CN114703207B - Recombinant plasmid preparation method and recombinant virus - Google Patents

Recombinant plasmid preparation method and recombinant virus Download PDF

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CN114703207B
CN114703207B CN202210359526.3A CN202210359526A CN114703207B CN 114703207 B CN114703207 B CN 114703207B CN 202210359526 A CN202210359526 A CN 202210359526A CN 114703207 B CN114703207 B CN 114703207B
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陈瑞爱
谢文婷
徐婷
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Zhaoqing Institute Of Biotechnology Co ltd
South China Agricultural University
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South China Agricultural University
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Abstract

The invention belongs to the field of biology, and discloses a preparation method of a recombinant plasmid, wherein the recombinant plasmid is obtained by homologous recombination of an F1 fragment, an F2 fragment, an HA fragment or an HA-2P fragment and an F4 fragment. Through mutating the 403 th isoleucine and the 411 th valine on the C loop of the HA stem end of the hemagglutinin protein HA of the H9N2 subtype avian influenza into proline, and respectively inserting HA genes before and after mutation into a non-coding region between P genes and M genes of an rDHN3-mF virus genome, two newcastle disease gene VII type attenuated recombinant viruses rDHN3mF-HA-2P capable of stably expressing the HA protein of the H9N2AIV are obtained, and WB verification shows that the HA genes after double proline mutation have higher expression efficiency compared with the original HA genes. The recombinant virus is expected to be further developed into a bivalent vaccine against newcastle disease and H9N2 subtype avian influenza virus.

Description

Recombinant plasmid preparation method and recombinant virus
Technical Field
The invention relates to the field of biology, in particular to a preparation method of a recombinant plasmid and a recombinant virus.
Background
Avian Influenza (AI) is an acute respiratory infectious disease caused by Avian Influenza virus (Avian Influenza virus, AIV), abbreviated as Avian Influenza. Avian influenza viruses belong to the influenza A virus, and are generally characterized by a combination of their surface proteins, hemagglutinin (HA) and Neuraminidase (NA), resulting in a number of different subtypes, e.g., H1N1, H5N6 or H9N2[1]. Avian influenza can be classified into highly pathogenic avian influenza and less pathogenic avian influenza according to its pathogenicity. Among them, H9N2 subtype AIV is classified as low pathogenic avian influenza by both molecular characterization and pathological typing. In China, H9N2 has replaced H5N6 and H7N9 as the major AIV subtype for chickens and ducks [2]. The poultry infected with H9N2 subtype AIV generally presents subclinical symptoms such as cough, sneeze, royal, wheezing and other mild respiratory symptoms [3], can also obviously influence the immune system of the infected chicken, so that the immune suppression [4] and secondary infection of the infected chicken can be caused, the infected chicken and other pathogens can be easily combined to infect in broilers and young chickens, the death rate is increased, and the economic development of the poultry industry is threatened by high infection rate and high infectivity. In addition, H9N2 subtype viruses can also provide genes for novel influenza viruses through antigen drift or antigen transfer, and novel zoonosis viruses which can infect humans and other animals are generated, and the public health safety is seriously threatened [5,6]. AIV belongs to orthomyxoviridae, type a, and comprises a segmented negative-sense RNA genome encoding 10 core proteins and a plurality of helper proteins, wherein HA protein is the major component of influenza virus particle surface, the most important functions are receptor binding and membrane fusion, and it presents neutralizing antibody epitopes (neutralizing antibodies, NAbs) [7] of avian influenza virus, and also possibly plays a role in the process of viral particle budding and morphogenesis, and is also a main object of recognition by the host acquired immune system, and is a main target antigen for influenza virus vaccine development [8]. The HA protein is a typical type I viral envelope glycoprotein, and after intracellular translation into the polypeptide precursor HA0, each monomer in the HA0 trimer is cleaved by proteases in the cell to form two disulfide-linked subunits HA1 and HA2, constituting the receptor binding region and the membrane fusion domain, respectively. Upon entering the cell surface, HA1 attaches to the cell surface through interactions with sialic acid moieties in the plasma membrane. Subsequently, the virions are endocytosed and transported into advanced endocytosis. Acidification of endosomes triggers conformational changes in HA, leading to fusion of the virus and endosomal membrane, releasing viral genetic material into the host cell [9]. During this process, the HA protein undergoes pre-, intermediate-, post-fusion states. Studies have shown that in most cases, the relevant NAb epitope appears to be optimal or only when the trimer is in the pre-fusion conformation, whereas the post-fusion or other aberrant protein conformation induces predominantly non-neutralizing antibodies with no or limited protective capacity [10]. For type I viral envelope glycoproteins, this can be achieved by means designed to increase their stability, to increase the yield during production and to present their critical NAB epitopes in order to allow their antigen proteins to exert optimal immunological properties. Among these, the approach used to stabilize the protein conformation is to introduce a structure-directed proline substitution at a specific position to maintain the pre-fusion trimeric form [11].
Newcastle disease (Newcastle disease, ND) is a high contagious disease of poultry caused by newcastle disease virus (Newcastle disease virus, NDV) which can cause injuries to respiratory system, nervous system, digestive tract and viscera, and the mortality rate can reach 100%. With the widespread use of reverse genetics, scientists worldwide have demonstrated the potential of newcastle disease virus as a vaccine vector candidate for controlling human and animal disease. We have previously developed an attenuated strain rDHN3-mF which is effective against epidemic strains of the type VII gene.
The problems to be solved in the scheme are as follows: how to increase the expression effect of the virus.
The relevant documents related to the scheme are as follows:
[1].Peacock,T.,et al.,A Global Perspective on H9N2 Avian Influenza Virus.Viruses,2019.11(7).
[2].Bi,Y.,et al.,Dominant subtype switch in avian influenza viruses during 2016-2019in China.Nat Commun,2020.11(1):p.5909.
[3].Kim,J.A.,et al.,H9N2 influenza viruses isolated from poultry in Korean live bird markets continuously evolve and cause the severe clinical signs in layers.Vet Microbiol,2006.118(3-4):p.169-76.
[4].Bano,S.,K.Naeem and S.A.Malik,Evaluation of pathogenic potential of avian influenza virus serotype H9N2 in chickens.Avian Dis,2003.47(3Suppl):p.817-22.
[5].Lenny,B.J.,et al.,Replication Capacity of Avian Influenza A(H9N2)Virus in Pet Birds and Mammals,Bangladesh.Emerg Infect Dis,2015.21(12):p.2174-7.
[6].Quan,C.,et al.,New Threats from H7N9 Influenza Virus:Spread and Evolution of High-and Low-Pathogenicity Variants with High Genomic Diversity in Wave Five.J Virol,2018.92(11).
[7].Murin,C.D.,I.A.Wilson and A.B.Ward,Antibody responses to viral infections:a structural perspective across three different enveloped viruses.Nat Microbiol,2019.4(5):p.734-747.
[8].Skehel,J.J.and D.C.Wiley,Receptor binding and membrane fusion in virus entry:the influenza hemagglutinin.Annu Rev Biochem,2000.69:p.531-69.
[9].Das,D.K.,et al.,Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers.Cell,2018.174(4):p.926-937.e12.
[10].Sanders,R.W.and J.P.Moore,Virus vaccines:proteins prefer prolines.Cell Host Microbe,2021.29(3):p.327-333.
[11].Graham,B.S.,M.Gilman and J.S.McLellan,Structure-Based Vaccine Antigen Design.Annu Rev Med,2019.70:p.91-104.
[12] chinese patent application CN 202011642554-recombinant plasmid, recombinant gene VII type newcastle disease virus and culture method thereof;
[13] chinese patent application CN 202010730869-a gene VII type newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and vaccine.
Disclosure of Invention
The invention aims to provide a preparation method of recombinant plasmid and virus, which are characterized in that through mutating isoleucine 403 and valine 411 on a C-ring of a hemagglutinin protein HA stem end of H9N2 subtype avian influenza into proline, and respectively inserting HA genes before and after mutation into a non-coding region between P genes and M genes of rDHN3-mF virus genome, two newcastle disease gene VII type attenuated strain recombinant virus rN 3mF-HA-2P capable of stably expressing H9N2AIV HA protein is obtained, and WB verification shows that compared with original HA genes, the HA genes after double proline mutation have higher expression efficiency. The recombinant virus is expected to be further developed into a bivalent vaccine against newcastle disease and H9N2 subtype avian influenza virus.
The specific scheme of the invention is as follows: 1. a preparation method of recombinant plasmid comprises homologous recombination of F1 segment, F2 segment, HA segment or HA-2P segment and F4 segment to obtain recombinant plasmid;
the F1 fragment, the F2 fragment, the HA-2P fragment and the F4 fragment are respectively shown in the sequence table SEQ ID NO:1 to 5.
In the preparation method of the recombinant plasmid BR322-DHN3mF-HA, the recombinant kit is adopted to react for 30 minutes at 37 ℃ according to the following components, and the following components are added:
f1 fragment 30ng;
f2 fragment 97ng;
30ng of HA segment or HA-2P segment;
200ng of F4 fragment;
5xCE MultiS Buffer 4μl；
Exnase MultiS 2μl；
ddH2O was added to 20. Mu.l.
In the preparation method of the recombinant plasmid BR322-DHN3mF-HA, the preparation method of the F1 fragment comprises the following steps:
the pBR322-FDHN3 vector is used as a template, and primers FDHN3-F1 and FDHN3-SmaI-R1 are respectively used for amplification to obtain an F1 fragment;
the primers FDHN3-F1 and FDHN3-SmaI-R1 are respectively shown in the sequence table SEQ ID NO:6 to 7.
In the preparation method of the recombinant plasmid BR322-DHN3mF-HA, the preparation method of the F2 fragment comprises the following steps:
the pBR322-FDHN3 vector is used as a template, and primers FDHN3-SmaI-F2 and FDHN3-R2 are respectively used for amplification to obtain an F2 fragment;
the primers FDHN3-SmaI-F2 and FDHN3-R2 are respectively shown in the sequence table SEQ ID NO:8 to 9.
In the preparation method of the recombinant plasmid BR322-DHN3mF-HA, the preparation method of the HA segment comprises the following steps:
an HA segment obtained by amplification of primer HA-F, HA-R with pXJ flag-HA carrier as a template;
the preparation method of the HA-2P fragment comprises the following steps:
amplifying the obtained HA-2P fragment by using a pXJ flag-HA-2P carrier as a template and using a primer HA-F, HA-R;
the preparation method of the pXJ flag-HA-2P carrier comprises the following steps:
using pXJ Flag-HA plasmid as carrier, using primer Mutate-F, mutate-R to reversely amplify PCR fragment according to the instruction of the Mut Express II Fast Mutagenesis Kit V2 kit;
after Dpn I digestion, the digestion products are cyclized in vitro;
transforming the cyclized plasmid, and selecting monoclonal bacteria to sequence the HA segment;
selecting a colony with correct sequencing for propagation, and extracting plasmids to obtain a pXJ flag-HA-2P vector;
the HA-F, HA-R is respectively shown in a sequence table SEQ ID NO:10 to 11;
the primer Mutate-F, mutate-R is respectively shown in a sequence table SEQ ID NO:12 to 13.
In addition, the invention discloses a recombinant virus, and the plasmid prepared by any one of the methods is transfected into BHK-21 cells to obtain the virus rDHN3mF-HA or the virus rDHN3mF-HA-2P.
