CN113616784A - Preparation method of immune vaccine against porcine epidemic diarrhea virus variant shxx1902 - Google Patents

Preparation method of immune vaccine against porcine epidemic diarrhea virus variant shxx1902 Download PDF

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CN113616784A
CN113616784A CN202110920855.6A CN202110920855A CN113616784A CN 113616784 A CN113616784 A CN 113616784A CN 202110920855 A CN202110920855 A CN 202110920855A CN 113616784 A CN113616784 A CN 113616784A
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葛菲菲
沈海潇
于慧茹
沈莉萍
杨德全
李鑫
王建
鞠厚斌
刘健
葛杰
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Shanghai Animal Epidemic Prevention And Control Center (shanghai Veterinary Drug Feed Testing Institute And Shanghai Animal Husbandry Technology Promotion Center)
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Abstract

A method for preparing an immune vaccine against porcine epidemic diarrhea virus variant shxx1902, comprising a step of culturing vero cells; adding a cell culture solution of the porcine epidemic diarrhea virus variant shxx1902 into a vero cell culture solution, diluting and subculturing, and culturing for 72h for later use; a step of preparing immunogen, adding virus liquid into 0.2% formaldehyde, inactivating virus at 37 ℃; then added to a 96-well plate plated with vero cells in CO2Culturing in an incubator; harvesting cell supernatant, fixing the cells by cold acetone, adding the harvested supernatant into a 96-well plate paved with vero cells, and continuing culturing; discard the supernatant, fix the cells with cold acetone, and perform inactivation testAfter the product is qualified, the alumina gel adjuvant is added and mixed for use. The virus titer of the inactivated immunogen obtained by the method of the invention can generate an antibody with the level similar to that of a commercial vaccine after a mouse is immunized.

Description

Preparation method of immune vaccine against porcine epidemic diarrhea virus variant shxx1902
Technical Field
The invention belongs to the field of biology, and relates to a culture method of a strain, in particular to a preparation method of an immune vaccine against a porcine epidemic diarrhea virus variant shxx 1902.
Background
Porcine Epidemic Diarrheal (PED) is an acute, highly contagious enteric infectious disease caused by porcine epidemic diarrhea virus, PEDV. In 1971 the disease was first reported in the uk, followed by outbreaks in a number of countries such as belgium, uk, japan, etc. In 1984, Chinese scholars reported that PEDV was separated; PED is then generated in various places in China successively, and in 1995, a classical vaccine strain CV777 inactivated vaccine or attenuated vaccine is used for prevention and control, so that a good prevention and control effect is achieved. Since 2010, most swine farms across the country, including swine farms immunized with classical vaccine strains (e.g., CV777, DR13, etc.), have begun to have large-scale outbreaks of swine herd diarrheal diseases caused by PEDV variant virulent strains. The piglets which are suckling for 2-10 days are the most serious in morbidity, and the symptoms such as watery diarrhea, vomiting, dehydration and the like are caused, the morbidity reaches 100 percent, and the fatality rate reaches 50-90 percent. At present, the disease is still the most serious epidemic disease which endangers the pig industry, and has caused great loss to the pig industry in China and even all over the world. The invention discovers that the gene variation of the PEDV strain is the main reason for the failure of immunity and high mortality rate of piglets. Therefore, an effective vaccine of epidemic variant virulent strains of PEDV becomes a hotspot of the current invention. In 2019, 1 variant virulent strain is separated and obtained in the laboratory, an inactivated vaccine is prepared, and the mouse is immunized.
To date, the spread of PEDV remains challenging. In recent years, many PEDV strains isolated from clinical specimens have been difficult to culture in Vero cells. In addition, another invention successfully utilized porcine small intestine epithelial cells to isolate and propagate the PEDV strain and demonstrated that these cells are more suitable for isolation of PEDV than Vero cells. However, this cell line is not available in most laboratories.
It has also been reported that the first attempt to isolate virus in 68 clinical samples collected by the veterinary diagnostic laboratory at Iowa State university, which were positive for the U.S. variant of pedv S-INDEL, was unsuccessful in cell culture attempts. 3 pigs at 3 weeks of age were then inoculated with positive intestinal homogenate of the podv S-INDEL variant. A U.S. variant of pedv S-INDEL was successfully isolated from 3 pig small intestine homogenates and cecal contents. This may be due to a number of factors, such as low virus concentration in the sample, cytotoxicity of some samples, and changes in storage conditions after collection of clinical samples.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a preparation method of an immune vaccine against the porcine epidemic diarrhea virus variant shxx1902, and the preparation method of the immune vaccine against the porcine epidemic diarrhea virus variant shxx1902 aims to solve the technical problem that the immune effect of the immune vaccine against the porcine epidemic diarrhea virus variant shxx1902 in the prior art is poor.
The invention provides a preparation method of an immune vaccine against a porcine epidemic diarrhea virus variant shxx1902, which comprises the following steps:
1) a step of culturing vero cells, in which vero cells are added to a culture solution containing 10% by mass of fetal bovine serum and containing 5% by volume of CO at 37 ℃2Culturing in incubator, digesting, passaging to shake flask, and diluting with serum-free medium to cell density of 6 × 106When the strain is one/mL, the strain is ready for use;
2) a step of culturing the virus in vero cells to a cell density of 6X 106Adding 0.1L cell culture solution of porcine epidemic diarrhea virus variant shxx1902 to the cells per mL, diluting and passaging, inoculating with MOI of 0.1, pH of 7.4 + -0.1, shaking table speed of 120r/min, culturing temperature of 37 deg.C, and cell density of 3 × 10 after passage6Culturing for 72h for later use;
3) a step of preparing an immunogen to be titrated to 2X 106TCID50Adding 0.2 percent of formaldehyde into mL of the shxx1902 virus liquid of the porcine epidemic diarrhea virus variant, wherein the preservation number of the shxx1902 of the porcine epidemic diarrhea virus variant is CGMCC No.22380, and inactivating the virus at 37 ℃ for 28 h; then added to a 96-well plate plated with vero cells at 50. mu.L/well, 37 ℃ with 5% CO by volume2Culturing in an incubator for 2 d; harvesting cell supernatant, fixing the cells by cold acetone, adding the harvested supernatant into a 96-well plate paved with vero cells, and continuing culturing for 2 d; discarding the supernatant, fixing the cells with cold acetone, adding an alumina gel adjuvant after inactivation verification is qualified, and mixing according to the volume ratio of the antigen to the adjuvant of 1:1 for use.
The preservation number of the porcine epidemic diarrhea virus variant shxx1902 is CGMCC No.22380, and the porcine epidemic diarrhea virus variant is preserved in China general microbiological culture Collection center (CGMCC), and the addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The analysis of the whole gene sequence showed (SEQ ID NO. 1) that the shxx1902 strain belongs to the subgroup GII (c) including the PEDV strain circulating in our country since 2015. Since the end of 2010, highly virulent PEDV-GII genotype strains have emerged worldwide, causing severe losses to the pork industry in many countries. Phylogenetic invention shows that our isolates show inconsistent genotypes between spike gene (S)) and other genes. In addition, chimerism has been reported to confirm the GII (c) subgroup. This is consistent with our isolate, which belongs to GII-c by further analysis of Simplot v.3.5.1. At present, 2 commercial vaccine strains CV777 and AJ1102 mainly used in China belong to the GI group and the GII-b respectively, and the isolate strains belong to different gene groups, so that the isolate strains serve as vaccine candidate strains to provide a supplement for the prevention and control of the porcine epidemic diarrhea virus in China.
TABLE 1 CV777, nucleotide (amino acid) homology comparison of AJ1102 with SHXX1902
Figure BDA0003207351510000021
The invention aims to optimize a cell shake flask suspension batch culture porcine epidemic diarrhea virus (shxx1902) strain, and prepare an inactivated immunogen to obtain the maximum immunogenicity. According to the invention, Vero cells are cultured in a shake flask, and virus culture conditions such as shxx1902 strain virus culture temperature, MOI inoculation and initial cell density during virus inoculation are optimized, so that viruses and cells are adapted to each other, and the virus titer is improved. Culturing Vero cells in a 500mL cell shake flask in a serum-free suspension manner, diluting, passaging and inoculating, determining the optimal passage time in a cell culture stage and the optimal virus receiving time in a virus culture stage, culturing viruses by utilizing optimized conditions, inactivating the obtained virus liquid by formaldehyde, respectively adding an alumina gel adjuvant, preparing immunogen, immunizing BALB/c mice 2 times at intervals of 2 weeks, coating an ELISA plate by using N protein expressed by escherichia coli, and detecting the serum antibody titer of the mice.
