / SX / 2016, suggesting that rCH / SX / 2016, like its parent strain CH / SX / 2016, does not infect Vero ccl81 cells and cannot be successfully rescued.

[0051] Example 3 pBAC-CH / SX / 2016-S 2015 Construction of infectious clones and virus rescue

[0052] (1) pBAC-CH / SX / 2016-S 2015 Construction of infectious clones

[0053] pBAC-CH / SX / 2016-S 2015 A schematic diagram illustrating the specific construction of an infectious clone is shown below. Figure 6 As shown: First, the S2015 gene sequence (SEQ ID NO.1) was synthesized; the Bsu36I-BstBI fragment was amplified by overlap PCR, and the C-terminus of the S2015 gene was fused with the Bsu36I-BstBI fragment of CH / SX / 2016, and then ligated into the pBAC vector to construct pBAC-CH / SX / 2016-S 2015 -Bsu36I-BstBI; Perform overlap PCR again to amplify the AvrII-Bsu36I fragment, and ligate the C-terminus of the S2015 gene with the AvrII-Bsu36I fragment into pBAC-CH / SX / 2016-S 2015In the -Bsu36I-BstBI vector, pBAC-CH / SX / 2016-S was constructed. 2015 -AvrII-BstBI plasmid; then ligated with PacI-AvrII fragment to construct recombinant plasmid pBAC-CH / SX / 2016-S 2015 -PacI-BstBI, using the restriction enzyme sites PacI and BstBI, the entire latter half of the PacI-BstBI fragment is extracted from pBAC-CH / SX / 2016-S 2015 The PacI-BstBI clone was digested and ligated into the first half of the plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3', resulting in the full-length plasmid pBAC-CH / SX / 2016-S, which is the infectious clone of the CH / SX / 2016 genome. 2015 (SEQ ID NO.3).

[0054] (2) pBAC-CH / SX / 2016-S 2015 Transfection rescue

[0055] According to the method described in Example 2(2), pBAC-CH / SX / 2016-S 2015 To carry out the rescue.

[0056] (3) Saving strain rCH / CH / SX / 2016-S 2015 IFA identification

[0057] The rescue result was detected according to the IFA method described in Example 2 (3).

[0058] Saving strain rCH / CH / SX / 2016-S 2015 The IFA identification results are as follows Figure 7 As shown, rCH / SX / 2016-S was not detected using a specific polyclonal antibody against the PEDV N protein. 2015 Viral proteins in Vero ccl81 cells indicate rCH / SX / 2016-S 2015 The rescue was successful. The recombinant strain rCH / SX / 2016-S was successfully rescued. 2015 The growth curve is as follows Figure 8 As shown, the results indicate that the recombinant strain rCH / SX / 2016-S 2015 The growth curve is consistent with that of the parent strain CH / SX / 2016.

[0059] (4)rCH / SX / 2016-S 2015 Gene sequence sequencing

[0060] For the obtained rCH / SX / 2016-S2015 Gene sequencing revealed that rCH / SX / 2016-S 2015 Based on the full-length sequence of the parent CH / SX / 2016 strain, only the S gene (SEQ ID NO.2) of the parent CH / SX / 2016 strain was replaced with the S2015 gene shown in SEQ ID NO.1, and the other sequences were identical to those of the parent CH / SX / 2016 strain.

