CN106834282A - A kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full-length genome - Google Patents
A kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full-length genome Download PDFInfo
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Abstract
The invention belongs to biological technical field, a kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full genome are disclosed.The primer sets include 22 primer pairs, and each primer pair upstream and downstream primer is successively as shown in SEQ ID NO.1~SEQ ID NO.44.Methods described is inverted to cDNA to extract PEDV total serum IgEs, and pcr amplification reaction is carried out with the primer sets, and PCR primer is connected into cloning vector, chooses positive colony, and DNA sequencing identification is finally spliced sequence, obtains PEDV whole genome sequences.The present invention is expanded with carrying out all standing formula by designing 22 pairs of primer sets to PEDV, huge genome is subdivided into multistage, the nucleotides of case study particular section can be easy to change, including site mutation, nucleotides inserted and missing etc., more preferably to hold the change dynamic of PEDV genes, understand the change of strain, accurate molecular information is provided to solve the disease.
Description
Technical field
The invention belongs to biological technical field, in particular it relates to a kind of be segmented the full base of amplification Porcine epidemic diarrhea virus
Because of the primer sets and method organized.
Background technology
The cause of disease of pig epidemic diarrhea be Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus,
PEDV), it mainly induces pig intestines problem, and its Symptoms is vomiting, diarrhoea, loss of appetite, dehydration, and with significant
Age in days relies on phenomenon.Especially for the newborn piglet in 7 ages in days, in the case where effective maternal antibody is lacked, the death rate may be up to
100%.PED has been broken out on pig farm on a large scale in the end of the year 2010, China, and is not effectively controlled yet in recent years to China's pig industry
Cause huge loss.In April, 2013, PED epidemic situations, subsequent Asia other countries and part Europe were broken out successively in American States
PEDV epidemic situations are also broken out in succession in country, bring huge harm to worldwide pig industry again.Based in recent years popular
The appearance of street strain and it is very popular, traditional commodities vaccine lacks to variation strain and produces sufficient resistant function.Thus, it is right
The research of newfashioned PEDV variants and its molecular epidemiology is analyzed, can be the molecule machine for disclosing new hair PEDV Infectives
The development of system and specific diagnosis provides necessary some theoretical reference foundations.
Porcine epidemic diarrhea virus is have petal sample fine prominent with cyst membrane, and on cyst membrane in composition structure, nucleic acid type
It is linear, single-stranded positive, the RNA virus of non-segmented negative.In all RNA virus, the genome length of coronavirus is all relative
It is more long.And PEDV complete genome nucleotide there are about 28,038nt, its genome sequence is the same similar to mRNA, and 5 ' ends have
Cap sequence, 3 ' ends possess Poly (A) tail.From 5 ' ends to 3 ' ends, gene structure order is followed successively by 5 ' UTR-Replicase
(ORF1a/b)-S-ORF3-E-M-N-3’UTR；The structural proteins of main code have spike protein (Spike, S), memebrane protein
(Membrane, M), envelope protein (Envelope, E) and nucleocapsid protein (Nucleocapsid, N).And non-structural protein has
Replicase polyprotein (Open Reading Frame 1, ORF1a/b) and ORF3 albumen (Open Reading Frame 3,
ORF3).PEDV genomes 5 ' hold non-translational region (5 ' UTR) 296nt long, and 65nt-98nt targeting sequencings (L) are about comprising one,
Kozak sequences (GUUCAUGC) and a short and small ORFs (ORF).3 ' UTR length are about 334nt, possess an institute
Having coronavirus has, but different conservative 8 bases (GGAAGAGC) sequence in position.The replicase polyprotein ORF1a/ of PEDV
B (pp1ab) is collectively constituted by two subunit proteins ORF1a and ORF1b.In nucleotide sequence, ORF1a/b total lengths are about
20345nt, two big reading frames for being respectively 12354nt and 8037nt by comprising 46nt overlaps and length are constituted, and are accounted for
According to the nucleotides quantity of genome about 2/3.ORF1a/b non-structural proteins can perform various important biologies in virus infection early stage
Function is learned, wherein ORF1a mainly performs Protein cleavage effect and participates in duplication of RNA etc., and ORF1b main codes RNA is relied on
Property RNA polymerase.The spike protein S of coronavirus is jointly owned including being attached to target cell, and mediation cyst membrane melts with cell membrane
The basic biological function of conjunction etc..1383 amino that the S protein of classical strainses CV777 (AF353511) is encoded by about 4152nt
Acid composition.S protein belongs to the membrane glycoprotein of 1 type, is respectively signal peptide area (1-24aa), cell outskirt long from N to C-terminal
(25-1333aa), (1334-1356aa) and shorter cytoplasmic tail (1357-1383aa).Simultaneously according to other coronavirus
, can also be divided into S protein by the prominent Asia Protein S 1 (l-789aa) of the fibre for folding grappling and S2 (790- by the homology of S protein
1383aa).Wherein S1 albumen can also be sub-divided into the functional area S1-NTD (N-terminal with bind receptor
Domain, 19-252aa) and S1-CTD (C-terminal domain, 509-638aa).Up to the present, researcher is in S
Region COE (99-638aa) and 3 epitopes of induction neutralizing antibody generation that 1 induction neutralizing antibody is produced are identified on albumen,
It is respectively SS2 (748YSNIGVCK 755), SS6 (764LQDGQVKI 771)；2C10,1368GPRLQPY 1374.Research table