Compared with the prior art, the invention has the following advantages and effects:
according to the invention, the H9N2 subtype avian influenza HA gene is mutated into proline at the 403 rd isoleucine and the 411 th valine on the C ring of the HA stem end, and then the HA genes before and after mutation are respectively inserted between M and P genes of a Newcastle disease gene VII type DHN3mF attenuated strain by a DNA recombination method, so that infectious cDNA clones pBR322-DHN3mF-HA and pBR322-DHN3mF-HA-2P plasmids are respectively constructed. The two plasmids are respectively mixed with pXJ-NP, pXJ40-P, pXJ-L and pXJ-DE 3 plasmids, BHK-21 cells are transfected together, and two stable recombinant viruses rDHNmF-HA and rDHNmF-HA-2P which can effectively express H9N2 subtype avian influenza HA genes and NDV antigen proteins are successfully obtained. According to WB results, the expression efficiency of the HA gene after double proline mutation is higher than that of the HA gene before non-mutation. The recombinant virus is expected to be further developed into a bivalent vaccine against the avian influenza virus and the newcastle disease virus, and provides a novel defense means for controlling the infection of the avian influenza virus and the newcastle disease virus.
Drawings
FIG. 1 shows the sequencing result of avian influenza H9N2 subtype HA gene after double proline mutation.
FIG. 2 is a gel electrophoresis diagram of F1 gene fragment; wherein M: a DNA Marker;1-4: f1PCR fragment (1471 bp).
FIG. 3 is a gel electrophoresis diagram of F2 gene fragment; wherein M: a DNA Marker;1-4: f2PCR fragment (4815 bp);
FIG. 4 is a gel electrophoresis diagram of F3-HA gene fragment; wherein M: a DNA Marker;1-4: F3-HA PCR fragment (1731 bp).
FIG. 5 is a gel electrophoresis diagram of F3-HA-2P gene fragment; wherein M: a DNA Marker;1-4: F3-HA-2P PCR fragment (1731 bp);
FIG. 6 is a gel electrophoresis diagram of F4 gene fragment; wherein M: a DNA Marker;1-2: a DNA fragment after cleavage of pBR322-FDHN3 SmaI;
FIG. 7 is a diagram of PCR detection and identification of recombinant plasmid rDHN3-mF-HA single colonies, wherein M: DNA mark; 1-9: rDHN3-mF-HA single colony (positive colony: 2306 bp);
FIG. 8 is a diagram of PCR detection and identification of recombinant plasmid rDHN3-mF-HA-2P single colonies, wherein M: DNA mark; 1-9: rDHN3-mF-HA-2P single colony (positive colony: 2306 bp);
FIG. 9 is a diagram of the restriction enzyme identification of recombinant plasmids, wherein M: a DNA Marker;1-3: recombinant plasmid rDHN3-mF-HA;4-6: recombinant plasmid rDHN3-mF-HA-2P;
FIG. 10 is a schematic diagram of pBR322-FDHN3-HA plasmid;
FIG. 11 is a schematic diagram of pBR322-FDHN3-HA-2P plasmid;
FIG. 12 is an enlarged view of BHK-21 cells infected with recombinant virus allantoic fluid, wherein A: rDHN3mF-HA recombinant virus allantoic fluid; b: rDHN3mF-HA-2P recombinant virus allantoic fluid; c: SPF chick embryo allantoic fluid;
FIG. 13 is a diagram showing PCR detection and identification of recombinant virus HA and F genes, wherein M is DNA marker; left A: the amplified band size of the primer pair F-F/R for the recombinant virus rDHN3mF-HA is 815bp; left B: the amplified band size of the primer pair F-F/R for the recombinant virus rDHN3mF-HA-2P is 815bp; right a: the amplified band size of the primer pair HA-F/HA-R for the recombinant virus rDHN3mF-HA is 1731bp; right B: the amplified band size of the primer pair HA-F/HA-R for the recombinant virus rDHN3mF-HA-2P is 1731bp;
FIG. 14 is a schematic diagram showing the HA gene detection electrophoresis of recombinant virus rDHN3mF-HA chick embryo serial passage, wherein 1-3 are the 5 th, 10 th and 15 th generation recombinant virus HA genes (1731 bp) respectively;
FIG. 15 is a view of the HA gene detection electrophoresis of recombinant virus rDHN3mF-HA-2P chick embryos in serial passages, wherein 1-3 are the 5 th, 10 th and 15 th generation recombinant virus HA-2P genes (1731 bp) respectively;
FIG. 16 is a graph showing the results of NDV protein detection for recombinant viruses, wherein M: protein molecular weight; 1: a negative control; 2: a cellular protein sample transfected with pXJ flag-HA plasmid; 3: infecting rDHN3mF chick embryo allantoic fluid cell protein samples; 4: infecting rDHN3mF-HA allantoic fluid cell protein samples; 5: infecting rDHN3mF-HA-2P allantoic fluid cell protein samples;
FIG. 17 shows results of HA protein detection for recombinant viruses, wherein M: protein molecular weight; 1: a negative control; 2: a cellular protein sample transfected with pXJ flag-HA plasmid; 3: infecting rDHN3mF chick embryo allantoic fluid cell protein samples; 4: infecting rDHN3mF-HA allantoic fluid cell protein samples; 5: infecting rDHN3mF-HA-2P allantoic fluid cell protein samples;
FIG. 18 shows the results of detection of recombinant viral β -actin protein, wherein M: protein molecular weight; 1: a negative control; 2: a cellular protein sample transfected with pXJ flag-HA plasmid; 3: infecting rDHN3mF chick embryo allantoic fluid cell protein samples; 4: infecting rDHN3mF-HA allantoic fluid cell protein samples; 5: infecting rDHN3mF-HA-2P allantoic fluid cell protein samples;
fig. 19 is an isometric enlarged WB bar graph of fig. 17 and 18;
FIG. 20 is a map of the pMD19T-AIV-HA plasmid.
FIG. 21 is a gel electrophoresis diagram of a 1698bp fragment amplified with the primer AIV-HA-F/R using the pMD19T-AIV-HA plasmid as a template.
Detailed Description
The invention is further described below in connection with the examples, which are not to be construed as limiting the invention in any way, but rather as a limited number of modifications which are within the scope of the appended claims.
Example 1
Main instrument and equipment
Electrothermal constant temperature incubator HZ-100 (a constant scientific instrument Co., ltd., shanghai, china); three-hole electrothermal constant temperature water tank DK-8D (a constant scientific instrument Co., ltd., shanghai of China; sea BCD-579WE refrigerator (sea, shanghai, china); a CO2 incubator Forma 371 (Thermo corporation, usa); ultra clean bench SW-CJ-2FD (Antai air technologies Co., ltd., china, jiangsu), biosafety cabinet 1300SERIES A2 (Thermo Co., ltd., china, USA), inverted optical microscope (Nikon Co., ltd., USA), high-speed Centrifuge Centrifuge 5804R (Eppendorf Co., germany), pipettor Research plus (Eppendorf Co., germany), PCR instrument C1000 Touch (Bio-Rad Co., ltd., USA), electrophoresis instrument PowerPac Basic (Bio-Rad Co., ltd., USA), vertical electrophoresis tank MiniProtean Tetra (Bio-Rad Co., USA), gel imaging system 2500 (R) (Tanon Co., china Shanghai), ultra pure water instrument Milli-Q (Millipore Co., USA), ultra low temperature refrigerator Forma 994 (Thermo Co., USA), nucleic acid protein analyzer Nano Drop 2000 (Thermo Co., USA), biochemical incubator H-250 (A, china, science Sharpo, inc.), biotechnology, biosystem (Biotechnology, korsche) Lv-Share (Biotechnology, UK), vortex system (Biotechnology, korsche) 35, vortex, share, UK.S.35, share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Share.Kor.Kor.Co., USA.Kor.Kor.Kor.Co.Kor.Co.Co.Kor.Kor.Kor.Co.Co.USA.Kor.Kor.Kor.Kor.Kor.Kor.Kor.Kor.USA.Kor.Kor.Kor.Kor.USA.USA.USA.USL.USA.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.USL.J XJ.J.
Primary reagents and materials
TIAN prep Mini Plasmid Kit (DP 103-03) is available from Tiangen Biochemical technologies (Beijing); gel Extraction Kit (D2500-02) from OMEGA; hiScript III 1st Strand cDNA Sythesis Kit (+gDNA wind) (R312) was purchased from Nanjinopran biologicals Co.Ltd; agarose (E0301) is purchased from TSINGK;0.25% Trypsin-EDTA (25200-056), DMEM basic (C11995500 BT) available from Gibco; lipofectamine LTX and Plus Reagent (15338-100) from Invitrogen; FBS (10099-141C) was purchased from Gibco; premix-Taq (RR 902A) was purchased from TAKARA; pen Strep penicillin Streptomycin (15140-122) from Gibco; 2X Phanta Flash Master Mix (Dye Plus) (P520) was purchased from Nanjinopran biologicals Co., ltd; commonly used restriction enzymes are available from TAKARA; clonExpress Multis One Step Cloning Kit (C113) was purchased from Nanjinopran biologicals Co., ltd; trizol 15596-026 is purchased from Invitrogen; biochemical reagents such as chloroform, isopropanol, absolute ethanol and the like are purchased from Ningbo-extract chemical technology limited company; 10xSDS-PAGE electrophoresis was purchased from Biyun; SDS-PAGE gel preparation kit, beyoECL Plus (hypersensitive ECL chemiluminescent kit) was purchased from Biyun day; 0.22 μm NC film (3 meters/roll) was purchased from PALL company; goat Anti-Rabbit IgG H&L
Figure SMS_1
preadsorbed (ab 6940) was purchased from CST; rabbit anti-chicken IgG-HRP (SE 235) purchased from solabio; TBS buffer was purchased from Boschner Biolabs; AIV-HA polyclonal and NDV hyperimmune serum (homemade by the present laboratory); />
Figure SMS_2
Anti-beta-Actin Mouse Monoclonal Antibody (HC 201-01) was purchased from full gold corporation. pXJ40Flag-HA was constructed by the present laboratory; pBR322-DHN3mF, pXJ-NP, pXJ40-P, pX J40-L, pXJ-DE 3 are all available from the China center for Biotechnology institute of Zhaoqing; PMD 19T-Vector-Takara Bio-engineering (Dalian) Co.Ltd. PMD 19-T Vector Cloning Kit-Takara Biotechnology (Dalian) Co.Ltd.
The pXJ Flag-HA plasmid was constructed as follows:
amplification of HA Gene fragment of H9N2 subtype avian influenza
The experiment uses an H9N2 subtype AIV strain separated from a chicken farm in south China in 2015, allantoic fluid of infected chicken embryo is taken, AIV virus genome RNA is extracted by a TRIzol method, and then cDNA of genome is obtained by reverse transcription. The 1698bp fragment of interest was amplified by PCR using the AIV-HA-F/R primers in the Table with 2X Phanta Flash Master Mix (Dye Plus) enzyme, the amplified PCR product was identified with 0.8% agarose gel, the concentration of the fragment of interest was recovered by gel recovery and ligated into pMD19T-Vector with T4 ligase, designated pMD19T-AIV-HA, sequencing was sent to Bioengineering (Shanghai) Inc.
Preparing a PCR reaction solution:
Figure SMS_3
PCR reaction conditions:
Figure SMS_4
Figure SMS_5
the map of the pMD19T-AIV-HA plasmid is shown in FIG. 20.
(II) construction of pXJ Flag-HA plasmid
1. Amplifying 1698bp fragments by using pMD19T-AIV-HA plasmid as a template and using a primer AIV-HA-F/R, recovering the fragments by gel and measuring the concentration;
2. double digestion was performed with KpnI and BamHI enzyme pair pXJ-Flag plasmid and 1698bp fragment (SEQ ID NO: 16) amplified with primer AIV-HA-F/R;
3. the 1698bp fragment was ligated into the linear pXJ-Flag plasmid using T4 ligase to construct the pXJ Flag-HA plasmid.
FIG. 21 is a gel electrophoresis diagram of a 1698bp fragment amplified with the primer AIV-HA-F/R using the pMD19T-AIV-HA plasmid as a template
The pXJ-Flag plasmid can be seen as ZL201910894754.9 entitled the 146-153 paragraph of the description of the method of infectious recombinant cloning of type II, type VII, epidemic NDV strain DHN3, regarding the source channel of pXJ-Flag.