[0061] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. sequence list <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences Northwest A&F University <120> A recombinant strain of porcine epidemic diarrhea virus, its construction method and application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4149 <212> DNA <213> Artificial Sequence <400> 1 atgacgcctt taatttactt ctggttgttc ttaccagtac ttctaacact tagcctacca 60 caagatgtca ctaggtgcca gtctactatt aactttaggc ggttcttttc aaaatttaat 120 gttcaggcac ctgccgtcgt tgttttgggt ggttatctac ctagtatgaa ctcttctagc 180 tggtactgtg gcacaggcat tgaaactgat agtggcgttc atggtatttt tctcagttac 240 atcgattctg gtcagggctt tgagattggc atttcgcaag agccgtttga tcctagtggt 300 taccagcttt atttacacaa ggccactaat ggtaacacta gtgctattgc acgactgcgc 360 atttgccagt ttccagataa taaaacattg ggccctactg ttaatgatgt tacaacaggt 420 cgtaactgcc tattcaacaa agccattcca gctttgcagg atggaaaaaa tattgttgtc 480 ggcataacat gggataatga tcgtgtcact gtttttgctg acaagatcta tcatttttat 540 attaaaaatg attggtcccg tgttgcgaca agatgttaca ataaaagaag ttgtgccatg 600 caatatgttt atacacctac ctactacatg cttaatgtta ctagtgcagg tgaggatggc 660 atttactatg aaccttgtac agctaattgc agtggttacg ctgccaatgt atttgccact 720 gattccaatg gccatatacc agaaggtttt agttttaata attggtttct tttgtccaat 780 gactccactt tgttgcatgg taaagtggtt tcaaaccaac ctttgttggt caactgcctt 840 tgggccattc ctaagattta tggactaggc caatttttct cattcaatca aacgatggat 900 ggcgtttgta acggagccgc tgcgcagcgt gccccagagg ctctgaggtt taatattaat 960 gacacttttg tcattcttgc tgaaggctca attgtacttc atactgcttt aggaacaaat 1020 ctttcttttg tttgcagtaa ttcctcagat cctcacaaag ccatctttac catacctttg 1080 ggtgttactg aagtacccta ctattgcttt cttaaagtgg atacttacaa atccactgtt 1140 tataaattct tggctgtttt acctcctact gtcaaggaaa ttgtcatcac caagtacggt 1200 gatgtttatg tcaacgggtt tggctatttg catctcggtt tgttggatgc tgtcacaatt 1260 aatttcactg gtcatggcac tgacgatgac gtttcaggtt tctggaccgt agcatcgact 1320 aattttgttg atgcactcat cgaggttcaa ggaactgcca ttcagcgtat tctttattgt 1380 gatgaccctg ttagccaact taagtgttct caggtttctt ttgaccttga tgatggtttt 1440 taccctattt cttctagaaa ccttctgagt catgaacagc caatttcttt tgttactttg 1500 ccatcattca atgatcattc ttttgttaat attactgtct ctgcggcttt tggtggtcat 1560 agtggtgcca acctcattgc atctgacact actatcaatg ggtttagttc tttctgtgtt 1620 gacactagac aatttaccat tacactgttt tataacgtta caaacagtta tggttatgtg 1680 tctaagtcac aggatagtaa ttgccctttc accttgcaat ctgttaatga ttacctgtct 1740 tttagcaaat tttgtgtttc aaccagcctt ttggctggtg cttgtaccat agatcttttt 1800 ggttaccctg agttcggtag tggtgttaag tttacgtccc tttattttca attcacaaag 1860 ggtgagttga ttactggcac gcctaaacca cttcaaggtg tcacggacgt ttcttttatg 1920 actctggatg tgtgtaccaa gtatactatc tatggcttta aaggtgaggg tattattacc 1980 cttacaaatt ctagcttttt ggcaggtgtt tattatacat ctgattctgg acagttgtta 2040 gcctttaaga atgtcactag tggtgctgtt tattctgtta cgccatgttc tttttcagag 2100 caggctgcat atgttgatga tgatatagtg ggtgttattt ctagtttgtc taactccact 2160 tttaacaata ccagggagtt gcctggtttc ttctaccatt ctaatgatgg ctccaattgt 2220 acagagcctg tgttggtgta tagtaacata ggtgtctgta aatctggcag tattggctat 2280 gtcccacttc aggatggcca agtcaagatt gcacccatgg ttactgggaa tattagtatt 2340 cccaccaact ttagtatgag tattagaaca gaatatttac agctttacaa cacgcctgtt 2400 agtgttgatt gcgttacata tgtttgtaat ggtaactctc gttgtaaaca attactcacc 2460 2520 tctgttgaag ttaacttat gcttactatt tctgaagagg ctctacagtt agctaccatc 2580 agttcgttta atggtgatgg atataacttt actaatgtgc tgggtgtttc cgtgtacgac 2640 cctgcaagtg gcagggtggt acaaaaaggg tcttttttg aagacctgct tttaataaa 2700 gtggttacta atggccttgg tactgttgat gaagactata agcgctgttc taatggtcgc 2760 tctgtggcag atctagtctg tgcgcagtat tactctggtg tcatggtact acctggcgtt 2820 gttgacgctg agaagcttca catgtatagt gcgtctctca tcggtggtat ggcgctagga 2880 ggtcttacta ctgcagcggc attgcctttt agccatgctg ttcaagcgag gctcaattat 2940 cttgctttac agacggatgt tctacagcgc aaccagcaat tgcttgctga gtcttttaac 3000 tctgctattg gtaatataac ttcagccttt gagagtgtta aagaggctat tagtcaaact 3060 tccaatggtt tgaacactgt ggctcatgcg cttactaagg ttcaagaggt tgttaattcg 3120 cagggttcag ctttgaccca acttaccata cagctgcaac acaacttcca agccatttct agttctattg atgacattta ctcccgactg gacattcttt cagccgatgt tcaggttgat 3240. cgtctcatca ccggcagatt atcagcactt aatgcttttg ttgctcaaac cctcactaag tatactgagg ttcaggctag caggagcta gcacagcaaa aggttatga gtgcgtcaaa tcgcaatctc agcgttatgg tttttgtggt ggtgatggcg agcacatctt ctctctggta 3420 caggccgcac ctcagggcct gctgttctta catacagtac ttgtaccggg tgattttgta 3540. aatgttattg ccatcgatgg cttatgcgtt aatggtgata ttgccttgac tctacgtgag cctggcttag tcttgtttac gcatgaactt caaacttata ctgcgacgga atattttgtt 3660. tcatcgcgac gtatgtttga acctagaaaa cctaccgtta gtgattttgt tcaaattgag agttgtttgg tcacctatgt caatctgact agcgaccaac taccagatgt aatcccagat 3720. 3780. tacatcgatg ttaacaaaac acttgatgag attctagctt ctctgcccaa tagattggt cctagtcttc ccctagtgt ttttaatgcc acttatctta atctcactgg tgaaattgca gatttagagc agcgttcaga gtctctccgt aatactacag aagagctccg aagtctcata 3900 tataatatca acaacacact tgttgacctt gagtggctca accgagttga gacatatatc 3960 aagtggccgt ggtgggtttg gttgattatt tttattgttc tcatctttgt tgtgtcatta 4020 ttagtgttct gctgcatttc cacgggttgt tgtggatgct gcggttgttg cggtgcttgt 4080 ttttcaggtt gttgtagggg tcctagactt caaccttacg aagcttttga aaaggtccac 4140 gtgcagtga 4149 <210> 2 <211> 4161 <212> DNA <213> Porcine epidemic diarrhea virus <400> 2 atgaagtctt taacttactt ctggttgttc ttaccagtac tttcgacact tagcctacca 60 caagatgtca ccaggtgctc agctaacact aattttaggc ggttcttttc aaaatttaat 120 gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga aaaccagggt 180 gttaattcaa cttggtactg ttatggccaa catccaactg ctagtggcgt tcatggtatc 240 tttcttagcc atattagagg tggtcatggc tttgagattg gcatttcgca agagcctttt 300 gaccctagtg gttaccagct ttatttacat aaggctacta atggtaacac taatgctact 360 gcgcgattgc gcatttgcca gtttcccagc attaaaacat tgggccccac tgctgataac 420 gatgttacaa caggtcgtaa ctgcctattt aacaaagcca tcccagctca tatgagtgaa 480 catagtgttg tcggcataac atgggataat gatcgtgtca ctgtcttttc tgacaagatc 540 tatcattttt attttaaaaa tgattggtcc cgtgttgcga caaagtgtta caacagtgga 600 ggttgtgcta tgcaatatgt ttacgaaccc acttactaca tgcttaatgt tactagtgct 660 ggtgaggatg gtatttctta tcaaccctgt acagctaatt gccttggtta tgctgccaat 720 gtatttgcta ctgagctcaa tggccacata ccagaaggtt ttagttttaa taattggttt 780 cttttgtcca atgattccac tttggtgcat ggtaaggtgg tttccaacca accattgttg 840 gtcaattgtc ttttggccat gcctaagatt tatggactag gccaattttt ctccttcaat 900 caaacgatcg atggtgtttg taatggagct gctgtgcagc gtgcaccaga ggctctgagg 960 tttaatatta atgacacctc tgtcattctt gctgaaggcg caattgtact tcatactgct 1020 ttaggaacaa atctttcttt tgtttgcagt aattcttcag atcctcattt agctaccttc 1080 accatacctc tgggtgctac ccaagtaccc tattattgtt ttcttaaagt ggatacttac 1140 aactccactg tttataaatt tttggctgtt ttacctccta ccgtcaggga aattgtcatc 1200 accaagtatg gtgatgttta tgtcaatggg tttggatact tgcatctcgg tttgttggat 1260 gctgtcacaa ttaatttcac tggtcatggc actgacgatg atgtttctgg ttttggacc 1320 atagcatcga ctaattttgt tgatgcactc atcgaagttc aaggaactgc cattcagcgt 1380 attctttatt gtgatgatcc tgttagccaa ctcaagtgtt ctcaggttgc ttttgacctt 1440 gacgatggtt tttaccctat ttcttctaga aaccttctga gtcatgaaca gccaacttct 1500 tttgttactt tgccatcatt taatgatcat tcttttgtta atattactgt ctctgcttcc 1560 tttggtggtc atagtggtgc caaccttatt gcatctgaca ctactatcaa tgggtttagt 1620 tctttctgtg ttgacactag acaatttacc atttcactgt tttataacgt tacaaacagt 1680 tatggttatg tgtctaaaac acaggacagt aattgccctt tcaccttgca atctgtcaat 1740 gattacctgt cttttagcaa atttgtgtt tccaccagcc ttttggctag tgcctgtacc 1800 atagatcttt ttggttaccc tgagtttggt agtggtgtta agtttacgtc cctttacttt 1860 caattcacag agggtgagtt gattactggc acgcctaaac cacttgaagg tgtcacggac 1920 gttctttta tgactctgga tgtgtgtacc aagtatacta tctatggctt tamaggtgag 1980 ggtatcatta cccttacaaa ttctagcttt ttggcaggtg tttattacac atctgattct 2040 ggacagttgt tagcttttaa gaatgtcact agtggtgctg tttattctgt tacgccatgt 2100 tctttttcag agcaggctgc atatgttgat gatgatatag tgggtgttat ttctagtttg 2160 tctagctcca cttttaacag tactagggag ttgcctggtt tcttctacca ttctaatgat 2220 ggctctaatt gtacagagcc tgtgttggtg tatagtaaca taggtgtttg taaatctggc 2280 agtattggct acgtcccatc tcagtctggc caagtcaaga ttgcacccac ggttactggg 2340 aatatcagta ttcccaccaa ctttagtatg agtattagga cagaatattt acagctttac 2400 aacacgcctg ttagtgttga ttgtgccaca tatgtttgta atggtaactc tcgttgtaaa 2460 caattactca cccagtacac tgcagcatgt aagaccatag agtcagcatt acaactcagc 2520 gctaggcttg agtctgttga agttaactct atgcttacta tttctgaaga ggctctacag 2580 ttagctacca ttagttcgtt taatggtgat ggatataatt ttactaatgt gctgggtgtt 2640 tctgtgtatg atcctgcaag tggcagggtg gtacaaaaaa ggtcttttat tgaagacctg 2700 ctttttaata aagtggttac taatggcctt ggtactgttg atgaagacta taagcgctgt 2760 tctaatggtc gctctgtggc agatctagtc tgtgcacagt attactctgg tgtcatggta 2820 ctacctggtg ttgttgacgc tgagaagctt cacatgtata gtgcgtctct catcggtggt 2880 atggtgctag gaggctttac ttctgcagcg gcattgcctt ttagctatgc tgttcaagct 2940 agactcaatt atcttgctct acagacggat gttctacagc ggaaccagca attgcttgct 3000 gagtctttta actctgctat tggtaatata acttcagcct ttgagagtgt taaagaggct 3060 attagtcaaa cttccaaggg tttgaacact gtggctcatg cgcttactaa ggttcaagag 3120 gttgttaact cgcagggtgc agctttgact caacttaccg tacagctgca acacaacttc 3180 caagccattt ctagttctat tgatgacatt tactctcgac tggacattct ttcagccgat 3240 gttcaggttg accgtctcat caccggcaga