Bright, PEDV breedings in the cell need the assistance of proteolytic enzyme, and the process may relate to S protein.When virus is adhered to
After the cell membrane of host cell, under the trypsin acting of external source, S protein digested be fractured into two subunits (S1 and
S2), make the cell for being infected that more obvious lesion occurs.Additionally, S protein and in vitro culture virus adaptability and virus drop
The decay of degree has relation.ORF3 is the only auxiliary genes of PEDV, and its complete ORF3 gene has 675nt to encode 225aa.And in body
During outer passage, the missing of different patterns can occur in ORF3 nucleotides, fully be demonstrated by its diversity, with virus poison
Power is relevant.In classical vaccine CV777 and South Korea's DR13 vaccine strains, the missing of the 49nt that ORF3 genes are occurred in that can be as
In PCR diagnosis, the genetic marker of the wild poison of PEDV and cell attenuated IBDVs is distinguished.2012, Wang etc. had found ORF3 albumen first
As the 3a albumen of SARS-CoV, potassium-channel can be formed in cell surface, improve the titre of virus.The E bases of PEDV
Reason 231nt is constituted, and encodes 76 amino acid.The albumen plays key at the aspect such as composition of peplos and virion
Effect.However, in DR13 attenuated IBDVs, there is the missing of 21nt, E genes still have on the pathogenic and structure function of PEDV
It is to be studied.Research finds that E protein can be positioned at endoplasmic reticulum and kernel surface, cause er stress effect, it is impossible to influence intestines
The cell replication cycle of road cell IEC, but can be the expression for raising interleukin 8 and Apoptosis Bcl-2.M full length genes
681nt, in research at present, its missing for not depositing base, sequence conservation is only second to N genes.M albumen is main as PEDV
Cyst membrane structural proteins, it is played an important role in the assembling of virion and Budding process.Laude, H. send out in its research
Existing, TGEV can produce alpha-interferon (IFN-α) in cell infection with excitating organism.During M albumen can also stimulate body to produce
And antibody.In addition, Zhang Zhi's list etc. also identifies the linear epitope 195WAFYVR200 of M genes, and the epitope can not be by
TGEV positive serums are recognized.PEDV N genes are made up of 1326nt, encode 441 amino acid.In PEDV genomes, in nucleosides
On sour value volume and range of product, conservative highest.N protein, can be with cell nuclear phosphoprotein in infection cell used as a kind of rna binding protein
In nucleus, according to the outstanding reports of Lv Mao, it finds that N genes nucleolar localization signal and N protein kernel location mechanism are provided to common location
Foundation.The alkalescence of height is shown because it is rich in basic amino acid, such as lysine and arginine residues, in pH=9.0
Hypotonic non-ionic solvents in have maximum solubility.In coronavirus known structure albumen, only N protein is phosphorylation
Albumen.Serine residue is considered as potential phosphorylation site, and the N protein of PEDV CH/S strains possesses 35 silk ammonia altogether
Sour residue, accounts for the 7.9% of its total amino acid residue number, and be concentrated mainly on (57-190aa and 199- in two basic regions
391aa).PEDV N proteins are proved that the expression of Interferon regulatory factor-3 activity and interferon type Ⅰ can be suppressed, and thus influence
The immune system of body.Xu etc. research find N protein can extend the S cycles of infection cell, while cause er stress and
Raise the expression of interleukin 8.Through extensive prevalence study, variation PEDV strain S gene coding regions popular both at home and abroad are complete at present
4161nt long, and CV777 is only 4152nt as the S mrna lengths of classical strainses.Its reason is mainly variant in S genes
S1 fragments have 15nt to insert and 6nt missings.Investigated according to Chen etc., due to evolution difference, the S genes of PEDV present sequence length
With the diversity of nucleotide difference.In the sequence of ncbi database, S mrna lengths are from 3359-4255nt, up to 11 kinds length
Type.It is detected and the sequence of the length of 4161nt equally accounts for most (about 68.4%), and after being 2010 years, table
The strain of the species has obvious evolutionary edge before improving eyesight.In addition, MF3809/2008/South Korea
(KF779469), TC-PC177-P2, Tottori2/JPN/2014 and TTR-2/JPN/2014 (LC063828) etc. divide in recent years
There is the nucleotide deletion of large fragment in S genes from the variation strain for obtaining, also belong to rare.In sequence homology, variation
Strain is not high with CV777 strains, and there is insertion and the missing of nucleotides, points out PEDV variation strains more than 30 year
Preceding classical strainses show speed of mutation faster.In major antigen and in site, variant has many places with classical strainses
The mutation of amino acid.The change in these sites is probably that current vaccine strain can not produce the main original of good protection to variation strain
Cause.But insertion on these sites and amino acid that are mutated and missing on the influence of S structural genes whether to its antigenicity,
The biological functions such as virulence still need further exploration.It is numerous to lack in genome other positions except the change of S genes
Gene location and quantity all difference, also enrich the bio-diversity of PEDV strains, so as to also cause the research of the virus
In the presence of more uncertainties.For example in ORF1a/b genes, in 1036-1043 amino acids, there can be 8 amino acid to lack
Lose (strain such as Attenuated DR13)；Furthermore in E genes, on 67-87 nucleotide positions, there are 21 nucleotide deletions
(Attenuated DR13 strains)；In ORF3 genes, start in 246 nucleotides, there are 49 missings of nucleotides etc..