Through experiments, the optimum culture conditions of the porcine epidemic diarrhea virus (shxx1902) strain in a shake flask are that the temperature is 37 ℃, the MOI is inoculated to be 0.1, and the initial cell density of inoculation is 3 multiplied by 106The virus titer can reach 4.52 multiplied by 10 per mL after inoculation for 48 hours6TCID 50/mL. Inactivating virus solution, mixing with aluminum gel adjuvant, immunizing mouse, setting commercial vaccine (CV777) immune group and blank control group, immunizing mouse for 2 times, and 2 weeks apart, and monitoring first immunityAt day 49, the inactivated virus elicited similar levels of antibody as the commercial inactivated vaccine.
Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The virus titer of the inactivated immunogen is cultured by using a cell shake flask, and an antibody with the level similar to that of a commercial vaccine can be generated after a mouse is immunized. The invention lays a foundation for the improvement of the preparation process of the porcine epidemic diarrhea virus and the inactivated vaccine thereof in large-scale production.
Drawings
FIG. 1 shows the viral titers of shxx1902(P35) in 12h,24h,36h,48h,60h and 72h suspension vero cells.
FIG. 2 shows 24h shxx1902, shxx1912 and SH1302 immunofluorescence assays.
FIG. 3 shows PEDV shxx1902, CV777 and PBS (control) PEDV N protein-specific antibody responses.
Fig. 4 shows an evolutionary tree of the entire gene of porcine epidemic diarrhea virus strain (shxx 1902).
Fig. 5 shows an evolutionary tree of the S gene of a porcine epidemic diarrhea virus strain (shxx 1902).
Detailed Description
1 materials and methods
1.1 cells and virus vero suspension cells are purchased from commercial North City Nao Chuanglian Biotech Co., Ltd, the porcine epidemic diarrhea virus strain (shxx1902) is separated and stored in the laboratory, and the preservation number of the porcine epidemic diarrhea virus variant shxx1902 is CGMCC No. 22380.
1.2 analysis of the sequence of the entire Gene of shxx1902 (passage 5) viruses Genbank MN841671 was analyzed for the entire gene sequence.
1.3 Experimental animals SPF-grade BALB/c mice, female, 6 weeks old, 24, weight 18-22 g, purchased from Shanghai Prolitz Biotech, Inc.
1.4 main reagents and an anti-mouse monoclonal antibody marked by FITC of an instrument, wherein fetal calf serum is purchased from sigma company; DMEM medium, pancreatin purchased from GIBCO; the alumina gel adjuvant was purchased from bio-technology ltd; porcine epidemic diarrhea virus S1D12 monoclonal antibody was purchased from veteriary media research & reduction corporation; 500mL cell shake flasks were purchased from CORNING.
1.5 optimization of Shake flask culture conditions to resuscitate Vero cells, using culture medium containing 10% fetal bovine serum at 37 deg.C and 5% CO2In an incubator, then digesting, passaging to a cell shake flask, diluting with serum-free medium to reach a cell density of 6X 106one/mL.
1.6 optimal virus-killing time in the virus culture stage refers to the optimal condition in the shake flask virus culture. Culturing until the cell density reaches 6 × 106At one/mL, 0.1L of cell culture medium was added, and dilution and passage were carried out, with the cell density after passage being 3X 106one/mL. The inoculation MOI is 0.1, pH is 7.4 + -0.1, shaker speed is 120r/min, and culture temperature is (37 + -0.1) ° C. Repeat 3 times. Sampling every 12h, detecting the virus titer at 0,12,24,36,48,60 and 72 hours, drawing a virus one-step growth curve and determining the optimal virus recovery time. The harvested virus fluid was centrifuged at 1600 Xg to remove cell debris, and the supernatant was split and frozen at-80 ℃.
The growth kinetics of the virus are shown in FIG. 1, and the virus titers at 37 ℃ for 12,24,36,48,60, and 72h are as follows: 1.28X 105,8.78×105,2.01×106,4.52×106,5.41×106And 8.20X 106TCID50/mL。
1.7 Virus TCID50The detection adopts an indirect immunofluorescence staining method, and 5 multiplied by 10 is taken4One/well vero cells were added to 96-well plates and virus was serially diluted 10-fold in DMEM (10)-1～10-8) Then, adding the mixture into a 96-well plate in sequence, culturing the mixture for 48 hours at 37 ℃ in a 5% CO2 incubator at 50 mu L/well; discarding the supernatant, adding 80% cold acetone to fix the cells, standing overnight at-20 deg.C, and washing the cells with PBST for 3 times, each time for 5 min; adding S1D12 monoclonal antibody, incubating at 37 deg.C for 2h, and washing cells with PBST for 5min for 3 times; FITC labeled goat anti-mouse secondary antibody working solution with a concentration of 50 muL/well is kept away from light and kept flat in a 37 ℃ incubator for 1h, PBST is used for washing for 3 times, 5min each time, DAPI is added for incubation for 5min, and PBST is used for washing cells for 3 times, 5min each time; green fluorescence (virus), blue fluorescence (nuclei) with 1 or more fluorescently labeled wells "+", wells without green spots: "-".
1.8 immunogen preparation titerIs 2 x 109TCID50Adding 0.2% formaldehyde into the virus solution at a final concentration, and inactivating the virus at 37 ℃ for 28 h; adding into 96-well plate with vero cells, culturing at 37 deg.C and 5% CO2 incubator for 2d at 50 μ L/well; harvesting cell supernatant, fixing the cells by cold acetone, adding the harvested supernatant into a 96-well plate paved with vero cells, and continuing culturing for 2 d; discarding the supernatant, fixing the cells by cold acetone, inactivating by 1.7 methods, and using the cells and an alumina gel adjuvant according to the ratio of antigen to adjuvant of 1:1 after the cells are qualified by verification; 10 at the same time as vaccine CV7775TCID 50/mouse was formulated.
1.9 mice grouping and immunization BABL/c mice were randomly divided into 3 groups of 8 mice each, which were respectively a 2-time alumina gel adjuvant PEDV variant group, a 2-time immunization CV777 vaccine group, and a 2-time PBS control group. The mice in the immunization group were injected with 0.1mL of immunogen per hind limb muscle, and the control group was immunized with 0.1mL of LPBS. Immunizations were repeated after 2 immunization groups at interval 14 d. Blood was collected on days 0,7,14,21,28,35,42 and 49, serum was collected after centrifugation, antibody titer was measured using indirect ELISA, and ELISA plates were coated with N protein expressed by e.
As shown in fig. 3, it was always monitored that the shxx1902 and CV777 vaccine groups produced similar ELISA antibody titers at day 49 post-immunization. Mice at 3 weeks of age were inoculated subcutaneously at multiple points with inactivated PEDV shxx1902, CV777 vaccine and PBS. Serum samples were collected on days 0,7,14,21,28,35,42 and 49 and tested by indirect ELISA and data are expressed as mean OD450 ± SD.
1.10 immunofluorescence assay shxx1902 was detected using S protein monoclonal antibody, followed by incubation of FITC-labeled goat anti-mouse secondary antibody with DAPI for nuclear staining. Vero cells infected with shxx1902 stained positive for immunofluorescence. Mock was negative (fig. 2).
1.11 statistical analysis was performed using the statistical software GraphPad prism8.0.1 and experimental data were analyzed using the One-way ANOVA method, expressed as mean. + -. SD as the mean.
Taken together, the strains of pdv shxx1902 and CV777 induced a higher and similar N protein-specific antibody response against porcine epidemic diarrhea virus at day 49 after the first immunization (2 immunizations total). Thus, the discovery of the present invention will help to develop new isolation methods and vaccine strategies that are expected to be an important step in improving the prevention of PEDV pandemics.