ttatcagcac ttaatgcttt tgtttctcaa 3300 accctcacta agtatactga ggttcaggct agcaggaagc tagcacagca aaaggttaat 3360 gagtgcgtta aatcgcaatc tcagcgttat ggtttttgtg gtggtgatgg cgagcacatt 3420 ttctccctgg tacaggcagc acctcagggc ctgctgtttt tacatacagt acttgtaccg 3480 ggtgattttg tagatgttat tgccatcgct ggcttatgcg ttaacgatga aattgccttg 3540 actctacgtg agcctggctt agtcttgttt acgcatgaac ttcaaaatca tactgcgacg 3600 gaatattttg tttcatcgcg acgtatgttt gaacctagaa aacctaccgt tagtgatttt 3660 gttcaaattg agagttgtgt ggtcacctat gtcaatttga ctagagacca actaccagat 3720 gtaatcccag attacatcga tgttaacaaa acacttgatg agattttagc ttctctgccc 3780 aatagaactg gtccaagcct tcctttagat gtttttaatg ccacttatct taatctcact 3840 ggtgaaattg cagatttaga gcagcgttca gagtctctcc gtaatactac agaggagctc 3900 caaagtctta tatataatat caacaacaca ctagttgacc ttgagtggct caaccgagtt 3960 gagacatata tcaagtggcc gtggtgggtt tggttgatta ttttcattgt tatcatcttt 4020 gttgtgtcat tactagtgtt ctgctgcatt tccacgggtt gttgtggatg ctgcggctgc 4080 tgctgtgctt gtttttcagg ttgttgtagg ggtcctagac ttcaacctta cgaagttttt 4140 gaaaaggtcc acgtgcagtg a 4161 <210> 3 <211> 36159 <212> DNA <213> Artificial Sequence <400> 3 atcgaatata acttcgtata atgtatgcta tacgaagtta ttagcgatga gctcggactt 60 ccattgttca ttccacggac aaaaacagag aaaggaaacg acagaggcca aaaagctcgc 120 tttcagcacc tgtcgtttcc tttcttttca gagggtattt taaataaaaa cattaagtta 180 tgacgaagaa gaacggaaac gccttaaacc ggaaaatttt cataaatagc gaaaacccgc 240 gaggtcgccg ccccgtaacc tgtcggatca ccggaaagga cccgtaaagt gataatgatt 300 atcatctaca tatcacaacg tgcgtggagg ccatcaaacc acgtcaaata atcaattatg 360 acgcaggtat cgtattaatt gatctgcatc aacttaacgt aaaaacaact tcagacaata 420 caaatcagcg acactgaata cggggcaacc tcatgtccga gctcgcgagc tcgtcgacag 480 cgacacactt gcatcggatg cagcccggtt aacgtgccgg cacggcctgg gtaaccaggt 540 attttgtcca cataaccgtg cgcaaaatgt tgtggataag caggacacag cagcaatcca 600 cagcaggcat acaaccgcac accgaggtta ctccgttcta caggttacga cgacatgtca 660 atacttgccc ttgacaggca ttgatggaat cgtagtctca cgctgatagt ctgatcgaca 720 atacaagtgg gaccgtggtc ccagaccgat aatcagaccg acaacacgag tgggatcgtg 780 gtcccagact aataatcaga ccgacgatac gagtgggacc gtggtcccag actaataatc 840 agaccgacga tacgagtggg accgtggttc cagactaata atcagaccga cgatacgagt 900 gggaccgtgg tcccagacta ataatcagac cgacgatacg agtgggacca tggtcccaga 960 ctaataatca gaccgacgat acgagtggga ccgtggtccc agtctgatta tcagaccgac 1020 gatacgagtg ggaccgtggt cccagactaa taatcagacc gacgatacga gtgggaccgt 1080 ggtcccagac taataatcag accgacgata cgagtgggac cgtggtccca gtctgattat 1140 cagaccgacg atacaagtgg aacagtgggc ccagagagaa tattcaggcc agttatgctt 1200 tctggcctgt aaaaggac attaagtaagagagataaa cgtagactaa aacgtggtcg 1260 catcagggtg ctggctttc aagttcctta agaatggcct caatttctc tatacactca 1320 gttggaacac gagacctgtc caggttaagc accattttat cgcccttata caatactgtc 1380 gctccaggag caactgatg tcgtgagctt aaactagttc ttgatgcaga tgacgtttta 1440 agcacagaag ttaaaagt gataacttct tcagctca atcacccc agctttttc 1500 tgctcatgaa ggttagatgc ctgctgctta agtaattcct ctttatctgt aaggctttt 1560 tgaagtgcat cacctgaccg ggcagatagt tcaccggggt gagaaaaag agcaacaac 1620 gatttaggca atttggcggt gttgatacag cgggtaataa tcttacgtga atattttcc 1680 gcatcagcca gcgcagaaat atttccagca aattcattct gcaatcggct tgcataacgc 1740 tgaccacgtt cataagcact tgttgggcga taatcgttac ccaatctgga taatgcagcc 1800 atctgctcat catccagctc gccaaccaga acacgataat cactttcggt aagtgcagca 1860 gctttacgac ggcgactccc atcggcaatt tctatgacac cagatactct tcgaccgaac 1920 gccggtgtct gttgaccagt cagtagaaaa gaagggatga gatcatccag tgcgtcctca 1980 gtaagcagct cctggtcacg ttcattacct gaccataccc gagaggtctt ctcaacacta 2040 tcaccccgga gcacttcaag agtaaacttc acatcccgac cacatacagg caaagtaatg 2100 gcattaccgc gagccattac tcctacgcgc gcaattaacg aatccaccat cggggcagct 2160 ggtgtcgata acgaagtatc ttcaaccggt tgagtattga gcgtatgttt tggaataaca 2220 ggcgcacgct tcattatcta atctcccagc gtggtttaat cagacgatcg aaaatttcat 2280 tgcagacagg ttcccaaata gaaagagcat ttctccaggc accagttgaa gagcgttgat 2340 caatggcctg ttcaaaaaca gttctcatcc ggatctgacc tttaccaact tcatccgttt 2400 cacgtacaac attttttaga accatgcttc cccaggcatc ccgaatttgc tcctccatcc 2460 acggggactg agagccatta ctattgctgt atttggtaag caaaatacgt acatcaggct 2520 cgaacccttt aagatcaacg ttcttgagca gatcacgaag catatcgaaa aactgcagtg 2580 cggaggtgta gtcaaacaac tcagcaggcg tgggaacaat cagcacatca gcagcacata 2640 cgacattaat cgtgccgata cccaggttag gcgcgctgtc aataactatg acatcatagt 2700 catgagcaac agtttcaatg gccagtcgga gcatcaggtg tggatcggtg ggcagtttac 2760 cttcatcaaa tttgcccatt aactcagttt caatacggtg cagagccaga caggaaggaa 2820 taatgtcaag ccccggccag caagtgggct ttattgcata agtgacatcg tccttttccc 2880 caagatagaa aggcaggaga gtgtcttctg catgaatatg aagatctggt acccatccgt 2940 gatacattga ggctgttccc tgggggtcgt taccttccac gagcaaaaca cgtagcccct 3000 tcagagccag atcctgagca agatgaacag aaactgaggt tttgtaaacg ccacctttat 3060 gggcagcaac cccgatcacc ggtggaaata cgtcttcagc acgtcgcaat cgcgtaccaa 3120 acacatcacg catatgatta atttgttcaa ttgtataacc aacacgttgc tcaacccgtc 3180 ctcgaatttc catatccggg tgcggtagtc gccctgcttt ctcggcatct ctgatagcct 3240 gagaagaaac cccaactaaa tccgctgctt cacctattct ccagcgccgg gttattttcc 3300 tcgcttccgg gctgtcatca ttaaactgtg caatggcgat agccttcgtc atttcatgac 3360 cagcgttat gcactggtta agtgttcca tgagtttcat tctgaacatc ctttaatcat 3420 tgctttgcgt ttttttatta aatcttgcaa tttactgcaa agcaacaaca aaatcgcaaa 3480 gtcatcaaaa aaccgcaaag ttgtttaaaa taagagcaac actacaaaag gagataagaa 3540 gagcacatac ctcagtcact tattatcact agcgctcgcc gcagccgtgt aaccgagcat 3600 agcgagcgaa ctggcgagga agcaaagaag aactgttctg tcagatagct cttacgctca 3660 gcgcaagaag aatatccac cgtgggaaaa actccaggta gaggtacaca cgcggatagc 3720 caattcagag tataaactg tgataatcaa ccctcatcaa tgatgacgaa ctaacccccg 3780 atatcaggtc acatgacgaa gggaaagaga aggaaatcaa ctgtgacaaa ctgccctcaa 3840 atttggcttc cttaaaaatt acagttcaaa aagtatgaga aaatccatgc aggctgaagg 3900 aaacagcaaa actgtgacaa attaccctca gtaggtcaga acaaatgtga cgaaccacccc 3960 tcaaatctgt gacagataac cctcagacta tcctgtcgtc atggaagtga tatcgcggaa 4020 ggaaaatacg atatgagtcg tctggcggcc tttctttttc tcaatgtatg agaggcgcat 4080 tggagttctg ctgttgatct cattaacaca gacctgcagg aagcggcggc ggaagtcagg 4140 catacgctgg taactttgag gcagctggta acgctctatg atccagtcga ttttcagaga 4200 gacgatgcct gagccatccg gcttacgata ctgacacagg gattcgtata aacgcatggc 4260 atacggattg gtgatttctt ttgtttcact aagccgaaac tgcgtaaacc ggttctgtaa 4320 cccgataaag aagggaatga gatatgggtt gatatgtaca ctgtaaagcc ctctggatgg 4380 actgtgcgca cgtttgataa accaaggaaa agattcatag cctttttcat cgccggcatc 4440 ctcttcaggg cgataaaaaa ccacttcctt ccccgcgaaa ctcttcaatg cctgccgtat 4500 atccttactg gcttccgcag aggtcaatcc gaatatttca gcatatttag caacatggat 4560 ctcgcagata ccgtcatgtt cctgtagggt gccatcagat tttctgatct ggtcaacgaa 4620 cagatacagc atacgttttt gatcccggga gagactatat gccgcctcag tgaggtcgtt 4680 tgactggacg attcgcggc tattttacg ttcttgtga tgataccg ctgtttccgc 4740 catgacagat ccatgtgaag tgtgacaagt tttagatg tcacactaaa taaaaaagag 4800 tcaataagca gggataactt tgtgaaaaaa cagctcttc tgaggcaat ttgtcacagg 4860 gttaagggca atttgtcaca cakaggactg tcatttgagg gtgatttgtc acacgaaag 4920 ggcaatttgt cacaacct tcttagaac cagcatggat aaggcctac aaggcgctct 4980 aaaaaagaag atctaaaaac taaaaaaaaatataaaataatcccc gtggataagt 5040 gcataacccc aagggaagtt tttcaggca tcgtgtgtaa gcagaatata taagtgctgt 5100 tccctgtgc ttcctcgctc actcgatcga gggctcgcc ctgtcgctcg actgcggcga 5160 gcactactgg ctgtaaagg acgaccaca tcatgttct gtgttcatta ggttgttctg 5220 tccattgctg acataatccg ctccactca acgtaacacc gcacgaagat ttctattgtt 5280 cctgaaggca tattcaatc gtttcgtta ccgcttgcag gcatcatgac agaacactac 5340 ttcctataaa cgctacacag gctcctgaga ttaataatgc ggatctctac gataatggga 5400 gattttcccg actgtttcgt tcgcttctca gtggataaca gccagcttct ctgtttaaca 5460 gacaaaaaca gcatatccac tcagttccac atttccatat aaaggccaag gcatttattc 5520 tcaggataat tgtttcagca tcgcaaccgc atcagactcc ggcatcgcaa actgcacccg 5580 gtgccgggca gccacatcca gcgcaaaaac cttcgtgtag acttccgttg aactgatgga 5640 cttatgtccc atcaggcttt gcagaacttt cagcggtata ccggcataca gcatgtgcat 5700 cgcataggaa tggcggaacg tatgtggtgt gaccggaaca gagaacgtca caccgtcagc 5760 agcagcggcg gcaaccgcct ccccaatcca ggtcctgacc gttctgtccg tcacttccca 5820 gatccgcgct ttctctgtcc ttcctgtgcg acggttacgc cgctccatga gcttatcgcg 5880 aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg 5940 ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag 6000 gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga 6060 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg 6120 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta cggcctttttt aaagaccgta aagaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggaca cttcttcgcc cccgttttca ccatgggcaa attacktacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct we call caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggttgcta cgcctgata agtgataata agcggatga tggcagaat tcgatgata gctgtcaaac atgagaattg gtcgacggcc cgggcggccg caaggggttc gcgttggccg 6900 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 6960 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 7020 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 7080 catgattacg ccaagctatt taggtgacac tatagaatac tcaagctggg cccttgacat 7140 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 7200 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 7260 