CN102108359A - Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof - Google Patents
Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof Download PDFInfo
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- CN102108359A CN102108359A CN200910200481XA CN200910200481A CN102108359A CN 102108359 A CN102108359 A CN 102108359A CN 200910200481X A CN200910200481X A CN 200910200481XA CN 200910200481 A CN200910200481 A CN 200910200481A CN 102108359 A CN102108359 A CN 102108359A
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Abstract
The invention provides a modified coded nucleic acid of peste des petits ruminants virus N protein which has a sequence of SEQ ID NO:41, and also provides its PCR amplification method and a kit used for the method.
Description
Technical field
The present invention relates to biology field, specifically, relate to PPR virus (Peste des petitsruminants virus, the transformation of N gene PPRV) and synthetic.
Background technology
PPR virus (Peste des petits ruminants virus, PPRV) (Peste des petits ruminants PPR) is a kind of goat, sheep and some wild strong contact category-A transmissible diseases of ruminating beastly similar rinderpest symptom for a short time of mainly betiding to the PPR that causes.This disease from 1940 since the Ivory Coast is recorded and narrated first, successively in Africa, some countries of the Middle East take place.
PPRV belongs to Paramyxoviridae, Morbillivirus.Other members of generic also have rinderpest virus (RPV), canine distemper virus (CDV), sea dog pestivirus (PDV), dolphin pestivirus (DDV), ox Measles virus (MV2K1) and people Measles virus (MV).PPRV at present divides 4 groups, and wherein 3 groups come from Africa, and a group comes from the Asia, at present, only finds that this virus has only 1 serotype.
The PPRV virus particle is polymorphism, mostly is circular or oval, outward by thick cyst membrane, fine dashing forward is arranged on the cyst membrane, and fine dashing forward only contained hemagglutinin and impassivity propylhomoserin enzyme; Its genome does not have sections RNA for the sub-thread minus strand, holds to 5c end N-P-M-F-HN-L6 the gene that distributing successively, the corresponding 6 kinds of structural protein of encoding respectively from the 3c of RNA chain; Between each gene by certain intergenic sequence separately.
Wherein, N albumen is the abundantest and the strongest viral protein of immunogenicity of content among the PPRV, and its molecular weight is approximately 58ku.Only comprise 1 open reading frame in the N gene, 525 amino acid of encoding altogether.The proteic main effect of N is the degraded that protection virus is avoided RNA rnase I (RNaseI), and combines transcribing and translation process of participation virus with RNA.The proteic antigenicity of N is stable, occupy an leading position at the proteic antibody of N in the animal serum of virus infection, but this antibody-like does not have the effect of neutralization virus.Be broadly divided into 4 zones in the N protein structure, i.e. I district (N-terminal district), II district (123-144AA), III district (proteic central region), IV district (the C-terminal district of 421-525).Find that when calculating comparison I district and III district conservative property are higher, 75% ~ 90% homology is arranged approximately with other Morbillivirus member's sequence.And II district and IV district morph easily, and homology has only 17% ~ 40%.In addition, 65.8% homology is arranged, can distinguish RPV and PPRV strain with the proteic monoclonal antibody of anti-N, thereby carry out differential diagnosis for the N gene nucleotide of PPRV and RPV.Research N such as Buckload find during proteic epitope collection of illustrative plates, all at the corresponding epi-position of the proteic monoclonal antibody of Morbillivirus member's N (McAb) mostly in the IV district.Result of study shows that also the proteic C end of N is exposed to the outside more, and may be connected with envelope protein.
The chemosynthesis of traditional DNA generally is earlier synthetic shorter double-stranded DNA (about 100bp), adjacent two segment DNAs connect by the method for overlapping PCR again, or adopt enzyme mode even to connect, for the sufficient supplies of template, each small segment may all will be done one time molecular cloning.And then further carry out than the connection of long segment wasting time and energy with synthetic, and have complicated secondary structure and high G the fragment of C content just be not easy to synthesize.
Therefore, because the importance of N albumen in virus immunity research presses for a kind of Method and kit for that can obtain the proteic coding nucleic acid of total length N rapidly.
Summary of the invention
The purpose of this invention is to provide a kind of N encoding histone nucleic acid of improvement, it helps fast and simple PCR building-up reactions.