There is mutation probability higher compared to DNA viroids as single-stranded RNA viroid in PEDV.Although from the nineties just
Start using vaccine immunity PED, even if but the report such as Chen in 2006 has a small amount of pig farm that bigeminy vaccine has been immunized still to send out
Raw PED epidemic situations.Until the end of the year 2010, in China's pandemic, this shows PEDV strains always constantly making a variation to PED epidemic situations,
Existing vaccine is less and less to its resistant function.According to current, the serum of variation strain and CV777 strains in vitro test
It is not high to learn reaction result.Although G2 subgroups strain in recent years still constantly spreads, after 2006, G1 subgroups
The frequency that strain is reported is far less than G2 subgroups, and this should be that vaccine based on CV777 has played very big effect.So,
Enough PEDV gene informations are obtained, the epidemic situation to prevention and control and diagnosis and treatment PED will provide crucial diagnostic base.
The content of the invention
The technical problems to be solved by the invention are to overcome Porcine epidemic diarrhea virus gene information in the prior art few, difficult
With prevention and control and the defect and deficiency of diagnosis and treatment, there is provided a kind of segmentation expands the primer sets of Porcine epidemic diarrhea virus full-length genome.This
Invention is expanded with carrying out all standing formula by designing 22 pairs of primer sets to PEDV, conveniently amplifies PEDV full-length genes, will
Huge genome is subdivided into multistage, and the nucleotides of case study particular section can be easy to change, including site mutation, nucleotides
Insertion and missing etc., more preferably to hold the change dynamic of PEDV genes, understand the change of strain, are provided accurately to solve the disease
Molecular information.
In this scenario, the technical problem that the present invention need to primarily overcome is how to design matching primer sets, can realized
On the basis of amplification PEDV full-length gene information purposes are obtained, can by the control of the primer expanding fragment length of PEDV about 1600bp with
It is interior, to adapt to current relatively ripe and cheap first generation gene sequencing technology, and the tract obtained by primer amplification
It is intersegmental to join end to end, and have enough overlaps (35~931bp).And grope the 22 pairs of primers for designing each
Optimum annealing temperature and the time, explore optimal PCR system and program.
It is an object of the invention to provide a kind of primer sets for being segmented amplification Porcine epidemic diarrhea virus full-length genome.
It is a further object of the present invention to provide the side that Porcine epidemic diarrhea virus full-length genome is expanded using above-mentioned primer sets
Method.
Above-mentioned purpose of the invention is to give realization by the following technical programs：
A kind of primer sets for being segmented amplification Porcine epidemic diarrhea virus full-length genome, the primer sets include 22 primers
Right, each primer pair upstream and downstream primer is successively as shown in SEQ NO.1~SEQ NO.44.
Nucleotide sequence according to CV777 (AF353511) in GenBank databases of the invention, using bioinformatics side
Method, designs and has screened 22 pairs of primers for expanding PEDV full-length genome nucleotide informations, with molecular biology method
The Successful amplification out 22 pairs of amplified productions, using gene sequencing technology and bioinformatics technique, obtain PEDV full-length genes
Information.And 5 ' and 3 ' ends of the PEDV genomes all employ small fragment amplification design, amplified production is set to control in 700bp
Interior, mainly having taken into account will use unconventional primer to expand 5 ' UTR front ends and 3 ' UTR end sequences, such as use
SMARTerTMRACE cDNA Amplification amplification techniques expand 5 ' UTR foremost with 3 ' the accurate nucleosides of UTR least significant ends
Acid sequence.There is the overlap homologous sequence of 39~931bp between the two neighboring amplified fragments of primer sets of present invention design, be easy to
Sequencing splicing, while the amplified fragments of all primer sets are respectively less than 1600bp, it is easy to the carrying out of PCR reactions.
A kind of to be segmented the method for expanding Porcine epidemic diarrhea virus full-length genome, methods described is：PEDV total serum IgEs are extracted,
CDNA is inverted to, being utilized respectively above-mentioned 22 primer pairs carries out pcr amplification reaction, cloning and sequencing is carried out respectively and (is produced PCR
Thing connects cloning vector, chooses positive colony, DNA sequencing identification), finally splice sequence, obtain PEDV whole genome sequences.
Wherein, the PCR primer is respectively less than 1600bp, it is easy to the carrying out of PCR reactions.
The pcr amplification reaction is carried out in a thermograde PCR instrument simultaneously, by using same PCR reaction intervals
Sequence, different primers correspondence different annealing temperature, whole genome sequence amplification that is disposable, efficiently completing PEDV.
Preferably, the thermograde of the PCR instrument is 48 DEG C~57 DEG C.
Preferably, the pcr amplification reaction program is：94 DEG C of predegeneration 1min；94 DEG C of denaturation 6s, (48 under annealing temperature
DEG C~56.1 DEG C) annealing 20s, 72 DEG C of extension 20s, circulate 36 times；72 DEG C extend 7min eventually；The annealing temperature corresponds to 22
Each self-corresponding annealing temperature of primer pair.