Figure SMS_6
The related primer or other fragment sequences related to the scheme are shown in the following table 1:
table 1 sequence listing
Figure SMS_7
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Figure SMS_8
Experimental procedure
Construction of pXJ40flag-HA-2P plasmid
(1) Using a plasmid shown in pXJ Flag-HA (shown as a sequence table SEQ ID NO: 14) as a vector, and reversely amplifying a PCR fragment by using a primer Mutate-F/Mutate-R (the primer sequence is detailed in the attached table 1) according to the specification of a kit Mut Express II Fast Mutagenesis Kit V2;
(2) After Dpn I digestion, the digestion products are cyclized in vitro;
(3) Transforming the cyclized plasmid, and selecting monoclonal bacteria to sequence the HA segment;
(4) And selecting a colony with correct sequencing for propagation, and extracting plasmids.
2. Construction of recombinant plasmid pBR322-DHN3mF-AIV-HA and-pBR 322-DHN3mF-AIV-HA-2P for expressing H9N2 subtype AIV-HA Gene
(1) The pBR322-FDHN3 vector is used as a template, and primers, FDHN3-F1/FDHN3-SmaI-R1 are respectively used; FDHN3-SmaI-F2/FDHN3-R2 amplified F1 of 1471bp in length and F2PCR fragment of 4815bp in length.
pBR322-FDHN3 refers to CN 202011642554-recombinant plasmid, recombinant gene VII type newcastle disease virus and sequence table SEQ ID NO recorded in culture method thereof: 3 is shown in the figure;
the primer HA-F/HA-R is used for amplifying the HA fragment with the length of 1731bp and the RCR fragment of HA-2P with the same length by taking pXJ flag-HA or pXJ flag-HA-2P carrier as a template. (details of primer sequences see Table 1)
The preparation of the PCR reaction solution is shown in the following Table 2:
TABLE 2
Figure SMS_9
PCR reaction conditions:
Figure SMS_10
the pBR322-DHN3mF vector (supplied by the present laboratory) was digested with SmaI, and 13399bp DNA fragment F4 was purified by gel recovery.
The pBR322-DHN3mF vector can refer to CN 202010730869-a gene VII type newcastle disease recombinant virus and vaccine for expressing infectious bursal disease virulent strain VP2 protein, and the described pBR322-mFDHN3; pBR322-mFDHN3 and pBR322-DHN3mF are identical.
The preparation method can refer to CN 202010730869-a gene VII type newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and 97-103 th section of vaccine instruction book.
(2) The 4 fragments were subjected to homologous recombination (recombination kit purchased from nanjing novzan (ClonExpressMultis One Step Cloning Kit, C113), the formulation of which is shown in table 3 below:
TABLE 3 Table 3
Reagent(s) Dosage of
F1 fragment 30ng
F2 fragment 97ng
HA/HA-2P fragment 30ng
F4 fragment 200ng
5xCE MultiS Buffer 4μl
Exnase MultiS 2μl
ddH 2 O up to 20μl
The above system was prepared on ice and reacted at 37℃for 30 minutes.
(3) The recombinant product was transformed into Trans2-Blue competent bacteria.
(a) Thawing Trans2-Blue competent cells on ice, adding 10 μl of recombinant into 50 μl of competent cells, mixing with light elastic tube wall (mixing without shaking), and standing on ice for 30min.
(b) After heat shock in a water bath at 42 ℃ for 45sec, the mixture is immediately placed on ice for cooling for 2-3min.
(c) 1ml of SOC medium (without antibiotics) was added and the mixture was shaken at 37℃for 1h (rotation speed 200-250 rpm).
(d) The amp+ LB plate solid medium was preheated in a 37℃incubator.
(e) Mu.l of the bacterial liquid was gently smeared onto a plate containing AMP+ resistance with a sterile plating bar.
(f) Culturing in an incubator at 37 ℃ for 12-16 hours in an inverted mode.
(4) Colony PCR was identified using primers M-F2/P-R2, and a total of 8 colonies were screened, 8 were positive, and the correct product was 2300bp.
(5) HindIII was digested and plasmid identified.
The 3 positive colonies were amplified and cultured, respectively, to extract plasmid DNA.
Cleavage with HindIII (from TAKARA), respectively, is performed as shown in Table 4 below:
TABLE 4 Table 4
Figure SMS_11
Figure SMS_12
The correct plasmid should be digested with HindIII to yield 1 12382bp,5654bp,3294bp DNA fragment, respectively.
The cleavage results were shown in FIGS. 9-11,1-6 below to yield the correct 3 fragments. Sequencing verification is carried out on the No. 1 plasmid and the No. 4 plasmid, and the result proves that the plasmids contain correct insertion sequences, which are named pBR322-DHN3mF-HA and pBR322-DHN3mF-HA-2P respectively.
2. Transfection of BHK cells to rescue the virus
(1) BHK-21 cells were cultured in 30mm dishes, transfected with Lipofectamine LTX DNA Transfection Reagents kit within 24h, and transfected exactly as indicated by the kit.
(2) The transfection reagent was formulated as follows:
and (3) solution A: 150 μl, lipofectamine LTX Reagent μl of Opti-MEM Medium, and standing for 5min;
and (2) liquid B: opti-MEM DNA 175. Mu.l, pBR322-DHN3mF-HA (pBR 322-DHN3 mF-HA-2P) 4. Mu.g, pXJ-NP 2.5. Mu.g, pXJ-P1.25. Mu.g, pXJ-L1.25. Mu.g, pXJ-DE 3 3. Mu.g, PLUS reagent 3.5. Mu.l, and mixing by blowing and standing for 5min.
Mixing solution A and solution B150 μl each, and standing for 20min. Mu.l of the mixture was used to transfect cells.
(3) After 4 days, the transfected cells (including the culture solution) were frozen at-80 ℃.
(4) The frozen cells were repeatedly freeze-thawed 3 times and centrifuged at 4℃10000/rpm for 5 minutes. 200 μl of supernatant was used to infect DF-1 cells. After 24h a large number of cell deaths were visible. The following figures show: a is rDHN3mF-HA infected cells; b is rDHN3mF-HA-2P infected cells; c is a negative control cell without virus solution. Total RNA was extracted from A, B, and C cells, respectively, for the virus identification assay described below.
3. Identification of recombinant viruses
(1)RT-PCR
RT-PCR was performed using A, B and total C-cell RNA as described above.
RT was carried out using HiScript III 1st Strand cDNA Sythesis Kit (+gDNA wind) and following exactly the instructions.
The following mixed solutions of genomic DNA were prepared in a Microtube in a total amount of 8. Mu.l, and the results are shown in Table 5.
TABLE 5
Reagent name Usage amount
Template RNA 8μl
5×gDNA wiper Mix 2μl
Gently stirring and mixing with a pipette, and standing at 42deg.C for 2min.
B. The following reverse transcription reaction solutions were prepared in the above described Microtube, and the following are referred to in Table 6.
TABLE 6
Reagent name Usage amount
The above mixed solution 10μl
10×RT mix 2μl
HiScript III Enzyme Mix 2μl
Oligo(dT) 20 VN 1μl
RNase-free ddH 2 O 5μl
Placing at 37 ℃ for reaction for 45min and 85 ℃ for reaction for 5s.
And (2) PCR: the primers F-F/F-R and HA-F/HA-R were used for the identification, respectively.
Composition of PCR system reference table 7:
TABLE 7
Primer1 10pmol
Primer2 10pmol
cDNA template 2μL
Premix Taq 10μL
ddH 2 O up to 20μL
PCR reaction conditions:
Figure SMS_13
Figure SMS_14
mu.l of each PCR product was run and the results were shown in FIG. 13: the left panel shows F-F/F-R primer, the correct product should be 815bp; the right panel shows the HA-F/HA-R primer, the correct product should be 1731bp.
(2) Amplification of viruses
SPF chick embryos were inoculated with 200. Mu.l of the above-mentioned rDHN3mF-HA and rDHN3 mF-HA-2P-generation viruses and passaged 10 times in succession, and allantoic fluids of the chick embryos of each generation were harvested and stored at-80 ℃.
(3) RT-PCR and sequencing to verify the stability of rDHN3mF-HA and rDHN3mF-HA-2P recombinant viruses
RNA was extracted from allantoic fluid of F1, F5 and F10 chick embryos of recombinant toxins rDHN3mF-HA and rDHN3mF-HA-2P, respectively, and RT-PCR was performed using the same. The method is consistent with the foregoing.
And (2) PCR: the primer is HA-F/HA-R; the correct product should be 1731bp.
The results are shown in figures 14 and 15 below, with 3 samples of each virus producing PCR products of the correct size. The sequences were sent separately and the results confirmed the correct DNA sequence, suggesting that the recombinant virus was relatively stable.
Verification of expression level of recombinant viral HA protein by WB
Protein samples (1) of cells were inoculated with normal SPF chick embryo allantoic fluid, cell protein samples (2) transfected with pXJ flag-HA plasmid, rDHN3mF chick embryo allantoic fluid cell protein samples (3), rDHN3mF-HA allantoic fluid cell protein samples (4) and rDHN3mF-HA-2P allantoic fluid cell protein samples (5) were run with 10% protein gel and WB assays were performed. The results are shown below:
a protein of about 55kD size was detected with NDV hyperimmune serum, which showed the same size and morphology on the protein gel as the rDHN3-mF infection product.
FIG. 16 shows that a protein of about 75kDa size was detected with the rabbit anti-HA polyclonal antibody, which protein was consistent with the size of the HA protein.
FIG. 17 shows that a protein of about 40kDa size is detected with murine anti-beta-actin monoclonal antibody, which corresponds to the size of the actin protein.
The detailed method is as follows:
detection of NDV protein:
BHK-21 cells were cultured to 90% in 6-well plates, washed twice with PBS, and 2ml of 0.1% trypsin-DMEM incubation solution was added, and BHK-21 cells were infected with 2MOI DHN3 chick embryo allantoic fluid, or 2MOI rDHN3mF-HA-2P chick embryo allantoic fluid, respectively. 200 μl of SPF chick embryo allantoic fluid was added to the negative control. After 24h, cells were collected, 200. Mu.l of RIPA strong lysate (containing 1mM PMSF) was added, and after 10min of dry bath lysis at 95℃12X g was centrifuged for 10min, 20. Mu.l of the supernatant was subjected to 10% SDS-PAGE (Bio-Rad). The proteins were then electrotransferred to NC membrane. 5% skim milk was blocked for 2h, and TBST (0.05% Tween 20) was washed four times for 5min each. Adding 1: the 1000-diluted chicken NDV hyperimmune serum was incubated overnight at 4 ℃. TBST was washed four times for 5min each. Horseradish peroxidase (HRP) -labeled rabbit anti-chicken Ig G (LIC) was diluted with 1:10000 times TBST, respectively, and incubated for 1h at room temperature. TBST was washed four times for 5min each. Adding the hypersensitive ECL chemiluminescent developer. Imaging was performed using Azure Biosystems C600 multi-functional molecular imaging system.
Detecting HA protein and beta-actin protein:
BHK-21 cells were plated to 60% with 3.5cm diameter plates, washed twice with PBS, and pXJ 40.40 flag-HA plasmid was transfected into BHK-21 cells using Lipofectamine LTX DNA Transfection Reagents transfection kit. BHK-21 cells were cultured to 90% in 6-well plates, washed twice with PBS, and 2ml of 0.1% trypsin-DMEM incubation solution was added, and BHK-21 cells were infected with 2MOI DHN3 chick embryo allantoic fluid, or 2MOI rDHN3mF-HA-2P chick embryo allantoic fluid, respectively. 200 μl of SPF chick embryo allantoic fluid was added to the negative control. After 24h, cells were collected, 200. Mu.l of RIPA strong lysate (containing 1mM PMSF) was added, and after 10min of dry bath lysis at 95℃12X g was centrifuged for 10min, 20. Mu.l of the supernatant was subjected to 10% SDS-PAGE (Bio-Rad). The proteins were then electrotransferred to NC membrane. 5% skim milk was blocked for 2h, and TBST (0.05% Tween 20) was washed four times for 5min each. Respectively adding 1:2000 dilution of rabbit HA polyclonal antibody, murine beta-actin monoclonal antibody, overnight incubation at 4 ℃. TBST was washed four times for 5min each. And (3) respectively adding 1:10000 times of TBST for dilution, and incubating the goat anti-rabbit fluorescent secondary antibody and the goat anti-mouse fluorescent secondary antibody for 1h at room temperature. TBST was washed four times for 5min each. Imaging was performed using Azure Biosystems C600 multi-functional molecular imaging system.