Therefore, aspect first, it is characterized in that a kind of coding PPR virus N is provided proteic isolating nucleic acid, and it is the sequence of SEQ ID NO:41 of the present invention.
In another aspect of the present invention, a kind of method of the above-mentioned nucleic acid that increases is provided, this method comprises the following steps:
A) be template with the proteic isolating nucleic acid of above-mentioned coding PPR virus N, organize primer: SEQID NO:1-10, SEQ ID NO:11-20, SEQ ID NO:21-30, SEQ ID NO:31-40 carry out the PCR reaction respectively with each.Obtain 4 nucleic acid fragments;
B) with the nucleic acid fragment that obtains in the step a) as template, increase the amplification proteic isolating nucleic acid of PPR virus N that obtains encoding with SEQ ID NO:1,10,11,20,21,30,31,40 primer.
In an embodiment aspect this, the reaction conditions in the step a) is: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 50s, 25 circulations, extend 72 ℃ of 5min.
In another embodiment aspect this, the reaction system in the step a) is: 5 * damping fluid, 10 μ l, dNTP4 μ l, inboard primer 12 μ l, outside primer 3 μ l, taq 1 μ l, ddH2O 17 μ l.
Also having among the embodiment aspect this, the reaction conditions in the step b) is: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 2min, 25 circulations, 72 ℃ of 5min of extension.
In another embodiment aspect this, the reaction system in the step b) is: 5 * damping fluid, 10 μ l, dNTP4 μ l, each 100ng of part fragment, the outside 2 μ l, taq 1 μ l, ddH2O 17 μ l.
Aspect the 3rd of the present invention, a kind of test kit of the above-mentioned nucleic acid that is used to increase is provided, it contains the primer of SEQID NO:1-40, and SEQ ID NO:41's is nucleic acid-templated, dNTP, PCR damping fluid, carrier and label.
Aspect the 4th of the present invention, a kind of expression vector is provided, it contains the described nucleotide sequence of SEQ ID NO:41 and is used to express this expression of nucleic acids box.
Aspect the 5th of the present invention, a kind of host cell is provided, this host cell contains above-mentioned expression vector.Among the embodiment of this aspect, host cell is a prokaryotic cell prokaryocyte.
Description of drawings
Fig. 1 has shown pcr amplification product electrophoretogram (1% agarose).
Fig. 2 has shown the result of western blotting, has wherein shown N albumen and the proteic monoclonal antibody specific hybrid of anti-N of 75kd.
Embodiment
The contriver utilizes the preference form of gene codon, has transformed the gene order of PPR virus tibet strain, makes it be more suitable for expressing in prokaryotic cell prokaryocyte, thereby has avoided dangerous virus culture.Simultaneously, the contriver has also invented a kind of more effective gene amplification method, can simplify repeated cloning fragment and step of connecting.
In the present invention, term " PPR virus N albumen " is preferably selected from the albumen of tibet strain, its gene order such as Genbank accession number EU360596.Preferred this N albumen is full-length proteins, but also comprises its functional protein fragments, and has the functional protein variant that homology is 50-99% with it.It will be appreciated by those skilled in the art that the transformation that these protein variant also can be by its coding nucleic acid, and sequence alignment,, utilize PCR method of the present invention its nucleotide sequence that increases with reference to the position of the primer that is provided among the present invention on sequence.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and albumen, but same polynucleotide or albumen as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating nucleic acid " is meant that this nucleic acid is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use this nucleic acid of nucleic acid purification technology purifying of standard.Basically pure nucleic acid can produce single master tape on sepharose.
Nucleic acid of the present invention is polynucleotide, can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
According to the present invention, it will be understood by those skilled in the art that fragment, derivative and the analogue that also can synthesize the proteic coding nucleic acid of N with the primer of SEQ ID NOs:1-40.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of N albumen of the present invention or active albumen basically.Fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted albumen of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has the albumen of substituted radical, or (iii) maturation protein and another compound (such as the compound that prolongs the albumen transformation period, polyoxyethylene glycol for example) merges formed albumen, or (iv) additional aminoacid sequence is fused to this protein sequence and the albumen that forms (as leader sequence or secretion sequence or be used for this proteic sequence of purifying or proteinogen sequence, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
As mentioned above, can express the proteic derivative of these N, analogue and fragment with its coding nucleic acid.According to the sequence of SEQ ID NO:41, also can replace its encoding sequence with the procaryotic codon of preference, make it be more suitable in prokaryotic organism, expressing.