Preferably, the system of the pcr amplification reaction is：The μ L of sense primer (10 μM) 2.0, anti-sense primer (10 μM) 2.0 μ
L、cDNA 2.0μL、2×PrimerSTAR Max 25.0μL、ddH2O 19.0μL。
Preferably, 22 primer pairs be respectively primer pair 5', 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,
16th, 17,18,19,20,3', each primer pair upstream and downstream primer is successively as shown in SEQ NO.1~SEQ NO.44；
The annealing temperature of 22 primer pairs is respectively：
Primer pair 3', primer pair 9, primer pair 10, primer pair 11, primer pair 13, primer pair 16, the annealing temperature of primer pair 20
Degree is 56.1 DEG C；
Primer pair 1, primer pair 6, primer pair 7, primer pair 8, primer pair 14, primer pair 17, primer pair 18, primer pair 19
Annealing temperature is 55.2 DEG C；
Primer pair 5', primer pair 2, the annealing temperature of primer pair 5 are 54.1 DEG C；
The annealing temperature of primer pair 3 is 53.0 DEG C, and the annealing temperature of primer pair 4 is 50.8 DEG C, the annealing temperature of primer pair 12
It is 48.9 DEG C to spend, and the annealing temperature of primer pair 15 is 48.0 DEG C.
In addition, particularly preferably, in the above method, the specific method of the cloning and sequencing is to reclaim pcr amplification product
Purifying, the end of amplified production 3 ' adds base A, is then attached to pMD18-T carriers, converts the competent cells of Top 10, screening and
After identification recombinant bacterium, it is sequenced；
It is described splicing sequence specific method be：Sequencing result is using in the bioinformatics softwares of Lasergene 7.10
Editseq softwares are removed including the export-oriented terminal sequence including primer pair, the splicing of full length sequence and homology analysis；Together
When, with GenBank in NCBI quickly compare tools BLAST carry out sequencing result checking or compare search, complete sequence with
Existing sequence in database compares.
In addition, above-mentioned primer sets or/and amplification method are in terms of Porcine epidemic diarrhea virus gene or genome amplification
Using also in the scope of the present invention.
Meanwhile, the present invention also provides a kind of method for expanding specific region on Porcine epidemic diarrhea virus genome, described
Method is according to needing above-mentioned 22 primer pairs of any combination to carry out pcr amplification reaction.
In addition, application of the above method in Porcine epidemic diarrhea virus specific genome area research is also protected in the present invention
In the range of shield.
Preferably, the application is to detect answering in Porcine epidemic diarrhea virus mutational site, nucleotides inserted or missing
With.
The above method of the present invention is that a kind of operation is simple, quick, stable, it is full to expand Porcine epidemic diarrhea virus to sectional
The method of genome.CDNA first with PEDV as template, with 22 pairs of primers of designed, designed, by high-fidelity DNA polymerase,
Amplification obtains the full length sequence of PEDV.By 22 pairs of PCR primer connection cloning vectors of primer amplification, choose positive colony, DNA surveys
Sequence is identified, then row will be sequenced by software splicing, obtains PEDV full length nucleotide sequence informations.The main advantages of the present invention being
Can be inside a thermograde PCR instrument device, by using same PCR response procedures, different primers and different annealing temperature,
Disposably, the whole genome sequence amplification of PEDV is efficiently completed.Due to be all standing formula expand, and amplified fragments do not surpass
1600bp is crossed, so the specific region for also allowing for the research according to needed for carries out determining region amplification.
Compared with prior art, the invention has the advantages that：
(1) 22 pairs of primer pair amplifies specificity of the invention and sensitiveness are good, and PEDV can be expanded with carrying out all standing formula.
And 5 ' and 3 ' ends of primer sets all employ small fragment amplification design and (have mainly taken into account such as SMARTer to be usedTMRACE
CDNA Amplification amplification techniques expand 5 ' UTR foremost with the nucleotide sequence of 3 ' UTR least significant ends), amplified fragments
Length is controlled within 1600bp, is adapted to current relatively ripe and cheap first generation gene sequencing technology.22 pairs
Design of primers site is suitable, has between two neighboring amplified fragments and overlaps homologous sequence, is easy to sequencing to splice.
(2) primer sets of the present invention can realize perfect matching, and amplification method is by a thermograde PCR instrument
Identical PCR reaction systems and program are used simultaneously, disposably efficiently complete the whole genome sequence amplification of PEDV.
(3) because PEDV full-length genomes are larger, be subdivided into for huge genome many by primer sets of the invention and method
Section, can be easy to the nucleotides of case study particular section to change, including site mutation, nucleotides inserted and missing etc., Ke Yigeng
The change dynamic of PEDV genes is held well, the change of strain is understood, and accurate molecular information is provided to solve the disease.
Brief description of the drawings
Fig. 1 is location map of the 22 pairs of primers of present invention design in PEDV genomes.
Fig. 2 is 22 pairs of primer amplification agarose gel electrophoresis figures of present invention design.
Fig. 3 is 5 ' primer pairs sequencing fragment removal sense primer and its export-oriented terminal sequence procedure chart.
Fig. 4 is the 22 sections of splicing effect figures of removal primer information guided by reference sequences.
Specific embodiment
The present invention is made with reference to Figure of description and specific embodiment further being elaborated, the embodiment
It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Material, reagent for being used etc., are the reagent for commercially obtaining unless otherwise specified
And material.
Embodiment 1 Porcine epidemic diarrhea virus (PEDV) total length design of primers
1st, according to the nucleotide sequence of CV777 (AF353511) in GenBank databases, ground by substantial amounts of design screening
Study carefully, finally shown that 22 pairs of primers as shown in table 1, are overlapped for expanding PEDV full-length genomes, the primer sets between primer pair
Position and size as shown in table 2, the optimum annealing temperature and thermograde of primer are as shown in table 3.