Sequence listing
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ttgtagacct ctgtaccaag aacattggta ctgctggttt tcatgagttt tacattacgg 2340
cccatgaaca acaggatctg caagggttcg taaccacttg ttgcacgatg tcaggttttg 2400
agtgttttat gcctataatc ccacagtgtc cagcagtgct tgaagagatt gatggtggta 2460
gcatctggcg gtcttttatc actggtctta atacaatgtg ggatttttgc aagcatctta 2520
aagtcagctt tggactagat ggcattgttg tcactgtagc acgcaaattt aaacgacttg 2580
gtgctctctt ggcagaaatg tataacactt acctttcaac tgtggtggaa aacttggtac 2640
tggccggtgt tagcttcaag tattatgcca ccagtgtccc aaaaattgtt ttgggctgtt 2700
gtttttacag tgttaaaagt gttcttgcga gtgccttcca gattcctgtc caggcaggca 2760
ttgagaattt taaagtcttt cttaactgtg ttcaccctgt tgtaccacgc gtcattgaaa 2820
cttcttttgt ggaattagaa gagacgacat ttaaaccacc agcactcaat ggtagtattg 2880
ctattgttga tggctttgct ttctattatg atggaacact atactatccc accgatggta 2940
atagtgttgt tcctatctgc tttaagaaga aaggtggtgg tgatgtcaaa ttctctgatg 3000
aagtttctgt taaaaccatt gacccagttt ataaggtctc ccttgaattt gagttcgagt 3060
ctgagactat tatggctgtg cttaataagg ctgttggtaa ccgtatcaag gttacaggtg 3120
gttgggacga tgttgttgag tatatcaacg ttgccattga ggttcttaaa gatcacatcg 3180
atgtgcctaa gtactatatt tatgatgagg aaggtggcac cgatcctaat cttcccgtaa 3240
tggtttctca gtggccgctg aatgatgaca cgacctcaca ggatctgctt gatgtggaag 3300
ttgttacgga tgcaccaatt gactccgagg gtgatgaagt ggactcctct gaccctgata 3360
aggtggcaga tgtggctaac tctgagcctg aggatggtgt tcttaatgta gctcctgaaa 3420
caaatgtaga gtctgaagtt gaggaagttg ccgcaacctt gtcctttatt aaagatacac 3480
cttccacagt tactaaggat ccttttgctt ttgactttgc aagctatgga ggacttaagg 3540
ttttaagaca atctcataac aactgttggg ttacttctac cttggtgcag ctacaattgc 3600
ttggcatcgt tgatgaccct gcaatggagc tctttagtgc tggtagagtc ggtccaatgg 3660
ttcgcaaatg ctatgagtca caaaatgcta ttttgggatc tttgggtgat gtatcggctt 3720
gtctagagtc tcttactaag aacctacaca cacttaagat tacctgttct gtagtctgtg 3780
gttgtggtac tggtgaacgc atctatgagg gctgtgcttt tcgtatgacg ccaactttgg 3840
aactgttccc atatggtgct tgtgctcagt gtgctcaagt tttgatgcac acttttaaaa 3900
gtattgttgg caccggcatc ttttgtcgag atactactgc tctctccttg gattctttgg 3960
ttgtaaaacc tctttgtgcg gctgctttta taggcaagga tagtggtcat tatgtcacca 4020
acttttatga tgctgctatg gctattgatg gttatggtcg tcatcagata aagtatgaca 4080
cactgaacac catttgtgtt aaagacgtga attggacagc accttctgtc cttgacgttg 4140
cgcctgtatt gaagcctgtt gtcaaacctt tctattctta taagaatgtt gatttttacc 4200
aaggagattt tagtgacctt gttaaacttc catgtgactt tgttgttaat gctgcaaatg 4260
agaatttgtc tcacggtggc ggcatagcaa gggccattga tgtttatacc aagggcatgt 4320
tgcagaagtg ttcgaatgat tacattaaag cacacggtcc cattaaagtt ggacgtggtg 4380
tcatgttgga ggcattaggt cttaaggtct ttaatgttgt tggtccacgt aagggtaagc 4440
atgcacctga gcttcttgtt aaggcttata agtccgtttt tgctaattca ggtgttgctc 4500
ttacaccttt gattagtgtt ggaattttta gtgttccttt ggaagaatct ttatctgctt 4560
ttcttgcatg tgttggtgat cgccactgta agtgcttttg ttatagtgac aaagagcgcg 4620
aggcgatcat taattacatg gatggcttgg tagatgctat tttcaaagat gcgcttgttg 4680
atactattcc tgtccaggaa gatgttcaac aagtttcaca aaaaccagtt ttgcctaatt 4740
ttgaaccttt caggattgaa ggtgctcatg ctttctatga gtgtgaccct gaaggtttga 4800
tgtccttagg tgctgacaag ctggtgttgt ttacaaattc cactttggat ttttgtagcg 4860
ttggtaagtg tcttaacaat gtgaccggcg gtgcattgct tgaagccata aatgtattta 4920
aaaagagtaa caaaacagtg cctgctggca actgtgttac ttttgagtgt gcagatatga 4980
tttctattac tatggtagta ttgccagctg atggtgatgc taattatgac aaaaattatg 5040
cacgcgccgt tgttaaggta tctaagctta aaggcaagtt attgcttgct gttggtgatg 5100
ccacgttgta ttccaagttg tcccacctta gcgtggtagg tttcgtatcc acacctgatg 5160
atgtggagcg tttctacgca aataagagtg tggttattaa agtcactgag gatacacgta 5220
gtgttaaggc tgttaaagta gaatccactg ttacttatgg acaacaaatt ggaccttgtc 5280
ttgttaatga caccgttgtt acagacaaca aacctgttgt tgctgatgtt gtagctaagg 5340
ttgtaccaag tgctaattgg gattcacatt atggttttga taaggcaggt gagttccata 5400
tgctagacca tactgggttt gcctttctta gtgaagttgt taacggtagg cgtgtgctta 5460
aaaccacaga taataactgt tgggttaatg ttacatgttt acaattacag tttgctagat 5520
ttaggttcaa gtcagcaggt ctacaggcta tgtgggagtc ctattgtact ggtgatgttg 5580
ctatgtttgt gcattggttg tactggctta ctggtgttga caaaggtcag cctagtgatt 5640
cagaaaatgc acttaacatg ttgtccaagt acattgttcc tgctggttct gttactattg 5700
aacgtgtcac gcatgacggc tgttgttgta gtaaacgtgt tgtcactgca ccagttgtta 5760
atgctagcgt tttgaagctt ggtgtcgagg atggtctttg tccacatggt cttaactaca 5820
ttgacaaagt tgttgtagtt aaaggtacta caattgttgt caatgttgga aaacctgtgg 5880
tggcaccttc acacctcttt ctcaagggtg tttcctacac aacattccta gataatggta 5940
acggtgttgt cggccattat actgtttttg atcatgacac tggtatggtg tatgatggag 6000
atgcttttgt accgggtgac ctcaatgtat cccctgttac aaatatcgtt gtctcagagc 6060
agacggctgt tgtgattaaa gaccccgtga agaaagtaga gttagacgct acaaagctgt 6120
tagacactat gaattatgca tcggaaagat tcttttcctt tggtgatttc atgtcacgta 6180
atttaattac agtgtttttg tacatcctta gcattttggg tctctgtttt agggcctttc 6240
gtaagagaga tgttaaagtt ctagctggtg taccccaacg tactggtatt atattgcgta 6300
aaagtgtgcg ctataatgca aaggcgttgg gtgtcttttt caagctaaag ctttattggt 6360
tcaaagttct tggtaagttt agtttgggtg tttatgcatt gtatgcacta ctattcatga 6420
caatacgctt tacacctata ggtagccctg tttgtgatga tgttgttgct ggttatgcta 6480
attctagttt tgataagaat gagtattgca acagtgttat ttgtaaggtc tgtctatatg 6540
ggtaccagga actctcggac ttctcccaca cacaggtagt atggcaacac cttagagacc 6600
cattaattgg taatgtgatg cctttctttt atttggcatt tttggcaatt tttgggggtg 6660
tttatgtaaa ggctattact ctctatttta tttttcagta tcttaacatt cttggtgtgt 6720
ttttgggctt acaacagtcc atttggtttt tgcagcttgt gccttttgat gtttttggcg 6780
acgagatcgt cgtctttttc atcgttacac gcgtattgat gttccttaag catgttttcc 6840
ttggctgtga taaggcatct tgtgtggctt gctctaagag tgctcgtctt aagcgcgttc 6900
ctatccagac tatctttcag ggtactagca aatccttcta cgtacatgcc aatggtggtt 6960
ctaagttctg taagaagcac aatttctttt gtttaaattg tgattcttat ggtccaggct 7020
gcacttttat taatgacgtc attgcaactg aagttggtaa tgtcgtcaaa cttaatgtgc 7080
aaccgacagg tcctgccact attcttattg acaaggttga attcagtaat ggtttttact 7140
atctttatag tggtgacaca ttttggaagt acaactttga cataacagat agcaaataca 7200