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat aggactttc 7320 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 7380 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 7440 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 7500 atcgcatta ccatggtgat gcggttttgg fòacatca atgggcgtgg atagcggttt 7560 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 7620 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 7680 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtacttaa 7740 agagattttc tatctacgga tagttagctc tttctctaga ctcttgtcta ctcaattcaa 7800 ctaaacgaaa ttttgtcctt ccagtcgcat gtctatgctt ctggaagctg acgtggaatt 7860 tcattaggtt tgcttaagta gccatcgcaa gtgctgtgct gtcctctagt tcctggttgg 7920 cgttccgtcg ccttctacat actagacaaa cagccttcct ccggttccgt ctgggggttg 7980 tgtggataac tagttccgtc tagtttgaaa ccagtaactg tcggctatgg ctagcaacca 8040 tgttacattg gctgttgcca atgatgcaga aatttcagcc tttggctttt gcactgctag 8100 tgaagccgtc tcatactatt ctgaggccgc cgctagtgga tttatgcaat gccgtttcgt 8160 gtccttcgat ctcgttgaca ctgttgaggg attgcttccc gaagactatg tcatggttgt 8220 ggtcggcact accaagctta gtgcgtatgt ggacactttt ggtagccgcc ccagaaacat 8280 ctgtggttgg ctattatttt ctaactgtaa ttacttcctc gaagagttag agctcacttt 8340 tggtcgtcgt ggtggtaaca tcgtgccagt tgatcaatac atgtgtggcg ctgacgggaa 8400 acctgttctt caggaatccg agtgggagta tacagacttc tttgctgact ccgaggacgg 8460 tcaactcaac attgctggga tcacttatgt gaaggcctgg attgtggagc gatcggatgt 8520 ctcttatgcg agtcagaatt taacatctat taaatctatt acttattgtt caacctatga 8580 gcatactttt cctgatggta ccgccatgaa ggttgcacgt aatccaaaga tcaagaagaa 8640 tgttgtcttg tctgagccac ttgctactat ctacagggaa attggttctc cttttgtgga 8700 taatgggagc gatgctcgtt ctatcattaa gagaccagtg ttcctccacg cttttgttaa 8760 gtgtaagtgt ggtagttatc attggactgt tggtgattgg acttcctatg tctccacttg 8820 ctgtggcttt aagtgtaagc cagtccttgt ggcttcatgc tctgctacgc ctggttctgt 8880 tgtggttacg cgcgctggtg ctggcactgg tgttaagtat tacaacaaca tgttcctgcg 8940 ccatgtggca gacattgatg ggttggcatt ctggcgaatt ctcaaggtgc agtccaaaga 9000 cgacctcgct tgctctggta aattccttga acaccatgag gaaggtttca cagatccttg 9060 ctactttttg aatgactcga gcattgctac taagctcaag tttgacatcc ttagtggcaa 9120 gttttctgat gaagtcaaac aagctatctt tgctggtcat gttgttgttg gcagtgcgct 9180 cgttgacatt gttgacgatg cactgggaca gccttggttt atacgtaagc ttggtgacct 9240 tgcaagtgca gcttgggagc agcttaaggc tgtcgttaga ggccttaacc tcctgtctga 9300 tgaggtcgtg ctctttggca aaagacttag ctgtgccact cttagtattg ttaacggtgt 9360 ttttgagttc atcgccgaag tgccagagaa gttggctgcg gctgttacag tttttgtcaa 9420 cttcttgaat gagctttttg attctgcctg tgactgttta aaggtcggag gtaaaacctt 9480 taacaaggtt ggctcctatg ttctttttga caacgcattg gttaagcttg tcaaggcaaa 9540 agttcgcggc ccacgacagg caggtgtttg tgaagttcgt tacacaagcc ttgttattgg 9600 gagtactacc aaggtggttt ccaagcgcgt tgaaaatgcc aatgtgaatc tcgtcgttgt 9660 tgacgaggat gtgaccctca acaccactgg tcgtacagtt gttgttgacg gacttgcatt 9720 cttcgagagt gacgggtttt acagacatct tgctgatgct gatgttgtca ttgaacatcc 9780 tgtttataag tctgcttgtg agctcaagcc agtttttgag tgtgacccaa tacctgattt 9840 tcctatgcct gtggccgcta gtgttgcaga gctttgtgtg caaactgatc tgttgcttaa 9900 aaattacaac actccttata aaacttacag ctgcgttgtg agaggtgata agtgttgtat 9960 cacttgcacc ttacatttca cagcaccaag ttatatggag gctgctgcta attttgtaga 10020 cctctgtacc aagaatattg gtactgctgg ttttcatgag ttttacatta cggcccatga 10080 acaacaggat ctgcaagggt tcgtaaccac ttgttgcacg atgtcaggtt ttgagtgttt 10140 tatgcctata atcccacagt gtccagcagt gcttgaagag attgatggtg gtagcatctg 10200 gcggtcttt atcactggtc ttaatacaat gtgggatttt tgcaagcatc ttaaagtcag 10260 ctttggacta gatggcattg ttgtcactgt agcacgcaaa tttaaacgac ttggtgctct 10320 cttggcagaa atgtataaca cttacctttc aactgtggtg gaaaacttgg tactggccgg 10380 tgttggcttc aagtattatg ccaccagtgt cccaaaatt gttttgggct gttgttttca 10440 cagtgttaaa agtgttcttg caagtgcctt ccagattcct gtccaggcag gcattgagaa 10500 gttaaagtc ttccttaact gtgttcaccc tgttgtacca cgcgtcatcg aaacttcttt 10560 tgtggaatta gaagagacga catttaaacc accagcactc aatggtagta ttgctattgt 10620 tgatggcttt gctttctatt atgatggaac actatactat cccaccgatg gtaatagtgt 10680 tgtgcctatc tgttttaaga agaagggtgg tggtgatgtc aaattctctg atgaagtctc 10740 tgttagaacc attgacccag tttataaggt ctcccttgaa tttgagttcg agtctgagac 10800 tattatggct gtgcttaata aggctgttgg taatcgtatc aaggttacag gtggttggga 10860 cgatgttgtt gagtatatca acgttgccat tgaggttctt aaagatcaca tcgatgtgcc 10920 taagtactac atctatgatg aggaaggtgg caccgatcct aatctgcccg taatggtttc 10980 tcagtggccg ttgaatgatg acacgatctc acaggatctg cttgatgttg aagttgttac 11040 tgatgcgcca gttgatttcg agggtgatga agtagactcc tctgaccctg ataaggtggc 11100 agacgtggct aactctgaga ctgaggatga cggtcttaat gtagctcctg aaacaaatgt 11160 agagtctgaa gttgaggaag ttgccgcaac cttgtcctttt attaaagata caccttccac 11220 agttacaaag gatccttttg cttttgactt tgcaagctat ggaggactta aggttttaag 11280 acaatctcat aacaactgtt gggttacttc taccttggtg cagctacaat tgcttggcat 11340 cgttgatgac cctgcaatgg agctttttag tgctggtaga gtcggtccaa tggttcgcaa 11400 atgctatgag tcacaaaagg ctattttggg atctttgggt gatgtgtcgg cttgtctaga 11460 gtctcttact aagaacctac acacacttaa gattacctgt tctgtagtct gtggttgtgg 11520 tactggtgaa cgcatctatg agggttgtgc tttcgtatg acgccaactt tggaaccgtt 11580 cccatatggt gcttgtgctc agtgtgctca agttttgatg cacactttta aaagtattgt 11640 tggcaccggc atctttgtc gagatactac tgctctctcc ttggattctt tggttgtaaa 11700 acctctttgt gcggctgctt ttaggcaa ggatagtggt cattatgtca ccaactttta 11760 tgatgctgct atggctattg atggttatgg tcgtcatcag ataaagtatg acacactgaa 11820 caccatttgt gttaaagacg ttaattggac agcacctttt gtcccagacg ttgagcctgt 11880 attggagcct gttgtcaaac cgttctattc ttataagaat gttgattttt accaaggaga 11940 ctttagtgac cttgttaaac ttccatgtga ctttgttgtt aatgctgcaa atgagaattt 12000 gtctcacggt ggcggcatag caaaggccat tgatgtttat accaagggca tgttgcagaa 12060 gtgctcgaat gattacatta aagcacacgg tcccattaaa gttggacgtg gtgtcatgtt 12120 ggaggcatta ggtcttaacg tctttaatgt tgttggtcca cgtaagggta agcatgcacc 12180 tgagcttctt gttaaggctt ataagtccgt ttttgctaat ttaggtgttg ctcttacacc 12240 tttgattagt gttggaattt ttagtgttcc tttggaagaa tctttatctg cttttcttgc 12300 atgtgttggt gatcgccact gtaagtgctt ttgttatagt gacaaagagc gcgaggcgat 12360 cattaattac atggatggct tggtagatgc tattttcaaa gatgcgcttg ttgatactac 12420 tcctgtccag gaagatgttc aacaagtttc acaaaaacca gttttgccta attttgaacc 12480 tttcaggatt gaaggtgctc atgctttcta tgagtgcaac cctgaaggtt tgatgtcatt 12540 aggtgctgac aagctggtgt tgtttacaaa ttccaatttg gatttttgta gcgttggtaa 12600 gtgtcttaac aatgtgaccg gcggtgcatt gcttgaagcc ataaatgtat ttaaaaagag 12660 taacaaaaca gtgcctgctg gcaactgtgt tacttttgag tgtgcagaca tgatttctat 12720 tactatggta gtattgccat ctgatggtga tgctaattat gacaaaaatt atgcacgcgc 12780 cgtcgtcaag gtatctaagc ttaaaggcaa gttattgctt gttgttggtg atgccacgtt 12840 gtattccaag ttgtcccacc tcagcgtgat aggttcgta tccacacctg atgatgtgga 12900 gcgtttctac gcaaataaga gtgtggttat taaagtcact gaggatacac gtagtgttaa 12960 ggctgttaaa gtagaatcca ctgttactta tggacaacaa attggacctt gtcttgttaa 13020 tgacaccgtt gtcacagaca acaaacctgt tgttgctgat gttgtagcta aggttgtacc 13080 aagtgctaat tgggattcac attatggttt tgataaggct ggtgagttcc acatgctaga 13140 ccatactggg tttgccttc ctagtgaagt tgttaacggt aggcgtgtgc ttaaaaccac 13200 agataataac tgttgggtta atgttacatg tttacaatta cagtttgcta gatttaggtt 13260 caagtcagca ggtctacagg ctatgtggga gtcctattgt actggtgatg ttgctatgtt 13320 tgtgcattgg ttgtactggc ttactggtgt tgacaaaggt cagcctagtg attcagaaaa 13380 tgcacttaac atgttgtcca agtacattgt ttctgctggt tctgtcacta ttgaacgtgt 13440 cacgcatgac ggctgttgtt gtagtaagcg tgttgtcact gcaccagttg tgaatgctag 13500 cgtattgaag cttggcgtcg aggatggtct ttgtccacat ggtcttaact acattgacaa 13560 agttgttgta gttaaaggta ctacaattgt tgtcaatgtt ggaaaacctg tagtggcacc 13620 atcacacctc tttcttaagg gtgtttccta tacaacattc ctagataatg gtaacggtgt 13680 tgtcggccat tatactgttt ttgatcatga cactggtatg gtgcatgatg gagatgtttt 13740 tgtaccgggt gatctcaatg tatctcctgt cacaaatgtt gtcgtctcag agcagacggc 13800 tgttgtgatt aaagaccccg tgaagaaagt agagttagac gctacaaagc tgttagacac 13860 tatgaattat gcatcggaaa gattcttttc ctttggtgat tttatgtcac gtaatttaat 13920 tacagtgttt ttgtacatct ttagcatttt gggtctttgt tttagggcct ttcgtaagag 13980 ggatgttaaa gttctagctg gtgtacccca acgtactggt attatattgc gtaaaagtgt 14040 gcgctataat gcaaaggcgt tgggtgtctt cttcaagcta aaactttatt ggttcaaagt 14100 tcttggtaag tttagtttgg gtatttatgc attgtatgca ttactattca tgacaatacg 14160 ctttacacct ataggtggcc ctgtttgtga tgatgttgtt gctggttatg ctaattctag 14220 ttttgacaag aatgagtatt gcaacagtgt tatttgtaag gtctgtctct atgggtacca 14280 ggaactttcg gacttctctc acacacaggt agtatggcaa caccttagag acccattaat 14340 tggtaatgtg atgcctttct tttatttggc atttctggca atttttgggg gtgtttatgt 14400 aaaggctatt actctctatt ttatttttca gtaccttaac attcttggtg tgtttttggg 14460 cctacaacag tccatttggt ttttgcagct tgtgcctttt gatgtttttg gtgacgagat 14520 cgtcgtcttt ttcatcgtta cacgcgtatt gatgttcctt aagcatgttt tccttggctg 14580 cgataaggca tcttgtgtgg cttgctctaa gagtgctcgc cttaagcgcg ttcctgtcca 14640 gactattttt cagggtacta gcaaatcctt ctacgtacat gccaatggtg gttctaagtt 14700 ctgtaagaag cacaatttct tttgtttaaa ttgtgattct tatggtccag gctgcacttt 14760 tattaatgac gtcattgcaa ctgaagttgg taatgttgtc aaacttaacg tgcaaccgac 14820 aggtcctgcc actattctta ttgacaaggt tgaattcagt aatggttttt actatcttta 14880 tagtggtgac acattttgga agtacaactt tgacataaca gatagcaaat acacttgcaa 14940 agaatcactt