5. Analysis of biological Properties of recombinant viruses
(1) HA assay of recombinant viruses
Adding 0.025 mL/hole of physiological saline into 96-hole micro-reaction holes, adding 0.025 mL/hole of virus to be detected into the first hole of each row, sequentially performing multiple dilution, diluting until the 11 th hole is discarded to 0.025mL, taking the 12 th hole as a negative control, finally adding 0.025mL of 1% chicken erythrocyte suspension into each hole, uniformly mixing by a micro-oscillator, standing at room temperature for 20-25 min, judging the hemagglutination titer (HA), and repeating the measurement for three times for each sample to reduce experimental errors.
(2) TCID50 assay for recombinant viruses
BHK-21 cells were plated in 96-well plates one day in advance, and after culturing the cells to 90%, the next experiment was started, old medium in 96-well was discarded, and the cells were washed 2 times with PBS. The recombinant virus rDHN3mF-HA or rDHN3mF-HA-2P is diluted by 0.1% of trypsin-DMEM incubation liquid according to the ratio of the dilution to the dilution gradient of 10 < -1 > -10 < -10 >. The virus solutions of different dilutions were each labeled with 100 μl of infected cells, and three replicates were made for each dilution. After 5-7 d, the lesions were observed under an inverted microscope and virus samples were repeated three times to reduce errors. TCID50 was calculated using Reed-Muench method.
(3) EID50 assay of recombinant viruses
The recombinant strain rDHN3mF-HA or rDHN3mF-HA-2P allantoic fluid is diluted by 10 times of normal saline in sequence, the dilution gradient is 10-1 to 10-10, 5 SPF chick embryos of 9 days to 11 days old are respectively inoculated by five dilution gradients of 10-5 to 10-10, 100 mu L of each chick embryo is inoculated, and the chick embryos are placed in a constant temperature incubator at 37 ℃. Observing the dead embryo within 24 hours and discarding; the embryo was frozen three times daily in the morning, in the middle and at night, and the dead embryo was observed continuously for 7 days. And (3) standing the blank after 7d for more than 4 hours in a refrigerator at the temperature of 4 ℃ to collect allantoic fluid, determining HA, wherein positive is infection, and calculating the half-number infection quantity EID50 of the chick embryo by using a Reed-Muench method.
Figure SMS_15
Conclusion IV
The H9N2 subtype avian influenza HA gene is mutated into proline at the 403 rd isoleucine and the 411 th valine on the C ring of the HA stem end, and then HA genes before and after mutation are respectively inserted between M and P genes of a Newcastle disease gene VII type DHN3mF attenuated strain by a DNA recombination method, so that infectious cDNA clones pBR322-DHN3mF-HA and pBR322-DHN3mF-HA-2P plasmids are respectively constructed. The two plasmids are respectively mixed with pXJ-NP, pXJ40-P, pXJ-L and pXJ-DE 3 plasmids, BHK-21 cells are transfected together, and two stable recombinant viruses rDHNmF-HA and rDHNmF-HA-2P which can effectively express H9N2 subtype avian influenza HA genes and NDV antigen proteins are successfully obtained. According to WB results, the expression efficiency of the HA gene after double proline mutation is higher than that of the HA gene before non-mutation. The biological characteristics of the recombinant newcastle disease virus after the exogenous gene is inserted are determined to have small differences with the parental viruses. The recombinant virus is expected to be further developed into a bivalent vaccine against the avian influenza virus and the newcastle disease virus, and provides a novel defense means for controlling the infection of the avian influenza virus and the newcastle disease virus.
Sequence listing
<110> agricultural university of south China
Huanong (Zhaoqing) Biological Industry Technology Research Institute Co., Ltd.
<120> method for producing recombinant plasmid and recombinant virus
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1471
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
ctacccgtat tttttcttaa ggtgttgaag catggattcg caaggggggg ctcccggggg 60
attccgtgtg atgctgaagc gacaggagcc tgctatgagt tgtgacggcc atcaaccatt 120
cagcgcaagg cgcttgatct tcctgatctc ttcaattgac ttggctgcat ctagcttgct 180
caggagctta gccgaggagc ttggatgcat cgggcgtgag gtgattaatg cgcggacagt 240
gtccttctcc actcccatgt caggcccgct tgcggtggca gacttaatca attcagaagg 300
gtgctgcact ggttgtgaga gtttattgag cgtcatttca ccctcttgtg tcacgtaagg 360
agatgggtct ccggggcccg aaactaggac tgggtgggat cgggctactg cccggagatc 420
acttaaagat gaaatgttgg cacaaccagg gtccagaatt ttcatcatac ctaagttagc 480
ttccatgatc gcaacagatg tcttgagctg ttggatttca gatcgtatca taggaataga 540
ggatgtctgt ttcaagacca gatccagctg atgatcaact ttacttacct tctgagataa 600
tgcctccatc atagacatca tcgcctgcgc aaagtcggca ggtagctgga cacgatccac 660
aggtacagga atattgtctt ggctctgccc tgactggggc gcatgagggg ttgcaccagc 720
tgatggttgt gactccttcc gttgtccatg atatgctgtg ttctcgtcta tgccccggtt 780
tccagagacg gccttgacct ggtgctgtgg tcttccctgg ttgcttccat ggctcgactg 840
gttcccgtgt tgttgggccg gagattggtg atgcccttct tggggacccg accatgggcc 900
ctttttagca ttagacgatt tattgcttaa tttgtcgagc atggacagaa gggagttgct 960
tgctccggtt ttgagctgtg tgtcgctggt ctcgcttgcg gcctgggtgg agggcggttc 1020
ggtgggtgtg gtcggtgggt tgttgtgtgg ggttgcctgc tcaggtgtgg atggctgttt 1080
ttctgttctg tcttgttggt cggggacgtc ctgactggcg tgtggctggg cactcccgtg 1140
cttctcccgt gctgtgcttg gagctttggt cttgccctgc gggatcgcac ttcttccgac 1200
ggtctcggct gatttgccct gggccgtaat tatgctatca atgacagtcc cactggtctc 1260
aagtatgtca tctatctccg catctgtaaa ggtagccatc ttcaccctaa ctcaccgggt 1320
agaagggaga acagagagct tggtgacccg tcctggtctc tggatgtctc tcttctaccc 1380
gtattttttt ctaatacctt ggactctggg aggattgttg actggccgag ctgtgcctgt 1440
gttgtcccgg gtggatgggg tcgtgtggct g 1471
<210> 2
<211> 4815
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
cttggtgttg cttgaactca cccgggtgac acgactgcga gatatgttgt caaatactgc 60
agatacgggg ttaagcctcg cttgttcatc atcaagcatc gtcccgaaga cccctcgtag 120
agtatgattc ctatagaaga ttaagggata tggatcggta tagaccccag tgatgcatga 180
gttggggcac cttgctgatg cctggcaagg gacactacct ggccgagtga aagcattaaa 240
catgtaagga ctatggagtg tagccgtttt gttacttact gtcatgggat acaataaggc 300
aggggagaaa tatgaagacc ctcgttgata caagaagtga gatgtcccta ctgtgaggat 360
tctgccttcg gctcccatga gtgtgattgt attaggtgga atagtcagca ccgggtcctt 420
acccagggat gttgacactt tgatagatag gatagcttgc tgtacgcgct ttccaccaaa 480
tcgcccaggt ttatatgagg acttagccat ccgaatttgg taatcttgtt tatcggggca 540
tgtgttatta tggcgcttgt atattacata tttcccttct tgtgcgatgt cactgggtga 600
attaggtttg agccctccgt aaactgggaa ccatacacgg ccgtcaataa aagaccctcc 660
tcccaccccc gggtaatttg ccacccaatc cttaaataag accgtggtgt ctaagtcttt 720
ctcatggtat tgaccgtcaa atcctagcct tccgtgcacc attgatgtgg gggcaactga 780
cttgtaatcc tcctcttcag tccctgtgac cttagagcac agcatatcac aacctaaagg 840
ggttgcactc acactgcaag acttccgatt ttgagtgtca tctaaattga tggagcgcag 900
agtagaaaag aataccctcc ctgttgcaga tgtccgaagc acaccaagtg ctaggtattg 960
atgtgagtgt gagtgatctc tgcaaccaga tagtatcaca ttgtgagtat aacaataatg 1020
ggtggtgccc atgtcaaatg agggtatccg agtgcaacca gatcctgtag taggtgccgg 1080
gatgaaattc aagtgttctt gatatgcaga aggataaaat gatgcgacat tactaatgtc 1140
gtccactatg agttctttgc ctatcccccc gatataatct gggtcatgaa cgggcgcacc 1200
gcatccgcta ctgttctcag ccccgttaat ttgataagaa agagaggtta ttgcattcat 1260
aattacagat tcggtgttta atagtgccag cggggattca agagctacct gcttgtatat 1320
cttatctatc acatcttggc ttgaactgag taaagacgta accttatctt ctgtcttgga 1380
gatcacagtc gatatgcctg cgaggtcgtg tggcgtactg gcctccatgc tgtatgccag 1440
ggcagccgca gagatagcta gaatcattac cattaaaaat aagactgcga tccggaaaac 1500
taagcgccat gtgttctttg cttctctttc ttcattctcc agcatgactc tgttaaccac 1560
gcggtccatg attgactctt gttgttggtg gtgtggtaga acggatgttg tgaagcctaa 1620
ttttcaattg aagggtggct cctctgaccg ttctacccgt gtattgctct ttggttgctt 1680
gttcccagta attttttctt aagtctccta cttgacaggt tatcggaatt gacacacggg 1740
ctgccgttgg gtgtccgtcc acttcctatt tgcattcatg ctctcatggt ggctctcatc 1800
tgatcgaggg tattgttccc aagccataac agggtctttt gttgtgcctt ctgcttgtac 1860
atcaggtaac acgctaaaac cagactaagt gcaccgaaaa ttagggaaat gacagttaga 1920
acaatatagg taatgagagc ggatgtgcta gttagtctga cgttgacttt ttctagtttg 1980
ctgttgcttt ctgccaactt gtccaaggca ttgctgatcg aattattgac gtttccaagt 2040
tcagttgata tatcaagatt gcctgtcacg atgacctgag aatctaatat tgagatgttc 2100
ttttgataag ttgcatcaaa ttccccactg agcctcagag ttatgccgtc taatgataag 2160
acattgcacg agtgtctatc tatcagggat acagcttctc cataattttg cgatatgatg 2220
ccaggagggt ctgtacatct gcatgttgtt atcttacaat tagcaataac tgagccccta 2280
agggccatat acggcgtagt gagtgcgcct tcagtctttg aatacatgca ggctgatgtg 2340
ttgccactta aacaggaata aatacctgga gacatgggga atgtcactat tctagtacaa 2400
tataaatcca gatcggactc tatacagtat gaggtgtcaa gctcttctat cacagaaccg 2460
acttgtgtca ctactttcgg gaccagtgct gaggcatatc ctttggttgt acttacagat 2520
aaggtctcca gataggtggc acgcatatta tttaagttcc cgactgaggg cagattaact 2580
tgtatgccca agagttgagt atgtgagtca tacagtatag ggtaaccagt gatcaggccg 2640
ctaccaatta atgagctgag ttgattgttt cctataccta acttagttaa taagtagtcc 2700
atattgccac cagctaaatt atagagcgcc tggatggtta gctgagttaa tgcaggggag 2760
gtgatctgtg gcccgaatac tgtagttagc tcagttaggt ataggttgag ttctacaccg 2820
acctgttgtg tgatttttat gcagtctaat tctcgcgccg tgttattaaa ctggtcattg 2880
acaaactgtt gcatcttccc aactgccact gataattgtg ataatccgtc ggtgacttca 2940
tgcacagctt cattggtcgc agcaatgctc tccttaagcc ggaggatgtt ggcagcattc 3000
tgtttggctt gtattagggc cgcagctgct gttatctgtg ccgctgttgc aaccccgaga 3060
gctacactgc caataacagc acctataagg cgcccctgtc tccctcctcc ggacgtggat 3120
acagaccctt ggatcttgcg aatggagtcg ccaagaggag tgagcagagt agtcagtgtt 3180
ctgttatatg cctccaatgg ggctcttgca catgcctcct tatctctggg catattcggg 3240
agcaacttga ctatgattga ccctgtctga gacgaggtat atacattgac tgccttatct 3300
cctgttatta caattcctgc agctgcaagg ggcctgccgt caagagaact tgtcagacgg 3360
atgcagttca atatcagcat aattcgagtg attagcatta gaggtgctgg gatcctggta 3420
gaaggtttgg agcccatgct gtgtcctgag tgtgccggtc ggggtccaga ctcttctacc 3480
cgtgtttttt ctaatttgct ggacaggtga actaactaca agcagtcgag atggatcatt 3540
atctggtgtc atgatgattc gggaaagcag gtgaactgca gtcttaggga tgtaacctat 3600