cttgcaaaga ggcacttaaa aattgtggca taatcacaga ctttattgtt tttaacaata 7260
atggttccaa tgtaaatcag gttaagaatg catgtgtgta tttttcacag atgctttgta 7320
aacctgttaa attagtggac tcagcgttgt tggccagttt gtctgttgat tttggtgcaa 7380
gtttacatag tgcttttgtt agtgtgttgt cgaatagttt cggcaaagac ctgtcaagtt 7440
gtaatgacat gcaggattgc aagagcacat tgggttttga tgatgtacca ttggatacct 7500
ttaatgctgc tgttgctgag gctcatcgtt atgatgtcct cttgactgac atgtcattca 7560
acaattttac caccagttat gcaaaaccag aggaaaaatt tcccgtccat gacattgcca 7620
cgtgtatgcg tgtaggtgcc aagattgtta atcataacgt tcttgtcaaa gatagtatac 7680
ctgtggtgtg gctcgtacgt gatttcattg ccctttcgga agaaactagg aagtacatta 7740
ttcgtacgac taaagttaag ggtataacat ttatgctgac ctttaatgat tgtcgtatgc 7800
atactaccat acctactgtt tgcattgcaa ataagaaggg tgcaggtctt cctagttttt 7860
caaaggttaa gaaattcttt tggtttttgt gtctattcat agttgctgtt ttctttgcac 7920
taagctttct tgattttagt attcaggtta gcagtgatag cgattatgat ttcaagtata 7980
ttgagagtgg ccagttgaag acttttgaca atccacttag ttgtgtgcat aatgtcttta 8040
gtaacttcga ccagtggcat gatgccaagt ttggtttcac ccccgtcaac aatcctagtt 8100
gtcctatagt tgttggtgtg tcagacgaag ctcgcactgt tccaggtatc ccagcaggtg 8160
tttatttagc tggtaaaaca cttgtgtttg ctattaacac catttttggt acatctggtt 8220
tgtgctttga tgctagtggc gttgctgata agggcgcttg catttttaat tcggcttgca 8280
ccacattatc cggtttgggt ggaactgctg tctactgtta taagaatggt ctagttgaag 8340
gtgctaaact ttatagtgag ttggcacctc atagctacta taaaatggta gatggtaatg 8400
ctgtgtcttt acctgaaatt atctcacgcg gctttggcat ccgtactatc cgtacaaagg 8460
ctatgaccta ctgtcgcgtt ggccagtgtg tgcaatctgc agaaggtgtt tgttttggcg 8520
ccgatagatt ctttgtctat aatgcagaat ctggttctga ctttgtttgt ggcacggggc 8580
tcttcacatt gttgatgaac gttattagtg ttttttccaa gacagtacca gtaactgtgt 8640
tgtctggtca aatacttttt aattgcatta ttgcttttgc tgctgttgcg gtgtgtttct 8700
tatttacaaa gtttaagcgc atgttcggtg atatgtctgt tggcgttttc actgtcggtg 8760
cttgtacttt gttgaacaat gtttcttaca ttgtaacaca gaatacactt ggcatgttgg 8820
gctatgcaac tttgtacttt ttgtgcacta aaggtgttag atatatgtgg atttggcatt 8880
tgggattttt gatctcatat atacttattg caccatggtg ggttttgatg gtttatgcct 8940
tttcagccat ttttgagttt atgcctaacc tttttaagct taaggtttca acacaacttt 9000
ttgagggtga caagttcgta ggctcttttg aaaatgctgc agcaggtaca tttgtgcttg 9060
atatgcatgc ctatgagaga cttgccaatt ctatctcaac tgaaaaactg cgtcagtatg 9120
ctagtactta caataagtac aagtattatt caggcagtgc ttcagaggct gattacaggc 9180
ttgcttgttt tgcccatttg gccaaggcta tgatggatta tgcttctaat cacaacgaca 9240
cgttatacac accacccact gtgagttaca attcaactct acaggctggc ttgcgtaaga 9300
tggcacaacc atctggtgtt gttgagaagt gcatagttcg tgtttgctat ggtaatatgg 9360
ctcttaatgg cctatggctt ggtgatactg ttatctgccc acgccatgtt atagcgtcta 9420
gtactactag cactatagat tatgactatg ccctttctgt tttacgcctc cacaacttct 9480
ccatttcatc tggtaatgtt ttcctaggtg ttgtgggtgt aaccatgcga ggtgctttgt 9540
tgcagataaa ggttaatcaa aacaatgtcc acacgcctaa gtacacctat cgcacagtta 9600
gaccgggtga atcttttaat atcttggcgt gctatgatgg ttctgcagct ggtgtttacg 9660
gcgttaacat gcgctctaat tacactatta gaggctcgtt cattaatggc gcttgtggtt 9720
cacctggtta taacattaac aatggtaccg ttgagttttg ctatttacac cagcttgaac 9780
ttggttcagg ctgtcatgtt ggtagcgact tagatggtgt tatgtatggt ggttatgagg 9840
accaacctac tttgcaagtt gaaggcgcta gtattctgtt tacagagaat gtgttggcat 9900
ttctttatgc agcactcatt aatggttcta cctggtggct tagttcttct aggattgctg 9960
tagacaggtt taatgagtgg gctgttcata atggtatgac aacagtagtt aatactgatt 10020
gcttttctat tcttgctgct aagactggtg ttgatgtaca aagtttgttg gcctcaatcc 10080
agtctctgca taagaatttt ggtggaaagc aaattcttgg ctatacctcg ttgacagatg 10140
agtttactac aggtgaagtt atacgtcaaa tgtatggcgt taatcttcag agtggttatg 10200
tttcacgcgc ctgtagaaat gtcttgctgg ttggttcttt tctgactttc ttttggtctg 10260
aattagtttc ctacactaag ttcttttggg taaatcctgg ttatgtcaca cctatgtttg 10320
cgtgtttgtc attgctgtcc tcacttttga tgttcacact caagcataag acattgtttt 10380
tccaggtctt tctaatacct gctctgattg ttacatcttg cattaatttg gcatttgatg 10440
ttgaagtcta caactatttg gcagagcatt ttgattacca tgtttctctc atgggtttta 10500
atgcacaagg tcttgttaac atctttgtct gctttgttgt taccatttta cacggcacat 10560
acacaaggcg cttttttaac acacctgtga gttctgtcac ttatgtggta gctttgctga 10620
ctgcggcata taactatttt tacgctagtg acattcttag ttgtgctatg acactatttg 10680
ctagtgtgac tggcaactgg ttcgttggtg ctgtttgtta taaagctgct gtttatatgg 10740
ccttgagatt tcctactttt gtggctattt ttggtgatat taagagtgtt atgttctgtt 10800
accttgtgtt gggttatttt acctgttgct tctacggtat tctctactgg ttcaacaggt 10860
tctttaaggt tagtgtaggt gtctatgact atactgttag tgctgctgag tttaagtata 10920
tggttgctaa cggcctacgt gcaccaactg gaacacttga ttcactactt ctgtctgcca 10980
aattgattgg tattggtggt gagcggaata ttaagatttc ttccgttcag tctaaactga 11040
ctgatattaa gtgtagtaac gttgtgcttt taggctgtct ctctagcatg aatgtctcag 11100
caaattcaac agaatgggcc tattgtgttg acttgcataa caagatcaac ctgtgtaatg 11160
acccagaaaa agcgcaggaa atgctacttg ctttgttggc atttttcctt agtaagaata 11220
gtgcttttgg tttagatgac ttattggaat cctattttaa tgacaatagt atgttgcaga 11280
gtgttgcatc tacttatgtc ggtttgcctt cttatgtcat ttatgaaaat gcacgccaac 11340
agtatgaaga tgctgttaat aatggttctc cacctcagtt ggttaagcaa ttgcgccatg 11400
ctatgaatgt agcaaagagc gaatttgacc gtgaggcttc tactcagcgt aagcttgata 11460
gaatggcgga acaggctgca gcacagatgt acaaagaggc acgagcagtc aataggaagt 11520
ccaaagttgt aagtgctatg cattcactgc tttttggtat gttgagacgt ttggatatgt 11580
cttctgtaga caccattctc aacttggcaa aggatggggt tgtacctctg tctgtcatac 11640
cggcagtcag tgctactaag cttaacattg ttacttctga tatcgattct tataatcgta 11700
tccagcgtga gggatgtgtc cattacgctg gtaccatttg gaatataatt gatatcaagg 11760
acaatgatgg caaggtggta cacgttaagg aggtaaccgc acagaatgct gagtccctgt 11820
catggcccct ggtcctaggg tgtgagcgta ttgttaagct ccagaataat gaaattattc 11880
ctggtaagct gaagcagcgc tccattaagg cagaaggaga tggcatagtt ggagaaggta 11940
aggcacttta caataatgag ggtggacgta cttttatgta tgctttcatc tcggataaac 12000
cggacctgcg tgtagttaag tgggagttcg atggtggttg taacactatt gagctagaac 12060
caccacgtaa gtttttggtg gattctccta atggtgcaca gatcaagtat ctctactttg 12120