aaaaattgta gcataatcac agactttatt gtttttaaca ataatggttc 15000 caatgtaaac caggttaaga atgcatgtgt ttatttttca cagatgcttt gtaaacctgt 15060 taagttagtg gactcagcgt tgttggccag tttgtctgtt gattttggtg caagcttaca 15120 tagtgctttt gttagtgtgt tgtcgaatag ttttggcaaa gatctgtcaa gttgtaatga 15180 catgcaggat tgcaagagca cattgggttt tgatgatgta ccattggata cctttaatgc 15240 tgctgttgct gaggctcatc gttacgatgt cctcttgact gacatgtcgt tcaacaattt 15300 taccaccagt tatgcaaaac cagaggaaaa acttccccgtc catgacatag ccacgtgtat 15360 gcgtgtaggt gccaagattg ttaatcataa cgttcttgtc areatagta tacctgtggt 15420 gtggcttgta cgtgatttca ttgccctttc ggaagaaact aggaagatca ttatcgtac 15480 gactaaagtt aagggtataa ccttcatgtt gacctttaat gattgtcgta tgcatactac 15540 catacctact gtttgcattg caataagaa gggtgcaggt cttcctagtt tttcaaaggt 15600 tagaaattc ttctggttt tgtgtctgtt catagttgct gttttctttg cactaagctt 15660 tcttgatttt agtactcagg ttagcagtga tagcgattat gacttcaagt atattgagag 15720 tggccagttg aagacttttg acaatccact tagttgtgtg cataatgtct ttagtaactt 15780 cgaccagtgg catgatgcca agtttggttt cacccccgtc aacaatccta gttgtcctat 15840 agtcgttggt gtatcagacg aagcgcgcac tgttccaggt atcccagcag gtgtttattt 15900 agctggtaaa acacttgttt ttgctattaa caccattttt ggtacatctg gtttgtgctt 15960 tgatgctagt ggcgttgctg ataagggcgc ttgcattttt aattcggctt gcaccacatt 16020 atctggtttg ggtggaactg ctgtctactg ttataagaat ggtctagttg aaggtgctaa 16080 actttatagt gagttggcac ctcatagcta ctataaaatg gtagatggta atgctgtgtc 16140 tttacctgaa gttatctcac gcggctttgg catccgtact atccgtacaa aggctatgac 16200 ctactgtcgc gttggccagt gtgtgcaatc tgcagaaggt gtttgttttg gcgccgatag 16260 attctttgtc tataatgcag aatctggttc tgattttgtt tgtggcacag ggctctttac 16320 attgttgatg aacgttatta gtgttttttc caagacagta ccagtaactg tgttgtctgg 16380 tcaaatactt tttaattgca ttattgcttt tgctgctgtt gcggtgtgtt tcttatttac 16440 aaagtttaag cgcatgttcg gtgatatgtc tgttggcgtt ttcaccgtcg gtgcttgtac 16500 tttgttgaac aatgtttcct acattgtaac acagaacaca cttggcatgt tgggctatgc 16560 aactttgtac tttttgtgca ctaaaggtgt tagatatatg tggatttggc atttgggatt 16620 tttgatctca tatatactta ttgcaccatg gtgggttttg atggtttatg ccttttcagc 16680 catttttgag tttatgccta accttttaa gcttaaggtt tcaacacaac tttttgaggg 16740 tgacaagttc gtaggctctt ttgaaaatgc tgcagcaggt acatttgtgc ttgatatgca 16800 tgcctatgag agacttgcca actctatctc aactgaaaaa ctgcgtcagt atgctagtac 16860 ttacaataag tacaagtatt attcaggcag tgcttcagag gctgattaca ggcttgcttg 16920 ttttgcccat ttggccaagg ctatgatgga ttatgcttct aatcacaatg acacgttata 16980 cacaccaccc actgtgagtt acaattcaac tctacaggct ggcttgcgta agatggcaca 17040 accatctggt gttgttgaga agtgcatagt tcgtgtttgc tatggtaata tggctcttaa 17100 tggcctatgg cttggtgata ctgttgtgtg cccacgccat gttatagcgt ctagtactac 17160 tagcactata gattatgact atgccctttc tgttttacgc ctccacaact tctccatttc 17220 atctggtaat gttttcctag gtgttgtggg tgtaaccatg cgaggtgctt tgttgcagat 17280 aaaggttaat caaaacaatg tccacacgcc tagtacacc tatcgcacag ttagaccggg 17340 tgaatctttt aatatcttgg cgtgctatga tggtgctgca gctggtgttt acggcgttaa 17400 catgcgctct aattacacta ttagaggctc gttcattaat ggcgcttgtg gttcacctgg 17460 ttataacatt aacaatggta ccgttgagtt ttgctattta caccagcttg aacttggttc 17520 aggctgtcat gttggtagcg acttagatgg tgttatgtat ggtggttatg aggaccaacc 17580 tactttgcaa gctgaaggcg ctagtagtct gtttacagag aatgtgttgg catttcttta 17640 tgcagcactc attaatggtt ctacatggtg gcttagttct tctaggattg ctgtagacag 17700 gtttaatgag tgggctgttc ataatggtat gacaacagta gttaatactg attgcttttc 17760 tattcttgct gctaagacwg gygttgatgt acaacgtttg ttggcctcaa tccagtctct 17820 gcataagaat tttggtggaa agcaaattct tggctatacc tcgttgacag atgagtttac 17880 tacaggtgaa gttatacgtc aaatgtatgg cgttaatctt cagagtggtt atgtttcacg 17940 cgcctgcaga aatgtcttgc tggttggttc ttttctgact ttcttttggt cagaattagt 18000 ttcctacact aagttctttt gggtaaatcc tggttatgtc acacctatgt ttgcgtgttt 18060 gtcattgctg tcctcacttt tgatgttcac actcaagcat aagacattgt ttttccaggt 18120 ctttctaata cctgctctga ttgttacatc ttgcattaat ttggcatttg atgttgaagt 18180 ctacaactat ttggcagagc atttgatta ccatgtttct ctcatgggtt ttaatgcaca 18240 aggtcttgtt aacatctttg tctgctttgt tgttaccatt ttacacggca catacacatg 18300 gcgcttttt aacacacctg tgagttcagt cacttatgtg gtagctttgc tgactgcggc 18360 atataactat tttacgcta gtgacattct tagttgtgct atgacactat ttgctagtgt 18420 gactggcaac tggttcgttg gtgctgtttg ttataaagct gctgtttata tggccttgag 18480 atttcctact tttgtggcta tttttggtga tattaagagt gttatgttct gttaccttgt 18540 gttgggttat tttacctgtt gcttctacgg tattctctac tggttcaaca ggttctttaa 18600 ggttagtgta ggtgtctatg actatactgt tagtgctgct gagtttaagt atatggttgc 18660 taacggccta cgtgcaccaa ctggaacact tgattcacta cttctgtctg ccaaattgat 18720 tggtattggt ggtgagcgga atattaagat ttcttccgtt cagtctaaac tgactgatat 18780 taagtgtagt aatgttgtgc ttttaggctg tctctctagc atgaatgtct cagcaaattc 18840 aacagaatgg gcctattgtg ttgacttgca taacaagctc aacttgtgca atgacccaga 18900 aaaagcgcag gaaatgctac ttgctttgtt ggcatttttc cttagtaaga atagtgcttt 18960 tggtttagat gacttattgg aatcctattt taatgacaat agtatgttgc agagtgttgc 19020 atctacttat gtcggtttgc cttcttatgt catttatgaa aatgcacgcc aacagtatga 19080 agatgctgtt aataatggtt ctccacctca gttggttaag caattgcgcc atgctatgaa 19140 tgtggcaaag agtgaatttg accgtgaggc ttctactcag cgtaagcttg atagaatggc 19200 ggagcaggct gcagcacaga tgtacaaaga ggcaagagcg gttaatagga agtccaaagt 19260 tgtaagtgct atgcattcac tgctttttgg tatgttgaga cgtttggata tgtcttctgt 19320 agacactatt ctcaacttgg caaaggatgg ggttgtacct ctgtctgtca taccggcagt 19380 cagtgctact aagcttaaca ttgttacttc tgatatcgat tcttataatc gtatccagcg tgagggatgt gtccactacg ctggtaccat ttggatata attgatatca about tggcaaggtg gtacacgtta aggaggtac cgcacagaat gctgagtccc tgtcatggcc cctggtcctt gggtgtgagc gtattgtcaa gctccagaat aatgaatta ttcctggtaa gctgaagcag cgctccatta aggcagaagg agatggcata gttggagaag gtaaggcact ttacaataat gagggtggac gtacttttat gtatgctttc atctcggaca aaccggacct gcgtgtagtt aagtgggagt tcgatggtgg ttgtaacact attgagctag aaccaccacg 19800 tagttcttg gtggattctc ctaatggtgc acagatcaag tatctctact ttgttcgtaa ccttaacacg ttacgtaggg gtgctgttct cggttacata ggtgccactg tacgcttgca ggctggtaaa caaacagaac aggctattaa ctcttcattg ttgacacttt gcgctttcgc tgtggatcct gctaagacct acatcgatgc tgtcaaaagt ggtcacaaac cagtaggtaa ctgtgttaag atgttggcca atggttctgg taatggacaa gctgttacta atggtgtgga ggctaatact aaccaagatt catatggtgg tgcgtccgtg tgtctatatt gtagagcaca 20160 tgttgagcat ccatctatgg atggtttttg cagactgaaa ggcaagtacg tacaggttcc 20220 actaggtaca gtggatccta tacgttttgt acttgagaat gacgtttgca aggtctgtgg 20280 ttgttggctg gctaatggct gcacttgtga cagatccatt atgcaaagca ctgatatggc 20340 ttatttaaac gagtacgggg ctctagtgca gctcgactag agccctgtaa cggtactgat 20400 acacaacatg tgtatcgtgc ttttgacatc tataacaagg atgttgcttg tctaggtaaa 20460 ttcctcaagg tgaactgtgt tcgcctgaag aatttggata agcatgacgc attctatgtt 20520 gtcaaaagat gtaccaagtc tgcgatggaa cacgagcaat ccatctatag cagacttgaa 20580 aagtgtggag ccgtagccga acacgatttc ttcacttgga aggatggtcg tgcaatctat 20640 ggtaacgttt gtagaaagga tcttaccgag tatactatga tggatttgtg ttacgcttta 20700 cgtaactttg atgaaaacaa ttgcgatgtt cttaagagca ttttaattaa ggtaggcgct 20760 tgtgaggagt cctacttcaa taataaagtc tggtttgatc ctgttgaaaa tgaagacatt 20820 catcgtgtct atgcattgtt aggtaccatt gtttcacgtg ctatgcttaa atgcgttaag 20880 ttctgtgatg caatggttga acaaggtata gttggtgttg tcacattaga taatcaggat 20940 cttaatggtg atttttatga ttttggtgat tttacttgta gcatcaaggg aatgggtata 21000 cccatttgca catcatatta ctcttatatg atgcctgtta tgggtatgac taattgcctt 21060 gctagtgagt gttttgttaa gagtgatata tttggtgagg atttcaagtc atatgacctg 21120 ctggaatatg atttcacgga gcataagaca gcactcttca acaagtattt caagtattgg 21180 ggactgcaat accaccctaa ctgtgtggac tgcagtgatg agcagtgcat agttcactgt 21240 gccaacttca atacgttgtt ttccactact atacctatta cggcatttgg acctttgtgt 21300 cgcaagtgtt ggattgatgg tgttccactg gtaactacag ttggttatca ttttaaacag 21360 ttaggtatag tttggaacaa tgacctcaac ttacactcta gcaggctctc tattaacgaa 21420 ctactccagt tttgtagtga tcctgcattg cttatagcat catcaccagc tcttgttgat 21480 cagcgtactg tttgcttttc agttgcagcg ctaggtacag gtatgactaa ccagactgtt 21540 aaacctggcc atttcaataa ggagttttat gacttcttac ttgagcaagg tttcttctct gagggctctg agcttacttt aaagcacttc ttctttgcac agaagggtga tgcagctgtt 21660. aaggattttg actactatag gtataataga cctactgttc tggacatttg ccaagctcgc gtcgtgtatc aaatagtgca acgctatttt gatatttacg aaggtggttg tatcactgct aaagaagtgg ttgttacaaa ccttaacaag agcgcaggtt atcctttgaa caagtttggt aaagctggtc tttactatga gtctttatcc tatgaggac aggatgaact ttatgcttat actaagcgta acatcctgcc cactatgaca cagctcaacc ttaaatatgc tataagtggc aaagaacgtg cacgcacagt gggtggtgtt tcgcttttgt cacgac tactcggcag 22080. tatcatcaaa aacaccttaa gtccatagtt aatactaggg gcgcttcggt tgttattggt actactaagt tttatggtgg ttgggacaat atgcttaaga accttattga tggtgttgaa aatccgtgtc ttatgggttg ggactaccca aagtgcgaca gagcactgcc caatatgata 22200 cgtatgattt cagccatgat tttaggctct aagcacacca catgctgcag ttccactgac 22260 cgctttttca ggttgtgcaa tgaattggct caagtcctta ctgaggttgt ttattctaat 22320 ggaggttttt atttgaagcc aggtggcact acctctggtg atgcaaccac cgcatatgca 22380 aactcagttt tcaatatctt ccaagcagta agtgccaatg ttaacaaact tcttagtgtt 22440 gacagcaatg tctgtcataa tttagaagtt aagcaattgc agcgtaagct ttatgagtgc 22500 tgttatagat caactaccgt cgatgaccag ttcgtcgttg agtattatgg ttacttgcgt 22560 aaacattttt caatgatgat tctttctgat gatggcgttg tttgttataa caatgactat 22620 gcatcacttg gttatgttgc