The total length primer sequence table of table 1
Note：With respect to nucleotide site Reference strains SHQP/YM/2013 (GenBank indexed numbers：KJ196348).
The total length primer sequence overlap length table of table 2
Note：Primer pair 14 is 931 with the overlapping interval nucleotides quantity of primer pair 15, has mainly paid the utmost attention to primer pair
16th, primer pair 17, the combination of primer pair 18 can intactly expand the S genes of primary study sequence of interval in PEDV sequences.And
Primer pair 15 is promoted to elapse forward, so as to have more overlap with primer pair 14.
The total length primer optimum annealing temperature of table 3 and thermograde PCR instrument set
Note：Grads PCR is 8*12 hole counts, and thermograde is set to (48 DEG C~57 DEG C).
Embodiment 2 Porcine epidemic diarrhea virus (PEDV) whole genome amplification method
1st, the acquisition of PEDV prevalences strain
The pig intestinal tissue sample of clinically suspected infection PEDV is gathered, simultaneously several soya beans of clip are big for Cord blood
Tissue block is ground.Quartz sand is mixed into grinding, the PBS of about 5mL or so precoolings is added.Homogenate is collected into 10mL Ep pipes,
Put freeze thawing 3 times in -80 DEG C of ultra low temperature freezers.Sample is again through 10,000 × g, 4 DEG C of centrifugation 10min.Supernatant is taken, through 0.22 μm of filter membrane
Filtering.If resulting grinding filtrate is divided into main to freeze in -80 DEG C of ultra low temperature freezers, treat that nucleic acid extraction is used.
2nd, the extraction of tissue sample total serum IgE
With total serum IgE in the ultrapure RNA extracts kits extraction lapping liquids of Magen.Take the μ L of filtrate 250 and add 750 μ L's
In Trizol, the PBS of 250 μ L being taken in sterile centrifugation tube, adding 750 μ L TRIzol, acutely vibration, 5 points are acted at room temperature
Clock.Again plus 200 μ L chloroforms, acutely vibration, places 5 minutes at room temperature.4 DEG C of 12,000r/min are centrifuged 15 minutes.By on 500 μ L
Layer water is mutually moved on in another new sterilizing microcentrifugal tube, adds 500 μ L isopropanols, soft to mix, and -20 DEG C are placed 15 minutes.
12,000r/min centrifugations 15 minutes, incline supernatant.75% ethanol of 1,000 μ L ice precoolings is added, slow overturning shakes up.4℃
12,000r/min is centrifuged 15 minutes.Gently supernatant discarded, places 3-5min to air-dry precipitation.Add the RNase Free of 20 μ L
Distilled water dissolves RNA, is saved backup in -20 DEG C.
3rd, the synthesis of the virus chains of cDNA first
The improved high-efficiency Reverse Transcriptase kit ReverTra of (Shanghai) bio tech ltd is spun with reference to Japan
The specification of Ace (TRT-101) is carried out.Taking the RNA of extraction carries out the synthesis of the chains of cDNA first, is wrapped in the reverse transcription system of 20 μ L
Include following each component (such as table 4)：
The reverse transcription system of table 4
Above component is fully mixed, is placed in 1h is incubated in 42 DEG C of water-baths immediately after, resulting cDNA is placed in -20
DEG C preserve.
4th, PCR amplification method
With 22 pairs of total length primers, expanded (such as table 5) using following PCR reaction systems and response procedures：
Table 5PCR reaction systems
PCR amplification programs：
5th, the recovery of amplified production and purifying
All amplified productions are entered into row agarose gel electrophoresis, purpose fragment, reference are cut under the of short duration irradiation of uviol lamp
The quick QIAquick Gel Extraction Kits of Magen companies DNA carry out recovery purifying, and specific method is as follows：
(1) genes of interest DNA fragmentation is separated through 0.8~1% agarose gel electrophoresis；
(2) after electrophoresis terminates, after Ago-Gel is dyeed through EB, with clean, sharp operation under long-wave ultra violet lamp
Blade cuts the agar gel block containing genes of interest fragment, is put into a 1.5 or 2mL centrifuge tube for cleaning；
(3) by 1:3 ratio (gel quality:Sol solutionses volume (mg：ML sol solutionses (Extraction) is added
Buffer)；
(4) incubate 10-15min in 56 DEG C of waters bath with thermostatic control or metal Lip river, until gel piece melt, in incubation period every
2~3min runs to mixing once up and down；
(5) mixed liquor is transferred in recovery column, 6,000r/min, 1min is centrifuged, discard liquid in pipe；
(6) 500 μ L sol solutionses are added in recovery column, 12000r/inin, 1min is centrifuged, discard liquid in pipe；
(7) to 750 μ L cleaning solutions (Wash Buffer) are added in recovery column, 2~5min, 12,000r/ are stored at room temperature
Min, is centrifuged 1min, discards liquid in pipe；
(8) again in 12,000r/min, 2min is centrifuged, then recovery column is transferred in aseptic 1.5mL centrifuge tubes, beat
Uncap son, be stored at room temperature 2~5min, treat its natural air drying；
(9) to Jia 30 in centrifugal column~50 μ L eluents, ddH2O or TE solution, 5min is incubated in 37 DEG C；
(10) 12,000r/min, 3min is centrifuged, the liquid in centrifuge tube can be again added to centrifugal column if needed
It is interior, elute again, the liquid that bottom of the tube is centrifuged is the genes of interest fragment of recovery, can be directly used for coupled reaction, or in -20
DEG C save backup.