tccgtaacct taacacgtta cgtaggggtg ctgttcttgg ctacataggt gccactgtac 12180
gcttgcaggc tggtaaacaa acagaacagg ctattaactc ttcattgttg acactttgcg 12240
ctttcgctgt ggatcctgct aagacctaca tcgatgctgt caaaagtggt cacaaaccag 12300
taggtaactg tgttaagatg ttggccaatg gttctggtaa tggacaagct gttactaatg 12360
gtgtggaggc tagtactaac caggattcat atggtggtgc gtccgtgtgt ctatattgta 12420
gagcacatgt tgagcatcca tctatggatg gtttttgcag actgaaaggc aagtacgtac 12480
aggttccact aggtacagtg gatcctatac gttttgtact tgagaatgac gtttgcaagg 12540
tttgtggttg ttggctggct aatggctgca cttgtgacag atccattatg caaagtactg 12600
atatggctta tttaaacgag tacggggctc tagtgcagct cgactagagc cctgtaacgg 12660
tactgataca caacatgtgt atcgtgcttt tgacatctac aacaaggatg ttgcttgtct 12720
aggtaaattc ctcaaggtga actgtgttcg cctgaagaat ttggataagc atgatgcatt 12780
ctatgttgtc aaaagatgta ccaagtctgc gatggaacac gagcaatcca tctatagcag 12840
acttgaaaag tgtggagccg tagccgaaca cgatttcttc acttggaagg atggtcgtgc 12900
tatctatggt aacgtttgta gaaaggatct taccgagtat actatgatgg atttgtgtta 12960
cgctttacgt aactttgatg aaaacaattg tgatgttctt aagagcattt taattaaggt 13020
aggcgcttgt gaggagtcct actttaataa taaagtctgg tttgaccctg ttgaaaatga 13080
agacattcat cgtgtctatg cattgttagg taccattgtt tcacgtgcta tgcttaaatg 13140
cgttaagttc tgtgatgcaa tggttgaaca aggtatagtt ggtgttgtca cattagataa 13200
tcaggatctt aatggtgatt tttatgattt tggtgatttt acttgtagca tcaagggaat 13260
gggtataccc atttgcacat catattactc ttatatgatg cctgttatgg gtatgactaa 13320
ttgccttgct agtgagtgtt ttgttaagag tgatatattt ggtgaggatt tcaagtcata 13380
tgacctgctg gaatatgatt tcacggagca taagacagca ctcttcaaca agtatttcaa 13440
gtattgggga ctgcaatacc accctaactg tgtggactgc agtgatgagc agtgcatagt 13500
tcactgtgcc aacttcaata cgttgttttc cactactata cctattacgg catttggacc 13560
tttgtgtcgc aagtgttgga ttgatggtgt tccactggta actacagctg gttatcattt 13620
taaacagtta ggtatagttt ggaacaatga cctcaactta cactctagca ggctctctat 13680
taacgaacta ctccagtttt gtagtgatcc tgcattgctt atagcatcat caccagccct 13740
tgttgatcaa cgtactgttt gcttttcagt tgcagcgcta ggtacaggta tgactaacca 13800
gactgttaaa cctggccatt tcaataagga gttttatgac ttcttacttg agcaaggttt 13860
cttctctgag ggctctgagc ttactttaaa gcacttcttc tttgcacaga agggtgatgc 13920
agctgttaag gattttgact actataggta taatagacct actgttctgg acatttgcca 13980
agctcgcgtc gtgtatcaaa tagtgcaacg ctattttgat atttacgaag gtggttgtat 14040
cactgccaaa gaagtggttg ttacaaacct taacaagagc gcaggttatc ctttgaacaa 14100
gtttggtaaa gctggtcttt actatgagtc tttatcctat gaggaacagg atgaacttta 14160
tgcttatact aagcgtaaca tcctgcccac tatgacacag ctcaacctta aatatgctat 14220
aagtggcaaa gaacgtgcac gcacagtggg tggtgtttcg cttttgtcaa ccatgactac 14280
tcggcagtat catcaaaaac accttaagtc catagttaat actaggggcg cttcggttgt 14340
tattggtact actaagtttt atggtggttg ggacaatatg cttaagaacc ttattgatgg 14400
tgttgaaaat ccgtgtctta tgggttggga ctacccaaag tgcgacagag cactgcccaa 14460
tatgatacgc atgatttcag ccatgatttt aggctctaag cacaccacat gctgcagttc 14520
cactgaccgc tttttcaggt tgtgcaatga attggctcaa gtccttactg aggttgttta 14580
ttctaatgga ggtttttatt tgaagccagg tggtactacc tctggtgatg caaccaccgc 14640
atatgcaaac tcagttttca atatcttcca agcagtaagt gccaatgtta acaaacttct 14700
tagtgttgac agcaatgtct gtcataattt agaagttaag caattgcagc gtaagcttta 14760
tgagtgctgt tatagatcaa ctaccgtcga tgaccagttc gtcgttgagt attatggtta 14820
cttgcgtaaa catttttcaa tgatgattct ttctgatgat ggcgttgttt gttataacaa 14880
tgactatgca tcacttggtt atgttgctga tcttaacgca ttcaaggctg ttttgtatta 14940
ccagaacaat gtcttcatga gcgcctctaa atgttggatc gagcctgaca ttaataaagg 15000
tcctcatgaa ttttgttcgc agcatactat gcagattgtc gataaggatg gtacttatta 15060
ccttccttac cctgatcctt caagaatcct ctctgcaggt gtgtttgttg atgacgttgt 15120
taaaactgat gcagttgtat tgcttgaacg ttatgtgtca ttggctatag atgcctaccc 15180
gttatctaag catgaaaacc ctgaatataa gaaggtgttt tatgtgcttt tggattgggt 15240
taagcatctg tacaaaactt tgaatgctgg tgtgttagag tctttttctg tcacactttt 15300
ggaagattct actgctaaat tctgggatga gagcttttat gccaacatgt atgagaaatc 15360
tgcagtttta caatctgcag ggctttgtgt tgtttgtggc tctcaaactg ttttacgttg 15420
tggtgattgt ctacggcgtc ctatgctttg tactaagtgt gcttatgatc atgtcgttgg 15480
aacaactcac aagttcattt tggccatcac tccatatgtg tgttgtgctt cagattgtgg 15540
tgtcaatgat gtaactaagc tctacttagg tggtcttagt tattggtgtc atgaacacaa 15600
gccacgtctt gcattcccgt tgtgctctgc tggtaatgtt tttggcttgt acaaaaattc 15660
tgctaccggc tcacccgacg ttgaagactt taatcgcatt gctacatccg attggactga 15720
tgtttctgac tacaggttgg caaatgatgt caaggactca ttgcgtctgt ttgcagcgga 15780
aactatcaag gccaaggagg agagcgttaa gtcatcctac gcttgtgcaa cactacatga 15840
ggttgtagga cctaaagagt tgttgctcaa atgggaagtc ggcagaccca aaccacccct 15900
taatagaaat tcggttttca cttgttatca tataacgaag aacaccaaat ttcaaatcgg 15960
tgagtttgtg tttgagaagg cagaatatga taatgatgct gtaacatata aaactaccgc 16020
cacaacaaaa cttgttcctg gcatggtttt tgtgcttacc tcacataatg ttcagccatt 16080
gcgcgcaccg accattgcta atcaagaacg ttattccact atacataagt tgcatcctgc 16140
ttttaacata catgaagctt attctagctt agtgccctat taccaactga ttggtaagca 16200
gaagattaca actatccagg gacctcccgg tagtggtaaa tctcactgtg ttatagggct 16260
aggtttgtac tatccaggtg cacgtatagt gtttacagct tgttctcatg cagcggtcga 16320
ttcactttgt gtgaaagcct ccactgctta tagcaatgac aaatgttcac gcatcatacc 16380
acagcgtgct cgtgttgagt gttatgacgg tttcaagtct aataatacta gtgctcagta 16440
ccttttctcc actgtcaatg ctttgccaga gtgcaatgcg gacattgttg tggtggatga 16500
ggtttctatg tgcactaatt atgacttgtc tgtcataaat cagcgcatca gctataggca 16560
tgtagtatat gttggtgacc ctcaacagct gcctgcacca cgtgttatga tttcacgtgg 16620
tactttggaa ccaaaggact acaacgttgt cactcaacgc atgtgtgccc ttaagcctga 16680
tgttttcttg cacaagtgtt atcgttgtcc tgctgagata gtgcgcactg tgtctgagat 16740
ggtctatgaa aaccaattca ttcctgtgca cccagatagc aagcagtgtt ttaaaatctt 16800
ttgcaagggt aatgttcagg ttgacaatgg ttcaagcatt aatcgcaggc aattggatgt 16860
tgtgcgtatg tttttggcta aaaaccctag gtggtcaaag gctgttttta tttctcctta 16920
taacagccag aattatgttg ccagccgcat gctaggttta caaattcaga cagttgactc 16980