tgatcttaac gcattcaagg ctgttttgta ttaccagaac 22680 aatgtcttca tgagcgcctc taaatgttgg atcgagcctg acattaataa aggtcctcat 22740 gaattttgct cgcagcatac tatgcagatt gtcgataaag atggtactta ttaccttcct 22800 taccctgatc cttcaagaat cctctctgca ggtgtgtttg ttgatgacgt tgttaaaact 22860 gatgcagttg tattgcttga acgttatgtg tcattggcta tagatgccta cccgttatct 22920 aagcatgaaa accctgaata taagaaggtg ttttatgtgc ttttggattg ggttaagcat 22980 ctgtacaaaa ctttgaatgc tggtgtgtta gagtctttt ctgtcacact tttggaagat 23040 tctactgcta aattctggga tgagagcttt tatgccaaca tgtatgagaa atctgcagtt 23100 ttacaatctg cagggctttg tgttgtttgt ggctctcaaa ctgttttacg ttgtggtgat 23160 tgtctacggc gtcctatgct ttgtactaag tgtgcttatg atcatgtcat tggaacaact 23220 cacaagttca ttttggccat cactccatac gtgtgttgtg cttcagattg tggtgtcaat 23280 gatgtacta agctctactt aggtggtctt agttattggt gtcatgaaca caagccacgt 23340 cttgcattcc cgttgtgctc tgctggtaat gtttttggct tgtacaaaaa ttctgctacc 23400 ggctcaccg atgttgaaga ctttaatcgc attgctacat ccgattggac tgatgtttct 23460 gactacaggt tggcaaatga tgtcaaggac tcattgcgtc tgttcgcagc ggaaactatc 23520 aaggccaagg aggagagcgt taagtcatcc tacgcttgtg caacactaca tgaggttgta 23580 ggacctaaag agttgttgct caaatgggaa gtcggcagac ccaaaccacc ccttaataga 23640 aattcggttt tcacttgtta tcatataacg aagaacacca aatttcaaat cggtgagttt 23700 gtgtttgaga aggcagaata tgataatgat gctgtaacat ataaaactac cgccacaaca 23760 aaacttgttc ctggcatggt ttttgtgctt acctcacata atgttcagcc attgcgcgca 23820 ccgaccattg ctaatcaaga acgttattcc actatacata agttgcatcc tgcttttaac 23880 atacctgaag cttattctag cttagtgccc tattaccaac tgattggtaa gcagaagatt 23940 acaactatcc agggacctcc cggtagtggt aaatctcact gtgttatagg gctaggtttg 24000 tactatccag gtgcacgtat agtgtttaca gcttgttctc atgcagcggt cgattcactt 24060 tgtgtgaaag cctccactgc ttatagcaat gacaaatgtt cacgcatcat accacagcgt 24120 gctcgtgttg agtgttatga cggtttcaag tctaataata ctagtgctca gtaccttttc 24180 tccactgtca atgctttgcc agagtgcaat gcggacattg ttgtggtgga tgaggtttct 24240 atgtgcacta attatgactt gtctgtcata aatcagcgca tcagctatag gcatgtagta 24300 tatgttggtg accctcaaca gctgcctgca ccacgtgtta tgatttcacg tggtactttg 24360 gaaccaaagg actacaacgt tgtcactcaa cgcatgtgtg cccttaagcc tgatgttttc 24420 ttgcacaagt gttatcgctg tcctgctgag atagtgcgca ctgtgtctga gatggtctat 24480 gaaaaccaat tcattcctgt gcacccagat agcaagcagt gttttaaaat cttttgcaag 24540 ggtaatgttc aggttgacaa tggttcaagc attaatcgca ggcaattgga tgttgtgcgt 24600 atgtttttgg ctaaaaaccc taggtggtca aaggctgttt ttatttctcc ttataacagc 24660 cagaattatg ttgccagccg catgctaggt ttacaaattc agacagttga ctcatcccag 24720 ggtagtgagt atgactatgt catttataca caaacttcag atactgccca tgcctgtaat 24780 gttaacaggt ttaatgttgc catcacaagg gctaagaaag gcatattatg tataatgtgc 24840 gataggtccc tttttgatgt gcttaaattt tttgagctta aattgtctga tttgcaggct 24900 aatgagggtt gtggtctttt taaagactgt agcagaggtg atgatttgtt gccaccatct 24960 cacgctaaca ccttcatgtc tttagcggac aattttaaga ctgatcaaga tcttgctgtt 25020 caaataggtg ttaatggatc cattaaatat gagcatgtta tctcgtttat gggcttccgt 25080 tttgatatca acatacccaa ccatcacact ctcttttgca cacgcgactt tgccatgcgc 25140 aatgttagag gttggttggg ttttgacgtt gaaggagcac atgttgttgg ctctaacgtc 25200 ggtacaaatg tcccattgca attagggttt tctaacggtg ttgattttgt tgtcagacct 25260 gaaggttgcg ttgtaactga gtctggtgac tacattaaac cmgtcagagc tcgtgctcca 25320 ccaggggaac aatttgcaca ccttttgcct ctactcaaac gcggccaacc atgggatgtg 25380 gttcgtaagc gtatagtgca aatgtgtagt gactacctgg ctaacctatc agacatacta 25440 atttttgtgt tgtgggctgg tggtttggag ttgacaacta tgcgttactt tgtcaagatt 25500 ggaccaagca agagttgtga ttgtggtaag gttgctactt gttacaatag tgcgctgcat 25560 acgtactgtt gtttcaaaca tgcccttggt tgtgattacc tgtacaatcc atactgtatt 25620 gatatacagc agtggggata caagggatca cttagcctta accaccatga gcattgtaat 25680 gtacatagaa acgagcatgt ggcttctggt gatgccataa tgactcgctg tctagccata 25740 catgattgct ttgtcaagaa cgttgactgg tccatcacat acccatttat tggtaatgag 25800 gctgttatta ataagagcgg ccgaattgtg caatcacaca ctatgcggtc agttcttaag 25860 ttatacaatc caaaagccat atatgacatt ggcaacccta agggcattag atgtgccgta 25920 acggatgcta agtggttctg ctttgacaag aatcctacta attctaatgt caagacattg 25980 gagtatgact atataacaca cggccaattt gatgggttgt gcttgttttg gaattgcaat 26040 gtggacatgt atccggaatt ctctgtggtc tgtcggtttg acactcgctg taggtcacca 26100 ctcaacttgg agggttgtaa tggtggttca ctgtatgtta ataatcatgc attccataca 26160 ccggcttttg acaagcgtgc ttttgccaag ttgaagccaa tgccattttt cttctatgat 26220 gatactgagt gtgacaagtt acaggactct ataaactacg ttcctcttag ggctagtaat 26280 tgcattacta aatgtaatgt tggtggagct gtctgtagta agcactgtgc tatgtaccat 26340 agctatgtta atgcttacaa cacctttacg tcggcgggct ttacgatttg ggtgcccact 26400 tcgtttgaca cctacaatct gtggcagaca tttagtaaca acttgcaagg tcttgagaac 26460 attgctttca atgtcgtaaa gaaaggatct tttgttggtg ctgaaggtga gcttcctgta 26520 gctgtggtta atgacaaagt gctcgttaga gatggtactg ttgatactct tgttttcaca 26580 aacaagacat cactacccac taacgtagct tttgagttgt atgccaagcg taaggtagga 26640 ctcaccccac ccattacgat cctacgtaac ttgggtgttg tttgcacatc taagtgtgtc 26700 atttgggact atgaagccga acgtccactt actactttta caaaggatgt ctgtaaatat 26760 accgactttg agggtgacgt ttgcacactc tttgataaca gcattgttgg ttcattagag 26820 cgattctcta tgacccaaaa tgctgtgctt atgtcactta cagctgttaa aaagcttact 26880 ggcataaagt taacttatgg ttatcttaat ggtgtcccag ttaacacaca tgaagataaa 26940 ccttttactt ggtacattta cactaggaag aacggcaagt tcgaggacta tcctgatggc 27000 tattttaccc aaggtagaac aaccgctgat tttagccctc gtagtgacat ggaacgggac 27060 ttcctaagta tggacatggg tctttttatt aacaagtacg gactagaaga ttacggcttt 27120 gagcacgttg tgtatggtga tgtttctaaa accacccttg gtggtttaca tctactaatt 27180 tcgcaggtgc gtttggcctg tatgggtgtg cttaaaatag acgagtttgt gtctagtaat 27240 gatagcacgt taaagtcttg tactgttaca tatgttgata accctagtag taagatggtt 27300 tgcacgtata tggatctcct tcttgacgat tttgtcagca ttcttaaatc gttggatttg 27360 agtgttgtat ctaaagttca tgaagttatg gtcgattgta aaatgtggag gtggatgttg 27420 tggtgtaagg atcataaact ccagacattt tatccgcaac ttcaggccag tgaatggaag 27480 tgtggttatt ccatgccttc tatttacaag atacaacgta tgtgtttaga accttgcaat 27540 ctctataact atggtgctgg tattaagtta cctgatggca ttatgtttaa cgtagttaaa 27600 tatacacagc tttgtcaata tcttaatagc accacaatgt gtgtacccca tcacatgcgc 27660 gtgctacatc ttggtgctgg ctccgacaag ggtgttgcac ctggcacggc tgtcttacga 27720 cgttggttgc cactggatgc cattatagtt gacaatgata gtgtggatta cgttagcgat 27780 gctgattata gtgttacggg agattgctct accttatacc tgtcagataa gtttgactta 27840 gttatatctg atatgtatga tggtaagatt aaaagttgtg atggggagaa cgtgtctaaa 27900 gaaggcttct ttccctatat taatggtgtc atcactgaaa agttggcact tggtggtact 27960 gtagctatta aggtgacgga gtttagttgg aataagaagt tgtatgaact cattcagaag 28020 tttgagtatt ggacaatgtt ctgtaccagt gttaacacgt catcgtcaga ggcattttta 28080 attggtgtcc actatttagg tgattttgca agtggcgctg ttattgacgg caacactatg 28140 catgccaatt atatcttctg gcgtaattcc acaattatga ctatgtctta caatagtgta 28200 cttgatttaa gcaagttcaa ttgtaagcat aaggctacag ttgttattaa tttaaaggat 28260 tcatccatta gtgatgttgt gttaggtttg ttgaagaatg gtaagttgct agtgcgtaat 28320 aatgacgcca tttgtggttt ttctaatcat ttggtcaacg taaacaaatg acgcctttaa 28380 tttacttctg gttgttctta ccagtacttc taacacttag cctaccacaa gatgtcacta 28440 ggtgccagtc tactattaac tttaggcggt tcttttcaaa atttaatgtt caggcacctg 28500 ccgtcgttgt tttgggtggt tatctaccta gtatgaactc ttctagctgg tactgtggca 28560 caggcattga aactgatagt ggcgttcatg gtatttttct cagttacatc gattctggtc 28620 agggctttga gattggcatt tcgcaagagc cgtttgatcc tagtggttac cagctttatt 28680 tacacaaggc cactaatggt aacactagtg ctattgcacg actgcgcatt tgccagtttc 28740 cagataataa aacattgggc cctactgtta atgatgttac aacaggtcgt aactgcctat 28800 tcaacaaagc cattccagct ttgcaggatg gaaaaaatat tgttgtcggc ataacatggg 28860 ataatgatcg tgtcactgtt tttgctgaca agatctatca tttttatatt aaaaatgatt 28920 ggtccccgtgt tgcgacaaga tgttacaata aaagaagttg tgccatgcaa tatgtttata 28980 cacctaccta ctacatgctt aatgttacta gtgcaggtga ggatggcatt tactatgaac 29040 cttgtacagc taattgcagt ggttacgctg ccaatgtatt tgccactgat tccaatggcc 29100 atataccaga aggttttagt tttaataatt ggtttctttt gtccaatgac tccactttgt 29160 tgcatggtaa agtggtttca aaccaacctt tgttggtcaa ctgcctttgg gccattccta 29220 agatttatgg actaggccaa tttttctcat tcaatcaaac gatggatggc gtttgtaacg 29280 gagccgctgc gcagcgtgcc ccagaggctc tgaggtttaa tattaatgac acttttgtca 29340 ttcttgctga aggctcaatt gtacttcata ctgctttagg aacaaatctt tcttttgttt 29400 gcagtaattc ctcagatcct cacaaagcca tctttaccat acctttgggt gttactgaag 29460 taccctacta ttgctttctt aaagtggata cttacaaatc cactgtttat aaattcttgg 29520 ctgttttacc tcctactgtc aaggaaattg tcatcaccaa gtacggtgat gtttatgtca 29580 acgggtttgg ctatttgcat ctcggtttgt tggatgctgt cacaattaat ttcactggtc 29640 atggcactga cgatgacgtt tcaggtttct ggaccgtagc atcgactaat tttgttgatg 29700 cactcatcga ggttcaagga actgccattc agcgtattct ttattgtgat gaccctgtta 29760 gccaacttaa gtgttctcag gtttcttttg accttgatga tggtttttac cctatttctt 29820 ctagaaacct tctgagtcat gaacagccaa tttcttttgt tactttgcca tcattcaatg 29880 atcattcttt tgttaatatt actgtctctg cggcttttgg tggtcatagt ggtgccaacc 29940 tcattgcatc tgacactact atcaatgggt ttagttcttt ctgtgttgac actagacaat 30000 ttaccattac actgttttat aacgttacaa acagttatgg ttatgtgtct aagtcacagg 30060 atagtaattg ccctttcacc ttgcaatctg ttaatgatta cctgtctttt agcaaatttt 30120 gtgtttcaac cagccttttg gctggtgctt gtaccataga tctttttggt taccctgagt 30180 tcggtagtgg tgttaagttt acgtcccttt