6th, the end of amplified production 3 ' adds base A
Because high-fidelity enzymatic amplification PCR fragment out is flat end, and general T aq enzymes have in the end of PCR primer 3 '
Plus a characteristic of base A, it is possible thereby to be connected with the pMD18-T carriers of cohesive end, such that it is able to greatly improve fragment with
The efficiency of carrier connection.Specific reaction system such as table 6：
The end of 6 amplified production of table 3 ' adds base A reaction systems
Response procedures are 72 DEG C of 30min.
7th, PCR fragment and carrier are connected
The PCR primer that 3 ' ends add base A is carried out into concentration mensuration, and with 1:3/1:5 molar ratio is carried with pMD18-T
Body is attached (linked system such as table 7).
The linked system of table 7
Above-mentioned system is placed in connection instrument after fully mixing, and 16 DEG C of connections are overnight.Connection product is put into 65 DEG C by next day
Water-bath water-bath 5min is inactivated to ligase.Then product is subsequently connected or is placed in -20 DEG C of preservations.
8th, connection product transformed competence colibacillus cell
(1) preparation of competent cell
The preparation of the competent cells of Top 10 is with reference to J. Pehanorm Brookers (1998)《Molecular Cloning:A Laboratory guide》Method
Carry out.The single bacterium colonies of picking Top 10 are inoculated in 4mL LB nutrient solutions, 37 DEG C, 200~220r/min shaken cultivations 12 hours.Take
15 μ L bacterium solutions are added in 1.5mL LB fluid nutrient mediums, 200r/min shaken cultivations 3 hours, and now bacterium is exponential phase.
Ice bath 10 minutes, 4 DEG C of 4,000r/min are centrifuged 10 minutes, abandon supernatant, collects thalline.Add the 0.1M CaCl of 500 μ L precoolings2
Resuspended thalline.4 DEG C, supernatant, collects thalline, with the 0.1M CaCl of 500 μ L precoolings are abandoned in 4,000r/min centrifugations 10 minutes2It is resuspended
Thalline.Put 30 minutes on ice, 4 DEG C, 4100r/min is centrifuged 10 minutes, abandons supernatant, collects thalline, with 100 μ L 0.1M CaCl2
Resuspended thalline, ice bath is directly converted, or adds final concentration of 10% glycerine, -80 DEG C of preservations.
(2) connection product transformed competence colibacillus cell
The μ L of connection product 10 that will be obtained are added in the competent cells of 100 μ L Top 10, while set up being not added with any plasmid
Competent cell to do negative control and pET-32a plasmids be positive control.Gently mix, ice bath 30min, 42 DEG C of thermal shocks 90 seconds
Ice bath 10min at once, is subsequently adding in 750 μ L LB fluid nutrient mediums, 37 DEG C afterwards, 200r/min shaking cultures 1h.Shaken cultivation
4,000r/min centrifugations 5min, abandons supernatant afterwards, and precipitation is resuspended in 150 μ L LB nutrient solutions, and piping and druming is mixed.Above-mentioned bacterium solution is taken to apply
It is distributed on the LB agar plates containing ampicillin, after bacterium solution is fully absorbed, is inverted into 37 DEG C of incubator 12~18h of culture,
Observation result.
9th, the screening and identification of recombinant bacterium
The doubtful positive single bacterium colony that picking size is homogeneous, form is mellow and full from the LB agar plates that conversion is completed, by nothing
Each bacterium colony to be checked is chosen the sterile centrifugation tube of the 1.5mL to the LB fluid nutrient mediums containing 30 μ L ampicillins for bacterium operation
In, pipettor piping and druming is uniform into bacterial suspension, while doing template with this suspension carries out bacterium colony PCR identifications (PCR system such as table
8), primer pair sequence is as described in Table 1.Each section of gene magnification of full-length genome is expanded with corresponding primer respectively.
Table 8PCR systems
PCR amplification programs：
Amplified production carries out 1~2% agarose gel electrophoresis, and selection can expand the corresponding of strong and special purpose band
Bacterium is shaken in bacterium solution to test tube ampicillin LB liquid mediums, and 300 μ L bacterium solutions of aseptic absorption are served in 1.5mL Ep pipes
Hai Yingjun Bioisystech Co., Ltd is sequenced.
10th, genome sequencing result treatment and analysis
After sequencing result is returned, gone using the Editseq softwares in the bioinformatics softwares of Lasergene 7.10
Except including the export-oriented terminal sequence (such as shown in Fig. 3) including primer pair, the splicing (as shown in Figure 4) of full length sequence and homology point
Analysis.Meanwhile, quickly comparing tools BLAST with GenBank in NCBI carries out the checking of sequencing result or compares to search for.
The primer sets and method application case of the above-mentioned segmentation of embodiment 3 amplification Porcine epidemic diarrhea virus full genome
The popular PEDV street strains of the new hair for obtaining are successfully separated based on the present inventor research department, in order to more preferably conveniently
New strain is studied, therefore, inventor have developed said gene amplification method of the present invention.And Successful utilization primer of the present invention
Group and method have obtained the full length sequence information of the PEDV street strains sequence.