tttctgaaag gattgtattt agcaatggca tgcttcttct ctatcttagt ggaggttacc 3660
tcatgatcgg cggtcactgc aacagcacgc tgagtgccgg cttgaatgat gactttcacg 3720
ctccgcaggt gcgcggtctg gctccagagt atcttggcaa cctggggaga ggcatttgct 3780
ataggatagc aggctgtccc actgctagag aagaaaggag caagtagctt agtccgtgca 3840
cctctcgcct tcacaagcac agagggtccg agcacatcac tgagcccaac agatagattg 3900
agtctcctta tcttttcctc tatcttgtca aatgtcactt tctttcctct cttatctaca 3960
gtggacataa gtccgatatg cagaaaaaga ttcgcatagt acccgctatc ggacttagaa 4020
agggatttga ctaacgggct cttcggatcc acgtccacat caatagtgac attgagcgca 4080
agattgtaca ggctcgagcc agacactttc aatactgtag ttgggatcct gtagacatcc 4140
cttctcggca ccacagtcaa ggagacaaaa ttcactttat actctagggt tccgctccca 4200
gggatctttt cgggcgcctt cacatgcttc actgcattca ctgatgagta ccgatttgac 4260
acaaccatac aattttgcag cactcgaggt gcctgcacta ctgagaagac tattctctca 4320
gtgttagttg cactcttctt gcaggttacc accatagtga ggcaggctct cgccagctca 4380
acaagatctc cgttgttcgg gacactccct aagcagagca ttgcggaaga gagtagctca 4440
tgcctgggat tgtcattgat cacaccgaca gtggctcctt cattcccaac ttgaaagatg 4500
aacccgtagg tggtgataaa taccgagtct tccttactgt ctgtccacga atcaagatgc 4560
tggatcctgt attgtggagt gatttgtttc ttcccgtctc ctgtatcttg taagataatc 4620
ggaaatgcta ataggctact ggaaggaagg gcagaatcaa agtacagccc gattgtcctg 4680
gatgagtcca ttttggttca atcgaggcac tttgattcta cccgtgtttt ttcttaatct 4740
ctaatgctgc tagattggtt gcagttgtgc gatcatgcga gggattgggg atggtgagag 4800
agggctgttg tctgg 4815
<210> 3
<211> 1731
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
gagagagggc tgttgtctgg ttatatacaa atgttgcatc tgcaagaccc attggacatg 60
gcccagaaca agaaggcagc aaaccccatt gcaatcacaa gagatgaggc gacagtcgaa 120
taaatggtga ggattttgta agttccttca gattccagct tgaccccctc tattttctgt 180
ctttctaatt ttgactcctc ttgatacttc cttctgttgt aggtcccgtt ccgaattgtc 240
tccatgcatt ggtcatcaca tttgtggtat agctcgaaac atcctttccc atcttccacc 300
gcattggaac ccaacgccct ctttacttta ttatatagat tgtttacatt tgcgtcatgc 360
tcatcgagtg ttttctggtt ttcaagcaga actagcaatt ctgcattata tgcccatata 420
tcctggattt gatcatcaat cttattattg atcatgttaa gtctagtttc tacctcactg 480
aattcatgat caatgatttc atactgcttg ttcattttgt cgactatatt attcactttg 540
gatgttattt tatctattgc cttttgggtt gagtctctat ctgctgccat accaacccct 600
tggtcatttg aatgctggaa cccataccaa ccagcaacta gtcctgacca acctccctct 660
ataaatcctg ctatggcccc gaatagtcct ctactagatc tagaaggcac attcctcaga 720
ccaactgcaa gtttgagact ctttatgcca atgtattttg agcagtttcc aaatgcatac 780
ttacttacat tttggaatgg caatgttgtg tttaagccac ctttctctgt ctgacattgc 840
actgtgcagc tacccttttt taaatcagtc ttcagaattc ttccgtggct ctctcctgaa 900
agaatgtgtc cataccatgg agctattaga ttcccatcag attttattcg caatgtttga 960
cccggtttca ataccgacca ataataatca attcttccca tcaaaccgtt gacaagaggc 1020
cttggtccta tcaatggttt gaagatccta tttatttctt ctgttgccac actcgttgtt 1080
gtgtcggttc tcgtgtacag atctctctgc gtagtatcgg tgggtgggtg atttatgccc 1140
cacatgaaaa gaatgttctt cccttgatta tttgtgtatt gggcgtcttg aatagggtaa 1200
tcgctgttct tttgagtcaa ccatctcatg cttctgtaga atgaacctga gcatgctttg 1260
cttgtcccat cgtaagacac attccagatt gtgtctggga aaatctggat tctttgataa 1320
gacctagcag aactaaaaag tgaccttagc tcttctagat tttctacatt cccggggtaa 1380
cacaatccgt taacagctga tggtctctca acgatatagg accattctct tccttccagc 1440
aatagatcac aagaaggatt gccatagatt agcccttcaa tggtgcaggt gtctaaaata 1500
agaggttgtc ccaagcttgt tgcacacagc atcccattat gctctgtgtg gagcagttct 1560
ttggcatgtg tcacagggac attgttttct gttagtgtgt ccacagtttc tgtggagttt 1620
gttgattgat agccgatgca gattttatct gcattgctta ctgttgctac tagtagtata 1680
gttattagtg atactgtctc catggtggct tctacccgta ttttttctta a 1731
<210> 4
<211> 1731
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
gagagagggc tgttgtctgg ttatatacaa atgttgcatc tgcaagaccc attggacatg 60
gcccagaaca agaaggcagc aaaccccatt gcaatcacaa gagatgaggc gacagtcgaa 120
taaatggtga ggattttgta agttccttca gattccagct tgaccccctc tattttctgt 180
ctttctaatt ttgactcctc ttgatacttc cttctgttgt aggtcccgtt ccgaattgtc 240
tccatgcatt ggtcatcaca tttgtggtat agctcgaaac atcctttccc atcttccacc 300
gcattggaac ccaacgccct ctttacttta ttatatagat tgtttacatt tgcgtcatgc 360
tcatcgagtg ttttctggtt ttcaagcaga actagcaatt ctgcattata tgcccatata 420
tcctggattt gatcatcaat cttattattg atcatgttaa gtctagtttc gggctcactg 480
aattcatgat caatgggttc atactgcttg ttcattttgt cgactatatt attcactttg 540
gatgttattt tatctattgc cttttgggtt gagtctctat ctgctgccat accaacccct 600
tggtcatttg aatgctggaa cccataccaa ccagcaacta gtcctgacca acctccctct 660
ataaatcctg ctatggcccc gaatagtcct ctactagatc tagaaggcac attcctcaga 720
ccaactgcaa gtttgagact ctttatgcca atgtattttg agcagtttcc aaatgcatac 780
ttacttacat tttggaatgg caatgttgtg tttaagccac ctttctctgt ctgacattgc 840
actgtgcagc tacccttttt taaatcagtc ttcagaattc ttccgtggct ctctcctgaa 900
agaatgtgtc cataccatgg agctattaga ttcccatcag attttattcg caatgtttga 960
cccggtttca ataccgacca ataataatca attcttccca tcaaaccgtt gacaagaggc 1020
cttggtccta tcaatggttt gaagatccta tttatttctt ctgttgccac actcgttgtt 1080
gtgtcggttc tcgtgtacag atctctctgc gtagtatcgg tgggtgggtg atttatgccc 1140
cacatgaaaa gaatgttctt cccttgatta tttgtgtatt gggcgtcttg aatagggtaa 1200
tcgctgttct tttgagtcaa ccatctcatg cttctgtaga atgaacctga gcatgctttg 1260
cttgtcccat cgtaagacac attccagatt gtgtctggga aaatctggat tctttgataa 1320
gacctagcag aactaaaaag tgaccttagc tcttctagat tttctacatt cccggggtaa 1380
cacaatccgt taacagctga tggtctctca acgatatagg accattctct tccttccagc 1440
aatagatcac aagaaggatt gccatagatt agcccttcaa tggtgcaggt gtctaaaata 1500
agaggttgtc ccaagcttgt tgcacacagc atcccattat gctctgtgtg gagcagttct 1560
ttggcatgtg tcacagggac attgttttct gttagtgtgt ccacagtttc tgtggagttt 1620
gttgattgat agccgatgca gattttatct gcattgctta ctgttgctac tagtagtata 1680
gttattagtg atactgtctc catggtggct tctacccgta ttttttctta a 1731
<210> 5
<211> 13399
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
gggtggatgg ggtcgtgtgg ctgggggtgg gggagagggt gggggggagc ttttgttggg 60
ggggtcgtgc ggggttgtgg gtcggggggt gtgggggggt tttgggggag gggtttggtg 120
tggtcctatg gaggcaggct gggagtagtc gatcagtacc cccagtcagt gtcgttgtct 180
tgggatgccc caggggtcgg gggaggctct agatgagtgg tgctctgtgc aggattaggc 240
gattcccgca tgctgttcgc cactgttctc ataaaatcca ggaattgggt ctccccatcc 300
ccagcatctg gttgcccttg cggcttgttc gatccaccct gtggagttcg ggggccttcg 360
tcactgagcc cggtgaggac tcctgcttgt tgagtaggaa tgtccatgct gccgatttct 420
tcagatactc gttgggcagc agctgccagg cctctccttg ctgctggggt tagttttaac 480
tcagcagcca tgtcttcatt gatactactt ccctgagcct gagcatactc tactccaagt 540
cgccagaatg atgtgctcat aaagtccctg gcaaattggt acttgccagt tcccttatct 600
aagactgatg ccatgcccat ggcaaaagaa taaagttgtg catattcggc tggcgcaaag 660
ctcatctggt cactgtcacc taacaatgtc atgtatggtg cattttcacc tttcatccga 720
tataaacgca tgagctgttt cattttttgg atatcacctg tgaggctgct gagtgcgaga 780
gctgacgtct tggtattgat tccatatttg agtgttagga aaaacgcagt aagcccggta 840
tttctgatgt atgagtctac atccccgacc aaattgtaat atgtagagct cccacctgct 900
gtattacggc ccctcttgag ctcactaacc aggaaaatac ggactgccag agaatgtctg 960
attatgagtt gaattgcact cctgcataca ggatgaagga tgtacttctt ctggactcta 1020
ccctgctgca tatacttatt tattcttctt gtttctgact catctgctgt ctcatatgca 1080
gtcatggcct ttgctaccgt gacccatact tggacttgga cagataggat cctttccaga 1140
gtgtcagtga tatcttctgg tgcatcatct tcaaccccag ccgtgacaaa cggagtaccg 1200
ttgctgcatg cccgagggag agaccctgca attaccatga atctctgtgc tctctcctcg 1260
gacactccac tcctattgtt gaactggggc acattattag caaaaccatc gatctcaaga 1320
acagctagtg tggcctcgtt ctgttttcct gcaagggcaa catggtttct catcacctga 1380
gaatgggagc ataagaggga tataagagca ccttgcctga gtggtttgtt ggcatcctca 1440
ctaacggcaa tccgaagaca gaataccgca aagttccatc tatcctctgg atcatcactg 1500
tttagggtaa atactgggac ctcaactttt aaggtgctcc ctttctctcc ccctccatgg 1560
gctccgttag ggcgggtctg ggcagcaagg agctgctcgt attcgtcaaa aacagacgac 1620
atatcggtag aaggttccct caggttcaag cttcgatctc gcgttcgggg ttcacacctt 1680
ctacccgtac gatctcgatt gcttcttcgc cttttatcgt acctcacgga ttctctgttt 1740
ggtgggtcgg catggcatct ccacctcctc gcggtccgac ctgggcatcc gaaggaggac 1800
gtcgtccact cggatggcta agggagagct cggatccggc tgctaacaaa gcccgaaagg 1860
aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta 1920
aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatcg agctagcgcg 1980
ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg gagcactgtc cgaccgcttt 2040
ggccgccgcc cagtcctgct cgcttcgcta cttggagcca ctatcgacta cgcgatcatg 2100
gcgaccacac ccgtcctgtg gatcctctac gccggacgca tcgtggccgg catcaccggc 2160
gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct 2220
cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc 2280
gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac 2340
ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga 2400
ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact 2460
atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca 2520
gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg 2580
tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc 2640
accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc 2700
tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc 2760
gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac 2820
gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcact 2880
ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca 2940
tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg 3000
agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc 3060
caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc 3120
agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg 3180
ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct 3240
ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg 3300
actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg 3360
tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca 3420
tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc 3480
attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct 3540
cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct 3600
ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag 3660
tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa 3720
cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc 3780
ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg 3840
aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900
ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960
tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080
ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 4980
ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5040
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5100
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5160
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5220
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5280
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5340
tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5400
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5460
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5520
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5580
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5640
ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat 5700
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5760
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5820
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5880
gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 5940
ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6000
gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 6060
aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattct catgtttgac 6120
agcttatcat cgataagcgc ggccgctcca ttaatacgac tcactatagg accaaacaga 6180
gatttggtga atgacatagc tgtgattaga gacgactttg cgcatttttg cttaaagtta 6240
actaactgaa tccgaatacg agttccaaag agtcagaatt taactttttt ctaatctggt 6300
ataatcaagt caatgaggga gtcatcagtt aagaagataa ctgattggtg tacgattgcc 6360
tttaagagtc attattactg taatatccct tggcagcatt acctatagtt ttcatgtaga 6420
atttttgttg agcgcgagtc aagtacaata acgaagtacc tgtgaacatt tctttgagct 6480
tagtaatacc taggaccgct ttcaggtatt taggacaggt actgtgcttc aggtatgtcc 6540
ttaatgggat gaggtcctct agggaaatcg cacctctgag tactaagctg attacatcac 6600
ctattttatt gatgtctttg gctctgagac ccaatattgt gacttccaag atctgtttgg 6660
tgcactgctt aagtgatgtt ctctttttac cgtctgtgga tagattgtaa ggagttaata 6720
agaacactgt gtcggcgagg tcaccctcag cctccatgta aatcacggac cggatgactg 6780
tgtcaatgtg gctggcaatg atcagagttg tctgggtcgt atttgttagt gtgctcacca 6840
aactttctgc acaaaatgga cggacgggct gtcccccggc ttcaattagt gcagcatcaa 6900
tgttttcact taagagcctc attagcttcg gtaaagggct ggataggaga tctgttacac 6960
gacgctgctg tgaggtaaat agcttagtca atgtgatttc atctgattta gatagaagtg 7020
taccgtgtcg ttgtattaga gcttttgcca tccttaccac ttcgtgcacg aaagtgggcc 7080
cgcctaagca gcccataacg aatatcaggt aacactccat atcccctcta caggcgtagc 7140
cattggaaag tatgtatcct ttcgtggaac atggagtgaa taaattcatg agtaagtgga 7200
agtagtaccc cattgcatac agtaccttga tgatcactac cccgccctct ctcacagaat 7260
gcatggcaat cagggataaa ttagtagcca gttgatctaa taagctttga ctggaccctg 7320
gaggaatctc aatgtcacaa tgtagtaatg atacagacct gtagggtact acagatgtga 7380
tatatccaac tgccttatct gaggtcagat cactttcttc tgcattctct ctccataatg 7440
gatagaactc ctggacatat ccatctttac atggcacttc cgcttgtaga ttcctataaa 7500
cgaccgagtt tagaaactgt gttggtgtag gtccgaaatg ccgctgtgga gggttcatct 7560
cattcgagaa aagtgtattg taatagatgg tctcatgtgg tatatgcaat tcaagaagac 7620
tcatgatggc tccgcttcct tccgctaagt ataaagagtt cccatgtctt gcacacctaa 7680
cctcagatat ggatagaaga tgagacgcct tataccaaga agatgaagca gtccctactc 7740
ctctgaacaa gtatcgtact aggtaatctt cccttgggtt cggtagtgtg tgctcgaagc 7800
aaatcttact cagtgtaaac gcgtcgtacc ttgctggggc actcagatcc agctcttgta 7860
tgaaagatgc gggttgtcgg gtaaacgggt ctttcactcg agcaccaatg tcccgtactg 7920
ggcgaagctc aagcagtgac tcctgaggga acaacaacga caagatagta tctctgtcct 7980
ctcgttctct gatcaaatta aggctcttcc tagacatgta gtatagattg gctgggaacg 8040
tgattatgtt gggagacatg atagaactca attgttcgga cctaagcaac ggttttacag 8100
catccgacaa tagatactca gtgagtattg agcatttctc ttctgctgat aggcccctta 8160
tttttgggat ttctcttgtt gtagcaaaga gcactgtgta caagcagcat aaccgggaaa 8220
ttagttgaag catcttctca ttcaggttat catctaagac agatggaaac agcagatcat 8280
acttactccc tgcatataaa cctgagacca cgcgtcgagt gcaggagact aacaatttcg 8340
cagacacctc gacaaagtct atatctgcaa gctggtgtga cccgtcatga ttaattagac 8400
ctactgcatt caaccttgag tgaatgaggg ggtgggatat cgtggctgca atattggaga 8460
gtaggatccc tggcatgttc ttatataagt cattcatgta taggacgatg ttgtttagac 8520
cccttaccct cagatagtag agttgataag cacagtcgag gagcacttcg agtgctgcat 8580
actcaaacag gcggaccaca tctgagttct gtgcctcact aatccaattc cgtgtgttgt 8640
catacactat tatagcatca ttctttatag aagtatcttc atcataagaa accacagatt 8700
ggccaatcaa tttcccgcta gatatcgaaa gaatgttcat tagctccagc gtgggatatg 8760
attccaagtt aagctcataa ctcttgaata tagctaagtc aagtctcgca aaatccctct 8820
ctgatatagg gctaggatca tacatgaact tatttgaggt taccatcctt aattccggta 8880
ccagtccgag gagctcgaaa ggaactgcga caggcgcttc tcggatacag cagctaaatt 8940
tactgtggag gtgtaatgtg atctcatcgt atgttctggt tgttgtcatt gggaagagtg 9000
actcaattag agataaaccc aagagcataa tttgctggta aaccacgttt ccctctttga 9060
ccccttcttc ggtgaacagc ctttgagaat cattggatat gtgaatgtaa ggcgacactc 9120
tgtagagaga tgcaggggta aatgtcatct gggttatgcc atcatccaat ctatgttgga 9180
gattcccagc tgtgggcagg ggtgacaata gccgaagata ctctgagctt atgttgcatc 9240
gagaccttgc aatattaaga gcagcagtcc agttcacttc attgtcccca taagcccaga 9300
ttaacacgga tgatgccctt agtgctgctt tcacatgtgg tgacatatgg gctattttcg 9360
caagtgaggc ggctcttctc tcttgagtct tcgacccgag atatggcact ctcatcgggg 9420
gatttttgct ggtgtcatca gtcaactcta tattgcttgg aagatggaac caagtaaact 9480
gctcatctcc gctgtcacat tttgtgcacc ctccactgac gctgagaatc tctccctcca 9540
caagttctat ggtatcgggg ttggatacac ccagtatttt cctgccccct gtcaggggtg 9600
accagcttct gttccgagca taatctgcta gcgtcagcga gcacatatta gaagaaacta 9660
atgggtggtt ggatctagtg gataagaaaa tatcatccct gaacaacatt gcatgcatgc 9720
ttgagtaatt gattatccgc atcagtcttt tgataccgag gggcctccta gtcagtgcaa 9780
tcttaatcac agtgttcgtt gtgtcaacaa gcccttgaat ttgctttctc ctacccacag 9840
agcttgcttc catgatagca tgtgcgacac gtgggtgaat cacttcttga ttgagtaaga 9900
attcagccaa tgctttctct tctgcctcat tgtcctctgt atgtaccccg gataataaag 9960
ggtttgaaca tgtctcaaat aggactttct gtgtatgttt cttgaggaca atatttgggc 10020
ttgcaacagt ctcaaaatta aaagagtatg ggtcgttgca taggctggcc caatctccat 10080
tgccaggtgg cctggttaag atgttagtca tgatgctggg actcaacaac cccactgctt 10140
ctagtcgctt gacctctgca aaggcagtgg tccctgggtc gccaatattc cttgtgtaga 10200
gccttgagta ttgaaggtta ctcagtcccc ccagttgggc aggggttaac acgtatgagt 10260
gtacgaaaga gatatcctca atccaggact ggttggaatc tgactgtgag ctgtgagtaa 10320
tagaaaactc tgaatcaaaa tatgtctgca cgcaactcat taggtagttc aaatagtaac 10380
agaagtcctt aggaagccca ttctcacata gtcgtgctac agtggatgca atgttggcac 10440
aggacattac agtgttttcg ctaaggtcac ctgatattag caccaattta gatgaatttt 10500
tgaggacctg actgagtatt gctccatctt tgaatattcg tttgctgtat atgaagaatg 10560
tgtctgatct aatggtttca cgatccttca ggttatggcc aatcagatga ttgacatgaa 10620
tcaattcctt gaagaaatta tcactagcct gatgcaactg cgtcaatacc atatccgggg 10680
aatcatctga tctcacctct ctcgttacag ctattacttg attgtcacct tgtaccatgc 10740
aggcaactcg acaatgagat cttgctgcgg caagttggat tgcagcaatt gagatcatcg 10800
tccatagctt ctgacagagt ccctcaatgc cccctctagc actgacaata tatatatcat 10860
catttgggac tcttgataga tcacagtcgg tcgggtcact tggaggattg aaaggatccc 10920
ctacaaacat tgtagtgtcc atcagcctaa gatgaatcca ctcaaagaaa tgaggtaggc 10980
ccatcagctg attgatggca tgggcgaata gtttgactgt ctgatatctc cagttaagac 11040
aatacttttg taggtcagtc gtgataaagg tggcaactct tctacgattc ttgctcttgg 11100
gatcatgatt gcggtttgag gaaaccctct ctttgcagtc agcgatacgt ttcttattgc 11160
tgttaaagga cagttgactc attgctaaca tactctttgt caaggatatg ctatcttgaa 11220
tgaccccatt tccctggaag aaaggtgcaa tctggtcagc tagaattcct tctgccatta 11280
cctggcaatt ccttagtttc tttgttaatt tagcaaaaat ccgtccattc actttcacct 11340
ctttctcttt gagtgagtac gatactgcca cactgtcatc tcttaggtac tcgagggttg 11400
tcaggtattc catttcttta tatggatcaa aatcatttga ttctaagaac tctatcagga 11460
ggcggttagt tgaagttgcc tcctttatct gtttcttctg gtcctcagag agtaggttcc 11520
gcctaaatga ggcgagccag ttatcactag gatgtgcgat tgccttgtct tttaggaaca 11580
tgcttagatt ggtaacaggg tcataatcta tacatggctc aaattcaaga gcggataaac 11640
tcttgtactc cctcaacatg acatcatgtg agatctctgc tgaatcagca tgtagctgcc 11700
caatgatatt tccatatatt gtatctactt tgacgcgtgg ccacacacct gagttctttt 11760
ttctgtatcc attgatgatt gttcctttaa agaaagataa tacctggagg atcatatcaa 11820
agtctaccat ctttggtgcg cacatctggc tcctgactgc tcttgctgca ctacgagact 11880
caagcaatgg atgaccccac aaacgcagca agcacaacat ctcagctgct tggttctgtt 11940
ctaatccaga gaatacagtg gcaatagcgt gagttactgc ttccgctata ttattgggga 12000
gaagttcgat taacgtgttt ttgagctcct gtaggttaaa tgcaaagaaa tctcctgcaa 12060
atgtacctga tggctcaagc agctgaacgg caccgtatgc aaatccctcc attaatgcta 12120
caacatcata gacttgatta cctaagtcct ttgccagagc atctactaac cgcagaatat 12180
catcaatttt ctcggatagg ttcctgagat gtgctgctgt agaagatatt atattgacca 12240
tgtccctgcc ttccatcata tccgcataca tcaatacaag ttcctgggtg agacatgtga 12300
acttgttctc gtctgtgtgt gtcacaatga caagctcagg agtaataaag acatggccta 12360
tcttatgatt taatgttact aacttgttga ctgcagacct tgtccttgct gctacaatca 12420
gatgcctttg gacttgtttt atatggagcc acgcgaactt ggctctggac cattttgagt 12480
gaaaccagaa tgccggatct gtacggatgc tgttgaattc ctctgatcgt gggacattat 12540
tagacagaga tgaccctagc aatttcttct caacttgcac gcacagcttt gtgaacaggt 12600
ctccatatct tgtgttgtga atctggatct tcttttcaat cttccggaat ttgttagttg 12660
aatcagggac ctcaatacta gccagttctt ctaaacacct gggatggagc actccagtca 12720
tcttggaatt gtggttgaga gtctggtgca ccgcccgccc aagttttatc attctctctg 12780
tgtcaggagt ggctgactcc agtattctct tccattgcct gctgataatg agatgatcaa 12840
agtcgcattc atcaggaagc ggtagcccgg ttaatttcca gtagtataac aatttgtgct 12900
tgaccaatgg agaggataaa tgtgactctg gtaggatgat ctggtgctct gccctctcag 12960
gaccggagcc cgccatgtcc tacccgtgtt ctctcttgtt ggttattttc ccttatatgt 13020
caatgcttct tgtgtttttt cttaataaag tgacagtgag cgagactcag aacaatcatg 13080
tcagcattgt cggattgcgg ttgcccaaca acacgggatc ctgatttatg ctaatgttca 13140
gcttgactct aagcattatg ggagatgatt ggcgcaatgt cgtcctccca accatcctat 13200
gattggctca atcggccaag tctagcttct taaactctat catctttgag gatctcaact 13260
agcaagggaa cgatcctaaa ttccccgaat agggtattgg atatttctgc gatactaaga 13320
caataaactt tattagtctt gacaacttta aaacatgtcg atgtcgtgta tgctgccttg 13380
gtgttgcttg aactcaccc 13399
<210> 6
<211> 41
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
ctacccgtat tttttcttaa ggtgttgaag catggattcg c 41
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
agccacacga ccccatccac cc 22
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
cttggtgttg cttgaactca ccc 23
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