attttcaatt cacaaagggt gagttgatta 30240 ctggcacgcc taaaccactt caaggtgtca cggacgtttc ttttatgact ctggatgtgt 30300 gtaccaagta tactatctat ggctttaaag gtgagggtat tattaccctt acaaattcta 30360 gctttttggc aggtgtttat tatacatctg attctggaca gttgttagcc tttaagaatg 30420 tcactagtgg tgctgttat tctgttacgc catgttcttt ttcagagcag gctgcatatg 30480 ttgatgatga tatagtgggt gttatttcta gtttgtctaa ctccactttt aacaatacca 30540 gggagttgcc tggtttctc taccattcta atgatggctc caattgtaca gagcctgtgt 30600 tggtgtatag taacataggt gtctgtaaat ctggcagtat tggctatgtc ccacttcagg 30660 atggccaagt caagattgca cccatggtta ctgggaatat tagtattccc accaacttta 30720 gtatgagtat tagaacagaa tatttacagc tttacaacac gcctgttagt gttgattgcg 30780 ttacatatgt ttgtaatggt aactctcgtt gtaaacaatt actcacccag tacactgcag 30840 catgtaagac catagagtca gcattacaac tcagcgctag gcttgagtct gttgaagtta 30900 actctatgct tactatttct gaagaggctc tacagttagc taccatcagt tcgtttaatg 30960 gtgatggata taactttact aatgtgctgg gtgtttccgt gtacgaccct gcaagtggca 31020 gggtggtaca aaaagggtct tttattgaag acctgctttt tataaagtg gttactaatg 31080 gccttggtac tgttgatgaa gactataagc gctgttctaa tggtcgctct gtggcagatc 31140 tagtctgtgc gcagtattac tctggtgtca tggtactacc tggcgttgtt gacgctgaga 31200 agcttcacat gtatagtgcg tctctcatcg gtggtatggc gctaggaggt cttactactg 31260 cagcggcatt gccttttagc catgctgttc aagcgaggct caattatctt gctttacaga 31320 cggatgttct acagcgcaac cagcaattgc ttgctgagtc ttttaactct gctattggta 31380 atataacttc agcctttgag agtgttaaag aggctattag tcaaacttcc aatggtttga 31440 acactgtggc tcatgcgctt actaaggttc aagaggttgt taattcgcag ggttcagctt 31500 tgacccaact taccatacag ctgcaacaca acttccaagc catttctagt tctattgatg 31560 acatttactc ccgactggac attctttcag ccgatgttca ggttgatcgt ctcatcaccg 31620 gcagattatc agcacttaat gcttttgttg ctcaaaccct cactaagtat actgaggttc 31680 aggctagcag gaagctagca cagcaaaagg ttaatgagtg cgtcaaatcg caatctcagc 31740 gttatggttt ttgtggtggt gatggcgagc acatcttctc tctggtacag gccgcacctc 31800 agggcctgct gttcttacat acagtacttg taccgggtga ttttgtaaat gttattgcca 31860 tcgatggctt atgcgttaat ggtgatattg ccttgactct acgtgagcct ggcttagtct 31920 tgtttacgca tgaacttcaa acttatactg cgacggaata ttttgtttca tcgcgacgta 31980 tgtttgaacc tagaaaacct accgttagtg attttgttca aattgagagt tgtttggtca 32040 cctatgtcaa tctgactagc gaccaactac cagatgtaat cccagattac atcgatgtta 32100 acaaaacact tgatgagatt ctagcttctc tgcccaatag aattggtcct agtcttcccc 32160 tagatgtttt taatgccact tatcttaatc tcactggtga aattgcagat ttagagcagc 32220 gttcagagtc tctccgtaat actacagaag agctccgaag tctcatatat aatatcaaca 32280 acacacttgt tgaccttgag tggctcaacc gagttgagac atatatcaag tggccgtggt 32340 gggtttggtt gattattttt attgttctca tctttgttgt gtcattatta gtgttctgct 32400 gcatttccac gggttgttgt ggatgctgcg gttgttgcgg tgcttgtttt tcaggttgtt 32460 gtaggggtcc tagacttcaa ccttacgaag cttttgaaaa ggtccacgtg cagtgatgtt 32520 tcttggactt tttcaataca cgattgacac agttgtcaaa gatgtctcaa agtctgctaa 32580 cttgtctttg gatgctgtcc aagagttgga gctcaatgta gttccaatta gacaagcttc 32640 aaatgtgacg ggttttcttt tcaccagtgt ttttatctac ttctttgcac tgtttaaagc 32700 gtcttctttg agtcgcaatt atattatgtt ggcagcgcgt tttgctgtca ttgtccttta 32760 ttgcccactt ttatattatt gtggtgcatt tttagatgca actattattt gttgcacact 32820 tattggcagg ctttgtttag tctgctttta ctcctggcgc tataaaaatg cgctctttat 32880 tatttttaat actacgacac tttctttcct caatggtaaa gcagcttatt atgacggcaa 32940 atccattgtg attttagaag gtggtgacca ttacatcact tttggcaact cttttgttgc 33000 ttttgttagt agcatcgact tgtatctagc tatacgtggg cggcaagaag ctgacctaca 33060 gctgttgcga actgttgagc ttcttgatgg caagaagctt tatgtctttt cgcaacatca 33120 aattgttggc attactaatg ctgcatttga ctcaattcaa ctagacgagt atgctacaat 33180 33240. tagtgaatga taattgtcta gtagttaatg ttatactttg gcttttcgta ctctttttcc tgcttattat aagcattact ttcgtccaat tggttaatct gtgcttcact tgtcaccggt tgtgtaatag cgcagtttac acacctatag ggcgtttgta tagtttat aagtcttaca tgcaataga ccccctccct agtactgtta ttgacgtata aacgaatat gtctaacggt tctattcccg ttgatgaggt gattcaacc cttagaaact ggaatttcac atggaatatc atactgacga tactacttgt agtgcttcag tatggccatt acaagtactc tgcgttcttg tatggtgtca agatggctat tctatggata ctttggcctc ttgtgttagc actgtcactt tttgatgcgt gggctagctt tcaggtcaat tgggtctttt ttgctttcag catccttatg 33660. gcttgcatca ctcttatgct gtggataatg tactttgtca atagcattcg gttgtggcgc aggacacatt cttggtggtc tttcaatcct gaaacagacg cgcttctcac tacttctgtg atgggccgac aggtctgcat tccagtgctt ggagcaccaa ctggtgtaac gctaacactc cttagtggta cattgcttgt agagggctat aaggttgcta ctggcgtaca ggtaagtcaa ttacctaatt tcgtcacagt cgccaaggcc actacaacaa ttgtctacgg acgtgttggt 33960 cgttcagtca atgcttcatc tggcactggt tgggctttct atgtccggtc caaacacggc 34020 gactactcag ctgtgagtaa tccgagttcg gttctcacag atagtgagaa agtgcttcat 34080 ttagtctaaa cagaaacttt atggcttctg tcagttttca ggatcgtggc cgcaaacggg 34140 tgccattatc tctctatgcc cctcttaggg ttactaatga caaacccctt tctaaggtac 34200 ttgcaaataa tgctgtaccc actaataaag gaaataagga ccagcaaatt ggatactgga 34260 atgagcaaat tcgctggcgc atgcgccgtg gtgagcgaat tgaacaacct tccaattggc 34320 atttctacta cctcggaaca ggacctcacg ccgacctccg ctataggact cgtactgagg 34380 gtgttttctg ggttgctaaa gaaggcgcaa agactgaacc cactaacctg ggtgtcagaa 34440 aggcgtctga aaagccaatc attccaaatt tctctcaaca gcttcccagc gtagttgaga 34500 ttgttgaacc taacacacct cctacttcac gtgcaaattc acgtagcagg agtcgtggta 34560 atggcaacaa caggtccaga tctccaagta acaacagagg caataaccag tcccgcggta 34620 attcacagaa tcgtggaaat aaccagggtc gtggagcttc tcaagaaga ggaggcaata 34680 34740 gtggtgtaac atcacgcgat gatctggtgg ctgctgtcaa ggatgccctt aaatctttgg 34800 gcattggcga aaaccctgac aagcttaagc aacagcagaa gctcaaacaag gaaaggtctg 34860 acagcagcgg caaaaataca cctaagaaga acaaatccag agccacttcg aaagaacgtg 34920 accttaaaga catcccagag tggaggaagaa ttcccaaggg cgaaaatagc gtagcagctt 34980 gcttcggacc cagggggaggc ttcaaaaatt ttggagatgc ggaatttgtc gaaaaaggtg 35040 ttgatgcctc aggctatgcc cagatcgcca gtttagcacc aaatgttgca gcattgctct 35100 ttggtggtaa tgtggctgtt cgtgagctag cggactctta cgagattaca tacaattata 35160 gaatgactgt gccaaagtct gatccaaatg tagagcttct tgtttcacag gtggatgcat 35220 taaaaactgg gaatgcaaaa cccagagaa agaagaaaa gaacaag cgtgaaacca 35280 cgcagcagct gaatgaagat gccatctacg atgatgtggg tttgccatct gattcgactc 35340 atgccaattt ggaatgggac acagctgtag acggtggtga cacggccgtt gaaattatca 35400 acgagatctt cgacacagga attaaataa tgtttgactg gcttattctg gctatgtccc 35460 agggtagtgc cattacactg ttattactga gtgtttttct agcgacttgg ctgctgggct 35520 atggctttgc cctctaacta gcggtcttgg tcttgcacac aacggtaagc cagtggtaat 35580 gtcagtgcaa gaaggatatt accatagcac tgtcacgagg ggaacgcagt accttttcat 35640 ctaaaccttt gcacgagtaa tcaaagatcc gcttgacgag ctatatgga agagcgtgcc 35700 aggtatttga ctcaaggact gttagtaact gaagacctga cggtgttgat atggatacaa 35760 aaaaaaaaaa aaaaaaaaaaaaaag ggtcggcatg gcatctccac ctcctcgcgg 35820 tccgacctgg gcatccgaag gaggacgtcg tccactcgga tggctaaggg agagctcgga 35880 tccgcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc cccgtgcct 35940 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 36000 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 36060 ggggaggatt gggaagacaa tagcaggcat gctggggaga gctcgagctc tgtacatgtc 36120 cgcggtcgcg acgtacgcgc gccagctgca ggcggccgc 36159 <210> 4 <211> 1851 <212> DNA <213> Artificial Sequence <400> 4 ggcccgggcg gccgcaaggg gttcgcgttg gccgattcat taatgcagct ggcacgacag 60 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 120 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 180 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tatttaggtg 240 acactataga atactcaagc tgggcccttg acattgatta ttgactagtt attaatagta 300 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 360 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac 420 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt 480 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 540 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 600 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 660 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 720 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 780 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 840 tataagcaga gctcgtttag tgaaccgtac ttaaagagat tttctatcta cggatagtta 900 gctctttctc tagaggatcc cacgtgttaa ttaattcgaa agaacgtgac cttaaagaca 960 tcccagagtg gaggagaatt cccaagggcg aaaatagcgt agcagcttgc ttcggaccca 1020 ggggaggctt caaaaatttt ggagatgcgg aatttgtcga aaaaggtgtt gatgcctcag 1080 gctatgccca gatcgccagt ttagcaccaa atgttgcagc attgctcttt ggtggtaatg 1140 tggctgttcg tgagctagcg gactcttacg agattacata caattataga atgactgtgc 1200 caaagtctga tccaaatgta gagcttcttg tttcacaggt ggatgcattt aaaactggga 1260 atgcaaaacc ccagagaaag aaggaaaaga agaacaagcg tgaaaccacg cagcagctga 1320 atgaagatgc catctacgat gatgtgggtt tgccatctga ttcgactcat gccaatttgg 1380 aatgggacac agctgtagac ggtggtgaca cggccgttga aattatcaac gagatcttcg 1440 acacaggaaa ttaaataatg tttgactggc ttattctggc tatgtcccag ggtagtgcca 1500 ttacactgtt attactgagt gtttttctag cgacttggct gctgggctat ggctttgccc 1560 tctaactagc ggtcttggtc ttgcacacaa cggtaagcca gtggtaatgt cagtgcaaga 1620 aggatattac catagcactg tcacgagggg aacgcagtac cttttcatct aaacctttgc 1680 acgagtaatc aaagatccgc ttgacgagcc tatatggaag agcgtgccag gtatttgact 1740 caaggactgt tagtaactga agacctgacg gtgttgatat ggatacaaaa aaaaaaaaaaa 1800 aaaaaaaaaa aaaagggtcg gcatggcatc tccacctcct cgcggtccga c 1851