By PEDV street strains full length sequence by US National Biotechnology Information center (National Center
For Biotechnology Information, NCBI) DNA databases, with the analysis tool program of similarity system design
(Basic Local Alignment Search Tool, BLAST), carries out similitude sequence and compares with public database.
Through the Liang Zhu street strains that Primary Study, the present inventor research department find：CH/GDZH01/1311 strains and CH/
The nucleotide similarity of GDZH02/1401 strains is 99.7%.And they are isolated respectively at nineteen seventies
The nucleotide similarity of CV777 (AF3535111) strain is only respectively 96.8% and 96.6%.
In addition, after CH/GDZH02/1401 strains are passed on into 65 generations, as CH/GDZH02/1401-P65 strains, its phase
Amino acid mutation at up to 14 in gene order is there occurs for parent's strain CH/GDZH02/1401.Moreover, especially CH/
There is the missing of continuous 2 nucleotides in S gene cytoplasmic regions in GDZH02/1401-P65 strains, cause shifting to an earlier date for S protein translation
Terminate.There are some researches show the such this phenomenon of coronavirus can cause S gene overexpressions.If this result points out the passage
The responsible stimulation body of virus produces the spike protein S overexpression of major antigen, it would be beneficial in vaccine development.
Furthermore, CH/GDZH02/1401-P65 strains occur in that the missing of continuous 6 nucleotides, this position in ORF3 genes
The missing put is the new phenomenon that the strain on current ncbi database is not all disclosed.And the nucleotide deletion in this site is still
Not having correlation test proves its function, so this site is the discovery that a new discovery while also will be the present inventor research department
One of key object of follow-up study.
Finally, the present inventor is thin by isolated Liang Zhu PEDV street strains of this research department and wherein one plant street strain
The Genomic sequence information of born of the same parents' pass on strain is submitted to NCBI, and wherein CH/GDZH01/1311 strains reception number is KR153325, CH/
The reception number of GDZH02/1401 strains is KR153326, and the reception number for passing on strain CH/GDZH02/1401-P65 strains is
KX016034。
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>A kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full-length genome
<130> 2016
<160> 44
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> 5'F
<400> 1
acttaaagag attttctatc tacgg 25
<210> 2
<211> 19
<212> DNA
<213> 5'R
<400> 2
actggcacga tgttaccac 19
<210> 3
<211> 20
<212> DNA
<213> 1F
<400> 3
agccgtctca tactattctg 20
<210> 4
<211> 20
<212> DNA
<213> 1R
<400> 4
gaacataaga gccaaccttg 20
<210> 5
<211> 19
<212> DNA
<213> 2F
<400> 5
tgccactctt agtatcgtt 19
<210> 6
<211> 23
<212> DNA
<213> 2R
<400> 6
tcatagatgt agtacttagg cac 23
<210> 7
<211> 22
<212> DNA
<213> 3F
<400> 7
gagactatta tggctgtgct ta 22
<210> 8
<211> 18
<212> DNA
<213> 3R
<400> 8
tggcgatcac caacacat 18
<210> 9
<211> 19
<212> DNA
<213> 4F
<400> 9
attcaggtgt tgctcttac 19
<210> 10
<211> 18
<212> DNA
<213> 4R
<400> 10
accataccag tgtcttga 18
<210> 11
<211> 26
<212> DNA
<213> 5F
<400> 11
acaacattcc tagataatgg taacgg 26
<210> 12
<211> 20
<212> DNA
<213> 5R
<400> 12
tcatcaaaac ccaatgtgct 20
<210> 13
<211> 21
<212> DNA
<213> 6F
<400> 13
atagttttgg caaagacctg t 21
<210> 14
<211> 20
<212> DNA
<213> 6R
<400> 14
ggctgaaaag gcataaacca 20
<210> 15
<211> 25
<212> DNA
<213> 7F
<400> 15
gtttcttatt tacaaagttt aagcg 25
<210> 16
<211> 20
<212> DNA
<213> 7R
<400> 16
aaactcatct gtcaacgagg 20
<210> 17
<211> 21
<212> DNA
<213> 8F
<400> 17
gcttagttct tctaggattg c 21
<210> 18
<211> 20
<212> DNA
<213> 8R
<400> 18
ctattaactg ctcgtgcctc 20
<210> 19
<211> 18
<212> DNA
<213> 9F
<400> 19
ttgaccgtga ggcttcta 18
<210> 20
<211> 18
<212> DNA
<213> 9R
<400> 20
cgaccatcct tccaagtg 18
<210> 21
<211> 24
<212> DNA
<213> 10F
<400> 21
aaaagatgta ccaagtctgc gatg 24
<210> 22
<211> 22
<212> DNA
<213> 10R
<400> 22
tagtaccaat aacaaccgaa gc 22
<210> 23
<211> 20
<212> DNA
<213> 11F
<400> 23
gtgtttcgct cttgtcaacc 20
<210> 24
<211> 19
<212> DNA
<213> 11R
<400> 24
cgctgcaaac agacgcaat 19
<210> 25
<211> 21
<212> DNA
<213> 12F
<400> 25
ttaatcgcat tgctacatcc g 21
<210> 26
<211> 20
<212> DNA
<213> 12R
<400> 26
ccacaaccct cattagcctg 20
<210> 27
<211> 20
<212> DNA
<213> 13F
<400> 27
tataatgtgc gataggtccc 20
<210> 28
<211> 20
<212> DNA
<213> 13R
<400> 28