atcccagggt agtgagtatg actatgtcat ttatacacaa acttcagata ctgcccatgc 17040
ctgtaatgtt aacaggttta atgttgccat cacaagggct aagaaaggca tattatgtat 17100
aatgtgcgat aggtcccttt ttgatgtgct taaatttttt gagcttaaat tgtctgattt 17160
gcaggctaat gagggttgtg gtctttttaa agactgtagc agaggtgatg atttgttgcc 17220
accatctcac gctaacacct tcatgtcttt agcggacaat tttaagactg atcaagatct 17280
tgctgttcaa ataggtgtta atggatccat taaatatgag catgttatct cgtttatggg 17340
cttccgtttt gatatcaaca tacccaacca tcacactctc ttttgcacac gcgactttgc 17400
catgcgcaat gttagaggtt ggttgggttt tgacgttgaa ggagcacatg ttgttggctc 17460
taacgtcggt acaaatgtcc cattgcaatt agggttttct aacggtgttg attttgttgt 17520
cagacctgaa ggttgcgttg taactgagtc tggtgactac attaaacccg tcagagctcg 17580
tgctccacca ggtgaacaat ttgcacacct tttgcctcta ctcaaacgcg gccaaccatg 17640
ggatgtggtt cgtaagcgta tagtgcaaat gtgtagtgac tacctggcta acctatcaga 17700
catactaatt tttgtgttgt gggctggtgg tttggagttg acaactatgc gttactttgt 17760
caagattgga ccaagcaaga gttgtgattg tggtaaggtt gctacttgtt acaatagtgc 17820
gttgcatacg tactgttgtt tcaaacatgc ccttggttgt gattatctgt ataacccata 17880
ctgtattgat atacagcagt ggggatacaa gggatcactt agtcttaacc accatgagca 17940
ttgtaatgta catagaaacg agcatgtggc ttctggtgat gccataatga ctcgctgtct 18000
ggctatacat gattgctttg tcaagaacgt tgactggtcc atcacatacc catttattgg 18060
taatgaggct gttattaata agagcggccg aattgtgcaa tcacacacca tgcggtcagt 18120
tcttaagtta tacaatccga aagccatata tgatattggc aatcctaagg gcatcagatg 18180
tgccgtaacg gatgctaagt ggttttgctt tgacaagaat cctactaatt ctaatgtcaa 18240
gacattggag tatgactata taacacatgg ccaatttgat gggttgtgct tgttttggaa 18300
ttgcaatgta gacatgtatc cagaattttc tgtagtctgt cgttttgata ctcgctgtag 18360
gtcaccactc aacttggagg gttgtaatgg tggttcactg tatgttaata atcatgcatt 18420
ccatacaccg gcttttgaca agcgtgcttt tgctaagttg aagccaatgc catttttctt 18480
ttatgatgat actgagtgtg acaagttaca ggactccata aactatgttc ctcttagggc 18540
tagtaactgc attactaaat gtaatgttgg tggagctgtc tgtagtaagc attgtgctat 18600
gtatcatagc tatgttaatg cttacaacac ttttacgtcg gcgggcttta ctatttgggt 18660
gcctacttcg tttgacacct ataatctgtg gcagacattt agtaacaatt tgcaaggtct 18720
tgagaacatt gctttcaatg tcgtaaagaa aggatctttt gttggtgccg aaggtgaact 18780
tcctgtagct gtggttaatg acaaagtgct cgttagagat ggtactgttg atactcttgt 18840
ttttacaaac aagacatcac tgcccactaa cgtagctttt gagttgtatg ccaagcgtaa 18900
ggtaggactc accccaccca ttacgatcct acgtaacttg ggtgtagttt gtacatctaa 18960
gtgtgtcatt tgggactatg aagccgaacg tccacttact acttttacaa aggatgtttg 19020
taaatatacc gactttgagg gtgacgtctg tacactcttt gataacagca ttgttggttc 19080
attagagcga ttctctatga cccaaaatgc tgtgcttatg tcacttacag ccgttaaaaa 19140
gcttactggc ataaagttaa cttatggtta tcttaatggt gtcccagtta acacacatga 19200
agataaacct tttacttggt atatttacac taggaagaac ggcaaattcg aggactatcc 19260
tgatggctat tttacccaag gtagaacaac cgctgatttt agccctcgta gcgatatgga 19320
aaaggacttc ctaagtatgg atatgggtct gtttattaac aagtacggac ttgaagatta 19380
cggctttgag cacgttgtgt atggtgatgt ttcaaaaacc acccttggtg gtttacatct 19440
actaatttcg caggtgcgtc tgtcctgtat gggtgtgctt aaaatagacg agtttgtgtc 19500
tagtaatgat agcacgttaa agtcctgtac tgttacatat gctgataacc ctagtagtaa 19560
gatggtttgc acgtatatgg atctcctgct tgacgatttt gtcagcattc ttaaatcgtt 19620
ggatttgagc gttgtatcta aagttcatga agttatggtc gattgtaaaa tgtggaggtg 19680
gatgttgtgg tgtaaggatc ataaactcca gacattttat ccgcaacttc aggccagtga 19740
atggaagtgt ggttattcca tgccttctat ttacaagata caacgtatgt gtttagaacc 19800
ttgcaatctc tataactatg gcgctggtat taagttacct gatggcatta tgtttaacgt 19860
agttaaatac acacagcttt gtcaatatct caatagcacc acaatgtgcg taccccatca 19920
catgcgtgtg ctacatcttg gtgctggctc cgataagggt gttgcacctg gcacggctgt 19980
cttacgacgt tggttgccac tggatgccat tatagttgac aatgatagtg tggattacgt 20040
tagcgatgct gattatagtg ttacaggaga ttgctctacc ttatacctgt cagataagtt 20100
tgacttagtt atatctgata tgtatgacgg taagattaaa agttgtgatg gggagaacgt 20160
gtctaaagaa ggcttctttc cctatattaa tggtgtcatc actgaaaagt tggcacttgg 20220
tggtactgtg gctattaagg tgacggagtt tagttggaat aagaagttgt atgaactcat 20280
tcagaaattt gagtattgga caatgttctg taccagtgtt aacacgtcat cgtcagaggc 20340
atttttaatt ggtgttcact atttaggtga ttttgcaagt ggcgctgtta ttgacggcaa 20400
cactatgcat gccaattata tcttctggcg taattccaca attatgacta tgtcttacaa 20460
tagtgtactt gatttaagca agttcaattg taagcataag gctacagttg ttattaattt 20520
aaaggattca tccattagtg atgttgtgtt aggtttgttg aagaatggta agttgctagt 20580
gcgtaataat gacgccattt gtggtttttc taatcatttg gtcaacgtaa acaaatgaag 20640
tctttaactt acttctggtt cttcttacca gtactttcaa cacttagcct accacaagat 20700
gtcaccaggt gctcagctaa tactaatttt aggcggttct tttcaaaatt taatgttcag 20760
gcgcctgcag ctgttgtact gggcggttat ctacctattg gtgaaaacca gggtgttaat 20820
tcaacttggt actgttatgg ccaacatcca actgctagtg gcgttcatgg tatctttctt 20880
agccatatta gaggtggtca tggctttgag attggcattt cgcaagagcc ttttgaccct 20940
agtggttacc agctttattt acataaggct actaatggta acactaatgc tactgcgcga 21000
ttgcgcattt gccagtttcc cagcattaaa acattgggtc ccactgctaa taatgatgtt 21060
acaacaggtc gtaactgcct atttaacaaa gccatcccag cttatatgag tgaacatagt 21120
gttgtcggca taacatggga taatgatcgt gtcactgtct tttctgacaa gatctatcat 21180
ttttatttta aaaatgattg gtcccgtgtt gcgacaaagt gttacaacag tggaggttgt 21240
gctatgcaat atgtttacga acccacttac tacatgctta atgttactag tgctggtgag 21300
gatggtattt cttatcaact ctgtacagct aattgcattg gttatgctgc caatgtattt 21360
gctactgagc ccaatggcca cataccagaa ggttttagct ttaataattg gtttcttttg 21420
tccaatgatt ccactttggt gcatggtaag gtggtttcca accaaccttt gttggtcaat 21480
tgtcttttgg ccattcctaa gatttatgga ctaggccaat ttttttcatt caatcaaacg 21540
atggatggcg tttgtaatgg ggctgctgtg cagcgtgcac cagaggctct gaggtttaat 21600
attaatgaca gctctgtcat tcttgctgaa ggctcaattg tacttcacac tgctttagga 21660
acaaatcttt cttttgtttg cagtaattct tcagaccctc atttagctac cttcgccata 21720
cctctgggtg ctacccaagt accctattat tgttttctta aagtggatac ttacaacccc 21780
cctgtttata aatttttggc tgttttacct cctaccgtca gggaaattgt catcaccaag 21840