The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded albumen or proteic fragment, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded protein in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) is than hybridization under low ionic strength and the comparatively high temps and wash-out; Or (2) hybridization the time is added with denaturing agent etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the albumen of interfertile polynucleotide encoding has identical biological function and activity with PPR virus N albumen.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PGR) of nucleic acid to determine and/or to separate the polynucleotide of coding lipase.
Albumen among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
Coding nucleic acid of the present invention (SEQ ID NO:41) is fit to be cloned into prokaryotic vector expresses in prokaryotic host cell.Preferred prokaryotic host cell is intestinal bacteria.Should understand and also can use other prokaryotic organism host well known in the art to express N albumen.Can select for use promotor commonly used, enhanser or mark to come the construction expression box.
The method of round pcr DNA amplification/RNA of the present invention (Saiki, et al.Science1985; 230:1350-1354), employed reaction conditions is conventional reaction conditions in its each step.According to the requirement of different stringent conditions, also can regulate according to technology well known in the art.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The transformation of embodiment 1 PPRVN protein gene:
Gene order (Genbank accession number EU360596) according to NCBI PPR virus tibet strain is carried out the transformation of amino acid synonym.The principle of transforming is to make it be suitable for expressing in prokaryotic system, uses the ACC of high frequency of utilization, CTG, and CGT, ATC replaces ACA, CTA, AGA, ATA.Arg codon AGA, AGG, CGG, CGA, Ile codon AUA, Leu codon CUA, Gly codon GGA and Pro codon CCC then carry out synonym as far as possible and replace.Consider simultaneously gene inside can not introduce with the expression vector multiple clone site on restriction enzyme the restriction enzyme site of conflict, improved N protein nucleic acid sequence such as SEQ ID NO:41 are arranged:
ATGGCTACTTTATTAAAATCTTTAGCTTTATTTAAACGTAATAAAGATAAAGCTCCTACTGCTTCTGGTTCTGGTGGTGCTATTCGTGGTATTAAAAATGTTATTATTGTTCCTATTCCTGGTGATTCTTCTATTACTACTCGTTCTCGTTTATTAGATCGTTTAGTTCGTTTAGCTGGTGATCCTGATATTAATGGTTCTAAATTAACTGGTGTTATGATTTCTATGTTATCTTTATTTGTTGAATCTCCTGGTCAATTAATTCAACGTATTACTGATGATCCTGATGTTTCTATTCGTTTAGTTGAAGTTGTTCAATCTACTCGTTCTCAATCTGGTTTAACTTTTGCTTCTCGTGGTGCTGATTTAGATAATGAAGCTGATATGTATTTTTCTACTGAAGGTCCTTCTTCTGGTGGTA