ccagacaaca gccctctctc 20
<210> 10
<211> 51
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
ttaagaaaaa atacgggtag aagccaccat ggagacagta tcactaataa c 51
<210> 11
<211> 42
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
gagagagggc tgttgtctgg ttatatacaa atgttgcatc tg 42
<210> 12
<211> 54
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
catgaattca gtgaggggga aactagactt aacatgatca ataataagat tgat 54
<210> 13
<211> 51
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
ccctcactga attcatgatc aatcccttca tactgcttgt tcattttgtc g 51
<210> 14
<211> 5983
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
ctgctttaat aagatctggt accggttata tacaaatgtt gcatctgcaa gacccattgg 60
acatggccca gaacaagaag gcagcaaacc ccattgcaat cacaagagat gaggcaacag 120
tcgaataaat ggtgaggatt ttgtaagttc cttcagattc cagcttgacc ccctctattt 180
tctgtctttc taattttgac tcctcttgat acttccttct gttgtaggtc ccgtttcgaa 240
ttgtctccat gcattggtca tcacatttgt ggtatagctc gaaacatcct ttcccatctt 300
ccaccgcatt ggaacccaac gccctcttta ctttattata tagattgttt acatttgcgt 360
catgctcatc gagtgttttc tggttttcaa gcagaactag caattctgca ttatatgccc 420
atatatcctg gatttgatca tcaatcttat tattgatcat gttaagtcta gtttctacct 480
cactgaattc atgatcaatg atttcatact gcttgttcat tttgtcgact atattattca 540
ctttggatgt tattttatca attgcctttt gggttgagtc tctatctgct gccataccaa 600
ccccttggtc atttgaatgc tggaacccat accaaccagc aactagccct gaccaacctc 660
cctctataaa ccctgctatg gccccgaata gtcctctact agatctagaa ggcacattcc 720
tcagaccaac tgcaagtttg agactcttta tgccaatgta ttttgagcag tttccaaatg 780
catacttact tacattttgg aatggcagtg ttgtgtttaa gccacctttc tctgtctgac 840
attgcactgt gcagctaccc ctttttaaat cagtcttcag aattcttccg tggctctctc 900
ctgaaagaat gtgtccatac catggagcta ttagattccc atcagatttt attcgcagtg 960
tttgactcgg tttcaataca gaccaataat aatcaattct tcccatcaaa ccgttgacaa 1020
gaggccttgg tcctatcaat ggtttgaaga tcctatttat ttcttctgtt gccacactcg 1080
ttgttgtgtc gtttctcgtg tacagatttc tctgcgtagt atcggtgggt gggtgatgta 1140
tgccccacat gaaaagaatg ttcttccctt gattatttgt gtattgggcg tcttggacag 1200
ggtaatcgcc gttctttcga gtcaaccatc tcatgcttct gtagaatgaa cctgagcatg 1260
ctgtgcttgt cccatcgtaa gacacattcc agattgtgtc tgggaaaatc tggattcttt 1320
gataagacct agcagaacta aaaagtgacc ttagctcttc tagattttct acattcccgg 1380
ggtaacacaa tccgttgaca gctgatggtc tctcgacgat ataggaccat tctcttcctt 1440
ccagcgatag atcacaagaa ggattgccat agattagccc ttcaatggtg caggtgtcta 1500
aaataagagg ttgtcccaag cttgttgcac acagcatccc attatgctct gtgtggagca 1560
gttctttggc atgtgtcaca gggacattgt tttctgttag tgtgtccaca gtttctgtgg 1620
agtttgttga ttgatagccg atgcagattt tatctgcatt gcttactgtt gctactagta 1680
gtatagttat tagtgatact gtctcggatc cggacttatc atcgtcgtcc ttgtagtcca 1740
tggtgaattc gccctatagt gagtcgtatt acaattcttt gccaaaatga tgagacagca 1800
caataaccag cacgttgccc aggagctgta ggaaaaagaa gaaggcatga acatggttag 1860
cagaggggcc cggtttggac tcagagtatt ttatcctcat ctcaaacagt gtatatcatt 1920
gtaaccataa agagaaaggc aggatgatga ccaggatgta gttgtttcta ccaataagaa 1980
tatttccacg ccagccagaa tttatatgca gaaatattct accttatcat ttaattataa 2040
caattgttct ctaaaactgt gctgaagtac aatataatat accctgattg ccttgaaaaa 2100
aaagtgatta gagaaagtac ttacaatctg acaaataaac aaaagtgaat ttaaaaattc 2160
gttacaaatg caagctaaag tttaacgaaa aagttacaga aaatgaaaag aaaataagag 2220
gagacaatgg ttgtcaacag agtagaaagt gaaagaaaca aaattatcat gagggtccat 2280
ggtgatacaa gggacatctt cccattctaa acaacaccct gaaaactttg ccccctccat 2340
ataacatgaa ttttacaata gcgaaaaaga aagaacaatc aagggtcccc aaactcaccc 2400
tgaagttctc aggatcgatc cggagctttt tgcaaaagcc taggggtccg ttccaatgca 2460
ccgttcccgg ccgcggaggc tggatcggtc ccggtgtctt ctatggaggt caaaacagcg 2520
tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta tatagacctc 2580
ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg 2640
aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact 2700
tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat 2760
caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa 2820
ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg 2880
cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc 2940
cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat 3000
tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta 3060
tgtaacggac tctagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3120
gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt 3180
gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 3240
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 3300
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 3360
tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 3420
ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 3480
atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 3540
aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 3600
tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 3660
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 3720
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 3780
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 3840
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 3900
agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 3960
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4020
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4080
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4140
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4200
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4260
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4320
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4380
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4440
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4500
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4560
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 4620
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 4680
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 4740
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 4800
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 4860
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 4920
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 4980
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5040
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5100
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5160
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 5220
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5280
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5340
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5400
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5460
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 5520
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 5580
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 5640
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 5700
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 5760
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 5820
tgagcgagga agcggaagag tctagagtcg accagacatg ataagataca ttgatgagtt 5880
tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 5940
tattgcttta tttgtaacca ttataagctg caataaacaa gtt 5983
<210> 15
<211> 5983
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 15
ctgctttaat aagatctggt accggttata tacaaatgtt gcatctgcaa gacccattgg 60
acatggccca gaacaagaag gcagcaaacc ccattgcaat cacaagagat gaggcaacag 120
tcgaataaat ggtgaggatt ttgtaagttc cttcagattc cagcttgacc ccctctattt 180
tctgtctttc taattttgac tcctcttgat acttccttct gttgtaggtc ccgtttcgaa 240
ttgtctccat gcattggtca tcacatttgt ggtatagctc gaaacatcct ttcccatctt 300
ccaccgcatt ggaacccaac gccctcttta ctttattata tagattgttt acatttgcgt 360
catgctcatc gagtgttttc tggttttcaa gcagaactag caattctgca ttatatgccc 420
atatatcctg gatttgatca tcaatcttat tattgatcat gttaagtcta gtttctacct 480
cactgaattc atgatcaatg atttcatact gcttgttcat tttgtcgact atattattca 540
ctttggatgt tattttatca attgcctttt gggttgagtc tctatctgct gccataccaa 600
ccccttggtc atttgaatgc tggaacccat accaaccagc aactagccct gaccaacctc 660
cctctataaa ccctgctatg gccccgaata gtcctctact agatctagaa ggcacattcc 720
tcagaccaac tgcaagtttg agactcttta tgccaatgta ttttgagcag tttccaaatg 780
catacttact tacattttgg aatggcagtg ttgtgtttaa gccacctttc tctgtctgac 840
attgcactgt gcagctaccc ctttttaaat cagtcttcag aattcttccg tggctctctc 900
ctgaaagaat gtgtccatac catggagcta ttagattccc atcagatttt attcgcagtg 960
tttgactcgg tttcaataca gaccaataat aatcaattct tcccatcaaa ccgttgacaa 1020
gaggccttgg tcctatcaat ggtttgaaga tcctatttat ttcttctgtt gccacactcg 1080
ttgttgtgtc gtttctcgtg tacagatttc tctgcgtagt atcggtgggt gggtgatgta 1140
tgccccacat gaaaagaatg ttcttccctt gattatttgt gtattgggcg tcttggacag 1200
ggtaatcgcc gttctttcga gtcaaccatc tcatgcttct gtagaatgaa cctgagcatg 1260
ctgtgcttgt cccatcgtaa gacacattcc agattgtgtc tgggaaaatc tggattcttt 1320
gataagacct agcagaacta aaaagtgacc ttagctcttc tagattttct acattcccgg 1380
ggtaacacaa tccgttgaca gctgatggtc tctcgacgat ataggaccat tctcttcctt 1440
ccagcgatag atcacaagaa ggattgccat agattagccc ttcaatggtg caggtgtcta 1500
aaataagagg ttgtcccaag cttgttgcac acagcatccc attatgctct gtgtggagca 1560
gttctttggc atgtgtcaca gggacattgt tttctgttag tgtgtccaca gtttctgtgg 1620
agtttgttga ttgatagccg atgcagattt tatctgcatt gcttactgtt gctactagta 1680
gtatagttat tagtgatact gtctcggatc cggacttatc atcgtcgtcc ttgtagtcca 1740
tggtgaattc gccctatagt gagtcgtatt acaattcttt gccaaaatga tgagacagca 1800
caataaccag cacgttgccc aggagctgta ggaaaaagaa gaaggcatga acatggttag 1860
cagaggggcc cggtttggac tcagagtatt ttatcctcat ctcaaacagt gtatatcatt 1920
gtaaccataa agagaaaggc aggatgatga ccaggatgta gttgtttcta ccaataagaa 1980
tatttccacg ccagccagaa tttatatgca gaaatattct accttatcat ttaattataa 2040
caattgttct ctaaaactgt gctgaagtac aatataatat accctgattg ccttgaaaaa 2100
aaagtgatta gagaaagtac ttacaatctg acaaataaac aaaagtgaat ttaaaaattc 2160
gttacaaatg caagctaaag tttaacgaaa aagttacaga aaatgaaaag aaaataagag 2220
gagacaatgg ttgtcaacag agtagaaagt gaaagaaaca aaattatcat gagggtccat 2280
ggtgatacaa gggacatctt cccattctaa acaacaccct gaaaactttg ccccctccat 2340
ataacatgaa ttttacaata gcgaaaaaga aagaacaatc aagggtcccc aaactcaccc 2400
tgaagttctc aggatcgatc cggagctttt tgcaaaagcc taggggtccg ttccaatgca 2460
ccgttcccgg ccgcggaggc tggatcggtc ccggtgtctt ctatggaggt caaaacagcg 2520
tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta tatagacctc 2580
ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg 2640
aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact 2700
tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat 2760
caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa 2820
ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg 2880
cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc 2940
cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat 3000
tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta 3060
tgtaacggac tctagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3120
gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt 3180
gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 3240
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 3300
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 3360
tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 3420
ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 3480
atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 3540
aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 3600
tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 3660
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 3720
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 3780
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 3840
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 3900
agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 3960
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4020
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4080
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4140
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4200
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4260
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4320
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4380
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4440
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4500
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4560
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 4620
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 4680
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 4740
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 4800
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 4860
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 4920
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 4980
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5040
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5100
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5160
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 5220
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5280
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5340
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5400
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5460
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 5520
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 5580
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 5640
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 5700
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 5760
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 5820
tgagcgagga agcggaagag tctagagtcg accagacatg ataagataca ttgatgagtt 5880
tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 5940
tattgcttta tttgtaacca ttataagctg caataaacaa gtt 5983
<210> 16
<211> 1698
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 16
ctggtaccgg ttatatacaa atgttgcatc tgcaagaccc attggacatg gcccagaaca 60
agaaggcagc aaaccccatt gcaatcacaa gagatgaggc aacagtcgaa taaatggtga 120
ggattttgta agttccttca gattccagct tgaccccctc tattttctgt ctttctaatt 180
ttgactcctc ttgatacttc cttctgttgt aggtcccgtt tcgaattgtc tccatgcatt 240
ggtcatcaca tttgtggtat agctcgaaac atcctttccc atcttccacc gcattggaac 300
ccaacgccct ctttacttta ttatatagat tgtttacatt tgcgtcatgc tcatcgagtg 360
ttttctggtt ttcaagcaga actagcaatt ctgcattata tgcccatata tcctggattt 420
gatcatcaat cttattattg atcatgttaa gtctagtttc tacctcactg aattcatgat 480
caatgatttc atactgcttg ttcattttgt cgactatatt attcactttg gatgttattt 540
tatcaattgc cttttgggtt gagtctctat ctgctgccat accaacccct tggtcatttg 600
aatgctggaa cccataccaa ccagcaacta gccctgacca acctccctct ataaaccctg 660
ctatggcccc gaatagtcct ctactagatc tagaaggcac attcctcaga ccaactgcaa 720
gtttgagact ctttatgcca atgtattttg agcagtttcc aaatgcatac ttacttacat 780
tttggaatgg cagtgttgtg tttaagccac ctttctctgt ctgacattgc actgtgcagc 840
tacccctttt taaatcagtc ttcagaattc ttccgtggct ctctcctgaa agaatgtgtc 900
cataccatgg agctattaga ttcccatcag attttattcg cagtgtttga ctcggtttca 960
atacagacca ataataatca attcttccca tcaaaccgtt gacaagaggc cttggtccta 1020
tcaatggttt gaagatccta tttatttctt ctgttgccac actcgttgtt gtgtcgtttc 1080
tcgtgtacag atttctctgc gtagtatcgg tgggtgggtg atgtatgccc cacatgaaaa 1140
gaatgttctt cccttgatta tttgtgtatt gggcgtcttg gacagggtaa tcgccgttct 1200
ttcgagtcaa ccatctcatg cttctgtaga atgaacctga gcatgctgtg cttgtcccat 1260
cgtaagacac attccagatt gtgtctggga aaatctggat tctttgataa gacctagcag 1320
aactaaaaag tgaccttagc tcttctagat tttctacatt cccggggtaa cacaatccgt 1380
tgacagctga tggtctctcg acgatatagg accattctct tccttccagc gatagatcac 1440
aagaaggatt gccatagatt agcccttcaa tggtgcaggt gtctaaaata agaggttgtc 1500
ccaagcttgt tgcacacagc atcccattat gctctgtgtg gagcagttct ttggcatgtg 1560
tcacagggac attgttttct gttagtgtgt ccacagtttc tgtggagttt gttgattgat 1620
agccgatgca gattttatct gcattgctta ctgttgctac tagtagtata gttattagtg 1680
atactgtctc ggatccgg 1698

Claims (6)

1. A preparation method of recombinant plasmid is characterized in that F1 segment, F2 segment, HA-2P segment and F4 segment are subjected to homologous recombination to obtain recombinant plasmid;
f1 fragment, F2 fragment, HA-2P fragment and F4 fragment are respectively shown in the sequence table SEQ ID NO: 1.2, 4 and 5.
2. The method for preparing the recombinant plasmid according to claim 1, wherein the recombinant kit is adopted, and the following components are added after reacting for 30 minutes at 37 ℃, wherein the method comprises the steps of:
f1 fragment 30ng;
f2 fragment 97ng;
30ng of HA-2P fragment;
200ng of F4 fragment;
5xCE MultiS Buffer 4μl；
Exnase MultiS 2μl；
ddH2O was added to 20. Mu.l.
3. The method for preparing the recombinant plasmid according to claim 1, wherein the method for preparing the F1 fragment comprises the following steps:
amplifying the primers FDHN3-F1 and FDHN3-SmaI-R1 to obtain an F1 fragment;
the primers FDHN3-F1 and FDHN3-SmaI-R1 are respectively shown in a sequence table SEQ ID NO:6 to 7.
4. The method for preparing the recombinant plasmid according to claim 1, wherein the method for preparing the F2 fragment comprises the following steps:
amplifying the primers FDHN3-SmaI-F2 and FDHN3-R2 to obtain an F2 fragment;
the primers FDHN3-SmaI-F2 and FDHN3-R2 are respectively shown in a sequence table SEQ ID NO:8 to 9.
5. The method for preparing the recombinant plasmid according to claim 1, wherein the HA-2P fragment is prepared by the following steps:
amplifying the obtained HA-2P fragment by using a pXJ flag-HA-2P carrier as a template and using a primer HA-F, HA-R;
the preparation method of the pXJ flag-HA-2P carrier comprises the following steps:
using pXJ Flag-HA plasmid as carrier, using primer Mutate-F, mutate-R to reversely amplify PCR fragment according to the instruction of the Mut Express II Fast Mutagenesis Kit V2 kit;
after Dpn I digestion, the digestion products are cyclized in vitro;
transforming the cyclized plasmid, and selecting monoclonal bacteria to sequence the HA segment;
selecting a colony with correct sequencing for propagation, and extracting plasmids to obtain a pXJ flag-HA-2P vector;
the HA-F, HA-R is respectively shown in a sequence table SEQ ID NO:10 to 11;
the primer Mutate-F, mutate-R is respectively shown in a sequence table SEQ ID NO:12 to 13;
the pXJ Flag-HA plasmid is shown in a sequence table SEQ ID NO: 14.
6. A recombinant virus obtained by transfecting BHK-21 cells with a plasmid prepared according to any one of claims 1 to 5.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402071A (en) * 2018-11-08 2019-03-01 中国农业大学 A kind of recombinant herpesvirus of turkeys for expressing H9N2 subtype avian influenza virus H9 albumen
CN110128545A (en) * 2019-04-22 2019-08-16 肇庆大华农生物药品有限公司 A kind of fusion, recombinant expression carrier, antigen and its preparation method and application
CN110218706A (en) * 2019-05-14 2019-09-10 华南农业大学 Express the building and application of the recombinant herpesvirus of turkeys of H7N9 subtype highly pathogenic avian influenza virus HA albumen
CN112048484A (en) * 2020-07-27 2020-12-08 华南农业大学 Gene VII-type Newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and vaccine
CN112626123A (en) * 2020-12-31 2021-04-09 华农（肇庆）生物产业技术研究院有限公司 Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3395826B1 (en) * 2013-08-03 2020-10-14 Calder Biosciences Inc. Methods of making and using influenza virus hemagglutinin complexes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402071A (en) * 2018-11-08 2019-03-01 中国农业大学 A kind of recombinant herpesvirus of turkeys for expressing H9N2 subtype avian influenza virus H9 albumen
CN110128545A (en) * 2019-04-22 2019-08-16 肇庆大华农生物药品有限公司 A kind of fusion, recombinant expression carrier, antigen and its preparation method and application
CN110218706A (en) * 2019-05-14 2019-09-10 华南农业大学 Express the building and application of the recombinant herpesvirus of turkeys of H7N9 subtype highly pathogenic avian influenza virus HA albumen
CN112048484A (en) * 2020-07-27 2020-12-08 华南农业大学 Gene VII-type Newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and vaccine
CN112626123A (en) * 2020-12-31 2021-04-09 华农（肇庆）生物产业技术研究院有限公司 Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GenBank: AGQ81180.1;Lam,T.T. 等;《GenBank》;feature、origin部分 *
Rapid Development of an Effective Newcastle Disease Virus Vaccine Candidate by Attenuation of a Genotype VII Velogenic Isolate Using a Simple Infectious Cloning System;Nannan Wang 等;《Frontiers in Veterinary Science》;第7卷;第1-13页 *
表达ＩＢＤＶ ＶＰ２蛋白的基因Ⅶ型重组新城疫病毒的构建及鉴定;杜倩茹 等;《中国兽医学报》;第41卷(第8期);第1464-1469页 *

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