tatggtgatg tttatttcaa tgggttagga tacttgcatc tcggtttgtt ggatgctgtc 21900
acaattaatt tcactggtca tggcactgac gatgatgttt ctggtttttg gaccatagca 21960
tcgactaatt ttgttgatgc actcatcgaa gttcaaggaa ctgccattca gcgtattctt 22020
tattgtgatg atcctgttag ccaactcaag tgttctcagg ttgcttttga ccttgacgat 22080
ggtttttacc ccatttcttc tagaaacctt ctgagtcatg aacagccaat ttcttttgtt 22140
actctgccat catttaatga tcattctttt gttaacatta ctgtctctgc ttcctttggt 22200
ggtcatagtg gtgccaacct tattgcatct gacactacta tcaatgggtt tagttctttc 22260
tgtgttgaaa ctagacaatt taccatttca ctgttttata acgttacaaa cagttatggt 22320
tatgtgtcta actcacagga cagtaattgc cctttcacct tgcaatctgt taatgattac 22380
ctgtctttta gcaaattttg tgtttccacc agccttttgg ctagtgcctg taccatagat 22440
ctttttggtt accctgagtt tggtagtggt gttaagttta cgtcccttta ctttcaattc 22500
acaaagggtg agttgattac tggcacgcct aaaccacttg aaggtgtcac ggacgtttct 22560
tttatgactc tggatgtgtg taccaagtat actatctatg gctttaaagg tgagggtatc 22620
attaccctta caaattctag ctttttggca ggtgtttatt acacatccga ttctggacag 22680
ttgttagcct ttaagaatgt cactagtggt gctgtttatt ctgttacgcc atgttctttt 22740
tcagagcagg ctgcatatgt tgatgatgat atagtgggtg ttatttctag tttgtctaat 22800
tccactttta acagtactag ggagttgcct ggcttcttct accattctaa tgatggctct 22860
aattgtacag agcctgtgtt ggtgtatagc aacataggtg tttgtaaatc tggcagtatt 22920
ggctacgtcc catctcagtc tggccaagtc aagattgcac ccacggttac tgggaatatc 22980
agtattccca ccaactttag tatgagtatt aggacagaat atttacagct ttacaacacg 23040
cctgttagtg ttgattgtgc cacatatgtt tgtaatggta actctcgttg taaacaacta 23100
ctcacccagt acacttcagc atgtaagacc atagagtcag cattacaact cagcgctagg 23160
cttgagtctg ttgaagttaa ctctatgctt actatttctg aagaggctct acagttagct 23220
accattagtt cgtttaatgg tgatggatat aattttacta atgtgctggg tgtttctgtg 23280
tatgatcctg caagtggcag ggtggtacaa aaaaggtctt ttattgaaga cctgcttttt 23340
aataaagtgg ttactaatgg ccttggtact gttgatgaag actataagcg ctgttctaat 23400
ggtcgctctg tggcagatct agtctgtgca cagtattact ctggtgtcat ggtactacct 23460
ggtgttgttg acgctgagaa gcttcacatg tatagtgcgt ctctcatcgg tggtatggtg 23520
ctaggaggct ttacttctgc agcggcattg ccttttagct atgctgttca agccagactc 23580
aattatcttg ctctacagac ggatgttcta cagcggaacc agcaattgct tgctgagtct 23640
tttaactctg ctattggtaa tataacttca gcctttgaga gtgttaaaga ggctattagt 23700
caaacttcca agggtttgaa cactgtggct catgcgctta ctaaggttca agaggttgtt 23760
aactcgcagg gtgcagcttt gactcaactt accgtacagc tgcaacacaa cttccaagcc 23820
atttctagtt ctattggtga catttactct cgactggaca ttctttcagc cgatgttcag 23880
gttgaccgtc tcatcaccgg cagattatct gcacttaatg cttttgtttc tcaaaccctc 23940
actaagtata ctgaggtcca ggctagcagg aagctagcac agcaaaaggt taatgagtgc 24000
gttaaatcgc aatctcagcg ttatggtttt tgtggtggtg atggcgagca cattttctcc 24060
ctggtacagg cagcacctca gggtctgctg tttttacata cagtacttgt accgggtgat 24120
tttgtagatg ttattgccat cgctggctta tgtgttaacg atgaaattgc cttgactcta 24180
cgtgagcctg gcttagtctt gtttacgcat gaacttcaaa atcatactgc gacggaatat 24240
tttgtttcat cgcgacgtat gtttgaacct agaaaaccta ccgttagtga ttttgttcaa 24300
attgagagtt gtgtggtcac ctatgtcaat ttgactagag accaactacc agatgtaatc 24360
ccagattaca tcgatgttaa caaaacactt ggtgagattt tagcttctct gcccaataga 24420
actggtccaa gtcttccttt agatgttttt aatgccactt atcttaatct cactggtgaa 24480
attgcagatt tagagcagcg ttcagagtct ctccgtaata ctacagagga gctccaacgt 24540
tttatatata atatcaacaa cacactagtt gaccttgagt ggctcaaccg agttgagaca 24600
tatatcaagt ggccgtggtg ggtttggttg attatttcca ttgttctcat ctttgttgtg 24660
tcattactag tgttctgctg catttccacg ggttgttgtg gatgctgcgg ctgctgctgt 24720
gcttgttttt caggttgttg taggggtcct agacttcaac cttacgaagt ttttgaaaag 24780
gtccacgtgc agtgatgttt cttggacttt ttcagtacac gattgacaca gttgtcaaag 24840
atgtctcaaa gtctgctaac ttgtctttgg atgctgtcca agagttggag ctcaatgtag 24900
ttccaattag acaagcttca aatgtgacgg gttttctttt caccagtgtt ttcatctact 24960
tctttgcact gtttaaagcg tcttctttga ggcgcaatta tattatgttg gcagcgcgtt 25020
ttgctgtcat tgttctttat tgcccacttt tatattattg tggtgcattt ttagatgcaa 25080
ctattatttg ttgcacactt attggcaggc tttgtttagt ctgcttttac tcctggcgct 25140
ataaaaatgc gctctttatt atttttaata ctacgacact ttctttcctc aatggtaaag 25200
cagcttatta tgacggcaaa tccattgtga ttttagaagg tggtgaccat tacatcactt 25260
ttggcaactc ctttgttgct tttgttagta gcatcgactt gtatctagct atacgtgggc 25320
ggcaagaagc tgacctacag ctgttgcgaa ctgttgagct tcttgatggc aagaagcttt 25380
atgtcttttc gcaacatcaa attgttggca ttactaatgc tgcatttgac tcaattcaac 25440
tagacgagta tgctacaatt agtgaatgat aatggtctag tagttaatgt tatactttgg 25500
cttttcgtac tctttttcct gcttattata agcattactt tcgtccaatt ggttaatctg 25560
tgcttcactt gtcaccggtt gtgtaatagc gcagtttaca tacctatagg gcgtttgtat 25620
agagtttata agtcttatat gcaaatagac cccctcccta gtactgttat tgacgtataa 25680
acgaaatatg tctaacggtt ctattcccgt tgatgaggtg attcaacacc ttagaaactg 25740
gaatttcaca tggaatatca tactgacgat actacttgta gtgcttcagt atggccatta 25800
caagtactct gcgttcttgt atggtgtcaa gatggctatt ctatggatac tttggcctct 25860
tgtgttagca ctgtcacttt ttgatgcgtg ggctagcttt caggtcaatt gggtcttttt 25920
tgctttcagc atccttatgg cttgcatcac tcttatgctg tggataatgt actttgtcaa 25980
tagcattcgg ttgtggcgca ggacacattc ttggtggtct ttcaatcctg aaacagacgc 26040
gcttctcact acttctgtga tgggccgaca ggtctgcatt ccagtgcttg gagcaccaac 26100
tggtgtaacg ctaacactcc ttagtggtac attgcttgta gagggctata aggttgctac 26160
tggcgtacag gtaagtcaat tacctaattt cgtcacagtc gccaaggcca ctacaacaat 26220
tgtctacgga cgtgttggtc gttcagtcaa tgcttcatct ggcactggtt gggctttcta 26280
tgtccggtcc aaacacggcg actactcagc ggtgagtaat ccgagttcgg ttctcacaga 26340
tagcgagaaa gtgcttcatt tagtctaaac agaaacttta tggcttctgt cagttttcag 26400
gatcgtggcc gcaaacgggt gccattatcc ctctatgccc ctcttagggt tactaatgac 26460
aaaccccttt ctaaggtact tgcaaataat gctgtaccca ctaataaagg aaataaggac 26520
cagcaaattg gctattggaa cgagcaaatt cgctggcgca tgcgccgtgg tgagcgaatt 26580
gaacaacctt ctaattggca tttctactac ctcggaacag gacctcacgc cgacctccgc 26640
tataggactc gtactgaggg tgtcttctgg gttgctaaag aaggcgcaaa gactgaaccc 26700
actaacctgg gtgtcagaag ggcgtctgaa aagccaatca ttccaaattt ctctcaacag 26760