tgccacagat tataggtgtc 20
<210> 29
<211> 21
<212> DNA
<213> 14F
<400> 29
cttagggcta gtaactgcat t 21
<210> 30
<211> 22
<212> DNA
<213> 14R
<400> 30
tataatcagc atcgctaacg ta 22
<210> 31
<211> 20
<212> DNA
<213> 15F
<400> 31
aaaatgctgt gcttatgtca 20
<210> 32
<211> 18
<212> DNA
<213> 15R
<400> 32
tgtggtaggc taagtgtt 18
<210> 33
<211> 25
<212> DNA
<213> 16F
<400> 33
cattagtgat gttgtgttag gcttg 25
<210> 34
<211> 22
<212> DNA
<213> 16R
<400> 34
gaaggtttct agaagagata gg 22
<210> 35
<211> 22
<212> DNA
<213> 17F
<400> 35
cgactaattt tgttgatgca ct 22
<210> 36
<211> 22
<212> DNA
<213> 17R
<400> 36
tgcagcagta aaacctccta gc 22
<210> 37
<211> 20
<212> DNA
<213> 18F
<400> 37
tcacatgtat agtgcgtctc 20
<210> 38
<211> 23
<212> DNA
<213> 18R
<400> 38
gctgccaaca taatataatt gcg 23
<210> 39
<211> 20
<212> DNA
<213> 19F
<400> 39
atctacttct ttgcactgtt 20
<210> 40
<211> 20
<212> DNA
<213> 19R
<400> 40
atttgctggt ccttatttcc 20
<210> 41
<211> 21
<212> DNA
<213> 20F
<400> 41
attcagctgt gagtaatccg a 21
<210> 42
<211> 19
<212> DNA
<213> 20R
<400> 42
accgttgtgt gcaagacca 19
<210> 43
<211> 28
<212> DNA
<213> 3'F
<400> 43
gggtgcgcca tctgatgtga cccatgcc 28
<210> 44
<211> 20
<212> DNA
<213> 3'R
<400> 44
gtgtatccat atcaacaccg 20
Claims (10)
1. it is a kind of to be segmented the primer sets for expanding Porcine epidemic diarrhea virus full-length genome, it is characterised in that the primer sets include
22 primer pairs, each primer pair upstream and downstream primer is successively as shown in SEQ IDNO.1~SEQ IDNO.44.
2. a kind of method for being segmented amplification Porcine epidemic diarrhea virus genome, it is characterised in that methods described is：Extract to be measured
PEDV total serum IgEs, are inverted to cDNA, and being utilized respectively 22 primer pairs described in claim 1 carries out pcr amplification reaction, carries out respectively
Cloning and sequencing, finally splices sequence, obtains PEDV whole genome sequences.
3. method according to claim 2, it is characterised in that 22 primer pairs be respectively primer pair 5', 1,2,3,4,5,
6th, 7,8,9,10,11,12,13,14,15,16,17,18,19,20,3', each primer pair upstream and downstream primer is successively such as SEQ
Shown in IDNO.1~SEQ IDNO.44；
The annealing temperature of 22 primer pairs is respectively：
Primer pair 3', primer pair 9, primer pair 10, primer pair 11, primer pair 13, primer pair 16, the annealing temperature of primer pair 20 are equal
It is 56.1 DEG C；
Primer pair 1, primer pair 6, primer pair 7, primer pair 8, primer pair 14, primer pair 17, primer pair 18, the annealing of primer pair 19
Temperature is 55.2 DEG C；
Primer pair 5', primer pair 2, the annealing temperature of primer pair 5 are 54.1 DEG C；
The annealing temperature of primer pair 3 is 53.0 DEG C, and the annealing temperature of primer pair 4 is 50.8 DEG C, and the annealing temperature of primer pair 12 is
48.9 DEG C, the annealing temperature of primer pair 15 is 48.0 DEG C.
4. method according to claim 2, it is characterised in that the program of the pcr amplification reaction is：94 DEG C of predegenerations
1min；94 DEG C of denaturation 6s, anneal 20s under annealing temperature, 72 DEG C of extension 20s, circulates 36 times；72 DEG C extend 7min eventually；It is described to move back
Fiery temperature corresponds to 22 each self-corresponding annealing temperatures of primer pair.
5. method according to claim 2, it is characterised in that the system of the pcr amplification reaction is：10 μM of sense primers
2.0 μ L, 10 μM of μ L of anti-sense primer 2.0, cDNA 2.0 μ L, 2 × PrimerSTAR Max 25.0 μ L, ddH2O 19.0µL。
6. method according to claim 2, it is characterised in that the pcr amplification reaction is simultaneously in a thermograde PCR
Carried out in instrument, the thermograde of the PCR instrument is 48 DEG C~57 DEG C.
7. application of the primer sets described in claim 1 in terms of Porcine epidemic diarrhea virus gene or genome amplification.
8. application of any methods described of claim 2~6 in terms of Porcine epidemic diarrhea virus gene or genome amplification.
9. it is a kind of expand Porcine epidemic diarrhea virus genome on specific region method, it is characterised in that it is any as needed
Primer in primer sets described in combination claim 1 carries out pcr amplification reaction.
10. application of claim 9 methods described in Porcine epidemic diarrhea virus specific genome area research.
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