Claims

1. A recombinant strain of porcine epidemic diarrhea virus, characterized in that, The recombinant viral strain was obtained by replacing the S gene sequence of PEDV CH / SX / 2016 with the S2015 gene sequence shown in SEQ ID NO.1; the accession number of the PEDV CH / SX / 2016 strain is GenBank No. MT787025; the S gene sequence of the PEDV CH / SX / 2016 strain is shown in SEQ ID NO.

2.

2. The use of the recombinant porcine epidemic diarrhea virus strain as described in claim 1 in the preparation of PEDV diagnostic reagents and / or vaccines.

3. An infectious clone of PEDV strain CH / SX / 2016, characterized in that, The full-length cDNA sequence of the infectious clone of PEDV CH / SX / 2016 is shown in SEQ ID NO.

3.

4. The use of the infectious clone as described in claim 3 in the preparation of PEDV diagnostic reagents, and / or PEDV recombinant virus strains, and / or PEDV vaccines.

5. A recombinant strain of porcine epidemic diarrhea virus rCH / SX / 2016-S 2015 Its characteristics are, The recombinant virus strain rCH / SX / 2016-S 2015 It is obtained by rescuing infectious clones of the PEDV CH / SX / 2016 strain described in claim 3 transfected cells.

6. The recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S as described in claim 5 2015 Its characteristics are, The cells in question are Vero ccl81 kidney cells from African green monkeys.

7. The recombinant PEDV porcine epidemic diarrhea virus strain rCH / SX / 2016-S as described in claim 5 or 6. 2015 Application in the preparation of PEDV diagnostic reagents and / or vaccines.

8. The recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S as described in claim 5 or 6 2015 The preparation method of the [method] is characterized by, The method includes: pBac-CH / SX / 2016-S 2015 Construction of infectious clones: Using genetic engineering techniques, the S gene in the gene sequence of PEDV CH / SX / 2016 strain was replaced with the S2015 gene sequence shown in SEQ ID NO.1 to obtain the recombinant viral strain gene sequence, which was then ligated into the pBeloBac11 plasmid to construct pBac-CH / SX / 2016-S 2015 Infectious clones; pBac-CH / SX / 2016-S 2015 Transfection rescue: The obtained pBac-CH / SX / 2016-S 2015 Infectious clones were transfected into Vero ccl81 cells for rescue, yielding the recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S. 2015 .

9. The preparation method according to claim 8, characterized in that, pBac-CH / SX / 2016-S 2015 The method for constructing infectious clones is as follows: (1) PEDV CH / SX / 2016 restriction fragments: restriction sites were introduced into the full-length sequence of PEDV CH / SX / 2016 to digest it into 9 fragments: 5', XbaI-BamHI, BamHI-pmlI, PmlI-PacI, PacI-AvrII, AvrII-kasI, KasI-Bsu36I, Bsu36I-BstBI and 3'. (2) Construction of recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3': Construct the vector pUC17-CH / SX / 2016-5'-3' with the nucleotide sequence shown in SEQ ID NO.4; ligate the XbaI-BamHI, BamHI-pmlI, and PmlI-PacI fragments described in step (1) into the vector pUC17-CH / SX / 2016-5'-3' in sequence; ligate the digested fragments into the pBeloBac11 plasmid by digestion with RsrII and sfiI to obtain the recombinant plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3'; (3) Constructing the recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI: Amplify the Bsu36I-BstBI fragment described in step (1). During amplification, the upstream primers sequentially introduce restriction sites SfiI, PacI, AvrII, KasI, and Bsu36I, and the downstream primers sequentially introduce restriction sites BstB1 and RsrII. Using the restriction sites SfiI and RsrII, the Bsu36I-BstBI fragment is ligated into the pBeloBac11 vector to construct the vector pBAC-CH / SX / 2015-Bsu36I-BstBI. Then, the KasI-Bsu36I, AvrII-kasI, and PacI-AvrII fragments described in step (1) are sequentially ligated into the vector pBAC-CH / SX / 2015-Bsu36I-BstBI to obtain the recombinant plasmid pBAC-CH / SX / 2016-PacI-BstBI. (4) Construct pBac-CH / SX / 2016-S 2015 Infectious clones: The Bsu36I-BstBI fragment and AvrII-Bsu36I fragment described in step (1) were fused with the S2015 gene by overlap PCR to obtain the fused Bsu36I-BstBI fragment and AvrII-Bsu36I fragment; the fused Bsu36I-BstBI fragment, AvrII-Bsu36I fragment, and the PacI-AvrII fragment described in step (1) were sequentially ligated into the pBeloBac11 vector to construct the recombinant plasmid pBAC-CH / SX / 2016-S 2015 -PacI-BstBI; the PacI-BstBI fragment was obtained by enzyme digestion and ligated into the plasmid pBAC-CH / SX / 2016-5'-XbaI-PacI-3' described in step (2) to obtain the full-length plasmid pBAC-CH / SX / 2016-S, which is the infectious clone of the CH / SX / 2016 genome-wide cDNA. 2015 .

10. The preparation method according to claim 8, characterized in that, pBac-CH / SX / 2016-S 2015 The transfection rescue method is as follows: pBac-CH / SX / 2016-S 2015 Infectious clones were transfected into Vero ccl81 cells and cultured at 37°C in a 5% CO2 cell incubator until the virus stably induced cytopathic effects, thus obtaining the recombinant porcine epidemic diarrhea virus strain rCH / SX / 2016-S. 2015 .

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