cttcccagcg tagttgagat tgttgaacct aatacacctc ctacttcacg tgcaaattca 26820
cgtagcagga gtcgtggtaa tggcaacaac aggtccagat ctccaagtaa caacagaggc 26880
aataaccagt cccgcggtaa ttcacagaat cgtggaaata accagggtcg tggagcttct 26940
cagaacagag gaggcaataa taataacaat aacaagtctc gtaatcagtc caagaacaga 27000
aaccagtcaa atgaccgtgg tggtgtaaca tcacgcgatg atctggtggc tgctgtcaag 27060
gatgccctta aatctttggg cattggcgaa aaccctgaca agcttaagca acagcagaag 27120
cccaaacagg aaaggtctga cagcagcggc aaaaatacac ctaagaagaa caaatccaga 27180
gccacttcga aagaacgtga cctcaaagac atcccagagt ggaggagaat tcccaagggc 27240
gaaaatagcg tagcagcttg cttcggaccc aggggaggct tcaaaaattt tggagatgct 27300
gaatttgtcg aaaaaggtgt tgatgcctca ggctatgccc agatcgccag tttagcacca 27360
aatgttgcag cattgctctt tggtggtaat gtggctgttc gtgagctagc ggactcttac 27420
gagattacat acaattataa aatgactgtg ccaaagtctg atccaaatgt agagcttctt 27480
gtttcacagg tggatgcatt taaaactggg aatgcaaaac cccagagaaa gaaggaaaag 27540
aagaacaagc gtgaaaccac gcagcagctg aatgaagatg ccatctacga tgatgtgggt 27600
ttgccatctg attcgactca tgccaatttg gaatgggaca cagctgttga cggtggtgac 27660
acggccgttg agattatcaa cgagatcttc gatacaggaa attaaacaat gtttgattgg 27720
cttattctgg ctatgtccca gggtagtgcc attacactgt tattactgag tgtttttcta 27780
gcgacttggc tgctgggcta cggctttgcc ctctaactag cggtcttggt cttgcacaca 27840
acggtaagcc agtggtaatg tcagtgcaag aaggatatta ccatagcact gtcacgaggg 27900
gaacgcagta ccttttcatc taaacctttg cacgagtaat caaagatccg cttgacgagc 27960
ctatatggaa gagcgtgcca ggtatttgac ttaaggactg ttagtaactg aagacctgac 28020
ggtgttgata tggatacaca aaaaaaaaaa a 28051

Claims (2)

1. A preparation method of an immune vaccine for resisting a porcine epidemic diarrhea virus variant shxx1902 is characterized by comprising the following steps:
a step of culturing vero cells, adding the vero cells into a culture medium containing 10% by mass of fetal bovine serumIn liquid, the CO content is 5% by volume at 37 DEG C2Culturing in incubator, digesting, passaging to shake flask, and diluting with serum-free medium to cell density of 6 × 106When the strain is one/mL, the strain is ready for use;
a step of culturing the virus in vero cells to a cell density of 6X 106Adding 0.1L cell culture solution of porcine epidemic diarrhea virus variant shxx1902 to the cells per mL, diluting and passaging, inoculating with MOI = 0.1, pH 7.4 + -0.1, shaking table speed of 120r/min, culturing temperature of 37 deg.C, and cell density of 3 × 10 after passage6Culturing for 72h for later use;
a step of preparing an immunogen to be titrated to 2X 106 TCID50Adding 0.2 percent of formaldehyde into mL of the shxx1902 virus liquid of the porcine epidemic diarrhea virus variant, wherein the preservation number of the shxx1902 of the porcine epidemic diarrhea virus variant is CGMCC No.22380, and inactivating the virus at 37 ℃ for 28 h; then added to a 96-well plate plated with vero cells at 50. mu.L/well, 37 ℃ with 5% CO by volume2Culturing in an incubator for 2 d; harvesting cell supernatant, fixing the cells by cold acetone, adding the harvested supernatant into a 96-well plate paved with vero cells, and continuing culturing for 2 d; discarding the supernatant, fixing the cells with cold acetone, adding an alumina gel adjuvant after inactivation verification is qualified, and mixing according to the volume ratio of the antigen to the adjuvant of 1:1 for use.
2. A variant strain of porcine epidemic diarrhea virus has a preservation number of CGMCC No. 22380.
CN202110920855.6A 2021-08-11 2021-08-11 Preparation method of immune vaccine against porcine epidemic diarrhea virus variant shxx1902 Pending CN113616784A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117143207A (en) * 2023-09-05 2023-12-01 湖南派智生物科技有限公司 Recombinant epitope protein, encoding gene, recombinant plasmid, preparation method, engineering bacteria and application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108118032A (en) * 2017-12-12 2018-06-05 新疆希普生物科技股份有限公司 The culture of 2 type strain of Porcine epidemic diarrhea virus and the development of inactivated vaccine
AU2020103680A4 (en) * 2020-11-26 2021-02-04 Institute Of Animal Science And Veterinary Medicine, Shandong Academy Of Agricultural Sciences strain of porcine epidemic diarrhea virus strain MY01 and Its Application
CN112522212A (en) * 2020-12-16 2021-03-19 湖北省农业科学院畜牧兽医研究所 Porcine epidemic diarrhea virus strain and combined immunization method of inactivated vaccine and interferon thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108118032A (en) * 2017-12-12 2018-06-05 新疆希普生物科技股份有限公司 The culture of 2 type strain of Porcine epidemic diarrhea virus and the development of inactivated vaccine
AU2020103680A4 (en) * 2020-11-26 2021-02-04 Institute Of Animal Science And Veterinary Medicine, Shandong Academy Of Agricultural Sciences strain of porcine epidemic diarrhea virus strain MY01 and Its Application
CN112522212A (en) * 2020-12-16 2021-03-19 湖北省农业科学院畜牧兽医研究所 Porcine epidemic diarrhea virus strain and combined immunization method of inactivated vaccine and interferon thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FEI-FEI GE ET AL: "Novel Method for Isolation of Porcine Epidemic Diarrhea Virus with the Use of Suspension Vero Cells and Immunogenicity Analysis", J CLIN MICROBIO *
代洪波;岳丰雄;徐宏军;魏胜男;高鹏;胡来根;: "猪流行性腹泻病毒分离鉴定及其灭活疫苗免疫保护效果研究", 动物医学进展 *
庞丽丽;王孝彬;解倩倩;盖春云;张洪亮;张纯瑶;单虎;: "猪流行性腹泻病毒山东分离株SD-2016的S基因序列分析", 畜牧与兽医 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117143207A (en) * 2023-09-05 2023-12-01 湖南派智生物科技有限公司 Recombinant epitope protein, encoding gene, recombinant plasmid, preparation method, engineering bacteria and application
CN117143207B (en) * 2023-09-05 2024-03-12 湖南派智生物科技有限公司 Recombinant epitope protein, encoding gene, recombinant plasmid, preparation method, engineering bacteria and application

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