AAAAACGTATTAATTGGTTTGAAAATCGTGAAATTATTGATATTGAAGTTCAAGATCCTGAAGAATTTAATATGTTATTAGCTTCTATTTTAGCTCAAGTTTGGATTTTATTAGCTAAAGCTGTTACTGCTCCTGATACTGCTGCTGATTCTGAATTACGTCGTTGGGTTAAATATACTCAACAACGTCGTGTTATTGGTGAATTTCGTTTAGATAAAGGTTGGTTAGATGCTGTTCGTAATCGTATTGCTGAAGATTTATCTTTACGTCGTTTTATGGTTTCTTTAATTTTAGATATTAAACGTACTCCTGGTAATAAACCTCGTATTGCTGAAATGATTTGTGATATTGATAATTATATTGTTGAAGCTGGTTTAGCTTCTTTTATTTTAACTATTAAATTTGGTATTGAAACTATGTATCCTGCTTTAGGTTTACATGAATTTGCTGGTGAATTATCTACTATTGAATCTTTAATGAATTTATATCAACAATTAGGTGAAGTTGCTCCTTATATGGTTATTTTAGAAAATTCTATTCAAAATAAATTTTCTGCTGGTGCTTATCCTTTATTATGGTCTTATGCTATGGGTGTTGGTGTTGAATTAGAAAATTCTATGGGTGGTTTAAATTTTGGTCGTTCTTATTTTGATCCTGCTTATTTTCGTTTAGGTCAAGAAATGGTTCGTCGTTCTGCTGGTAAAGTTTCTTCTGTTATTGCTGCTGAATTAGGTATTACTGCTGAAGAAGCTAAATTAGTTTCTGAAATTGCTTCTCAAGCTGGTGATGAACGTACTGCTCGTGGTACTGGTCCTCGTCAAGCTCAAGTTTCTTTTTTACAACATCAAACTGGTGGTGGTGAATCTTCTGCTCCTGCTACTCGTGAAGGTGTTAAAGCTGCTATTCCTAATGGTTCTGAAGAACGTGATCGTAAACAAACTCGTCCTGGTCGTCCTCGTGGTGAAACTCCTGGTCAATTATTATTAGAAATTATGCCTGAAGATGAAGTTTCTCGTGAATCTGGTCAAAATCCTCGTGAAGCTCAACGTTCTGCTGAAGCTTTATTTCGTTTACAAGCTATGGCTAAAATTTTAGAAGATCAAGAAGAAGGTGAAGATAATAATCAAGTTTATAATGATAAAGATTTATTAGGTTAA ( N71.9％,100％ ) 。
Embodiment 2 PCR synthesize the N gene
Material: N protein nucleic acid sequence is from improved gene order.
Primer sequence SEQ ID NO:1-40 is synthetic by the biological company limited of Shanghai JaRa.
Test kit: PrimeSTAR HS archaeal dna polymerase test kit (Takara, catalog number (Cat.No.) DR010A)
The simple carrier of pM D19-T (Takara, catalog number (Cat.No.) D104A)
PCR instrument (S1000 thermal cycler) is available from Bio Rad company
Agarose is available from GENETECH (SHANGHAI) company limited (batch 111760)
PET-32a is preserved by this laboratory
Bl21 (DE3) Plyss (preserve in this laboratory)
The N protein monoclonal antibody is preserved by this laboratory
Two anti-for HRP goat anti-mouse IgG antibody (LP1002a) available from Abgent PrimaryAntibody company
Pre-dsred protein Marker (SM0671) is available from Fermentas company
1. design of primers
Designed primer with improved gene order for design template.Designed 7 groups of primers altogether, every group of primer is the 38-44 bar, and the length of primer is about 60bp, and total number is an even number.Reclose sequence between every adjacent two primers is 21-23bp.Adjacent two primer directions are opposite arbitrarily.Avoid the inner too space structures such as hair fastener of complexity that form of full gene.In the test, the 3rd group of primer can guarantee enough output and accuracy.The 3rd group of primer sequence such as following table:
| Numbering | The base number | Primer sequence |
| 1 | 54 | ATGGCTACTTTATTAAAATCTTTAGCTTTATTTAAACGTAATAAAGATAAAGCT |
| 2 | 60 | AATACCACGAATAGCACCACCAGAACCAGAAGCAGTAGGAGCTTTATCTTTATTACGTTT |
| 3 | 60 | GGTGGTGCTATTCGTGGTATTAAAAATGTTATTATTGTTCCTATTCCTGGTGATTCTTCT |
| 4 | 60 | ACGAACTAAACGATCTAATAAACGAGAACGAGTAGTAATAGAAGAATCACCAGGAATAGG |
| 5 | 60 | TTATTAGATCGTTTAGTTCGTTTAGCTGGTGATCCTGATATTAATGGTTCTAAATTAACT |
| 6 | 60 | AGATTCAACAAATAAAGATAACATAGAAATCATAACACCAGTTAATTTAGAACCATTAAT |
| 7 | 60 | TTATCTTTATTTGTTGAATCTCCTGGTCAATTAATTCAACGTATTACTGATGATCCTGAT |
| 8 | 60 | ACGAGTAGATTGAACAACTTCAACTAAACGAATAGAAACATCAGGATCATCAGTAATACG |
| 9 | 60 | GAAGTTGTTCAATCTACTCGTTCTCAATCTGGTTTAACTTTTGCTTCTCGTGGTGCTGAT |
| 10 | 60 | ACCTTCAGTAGAAAAATACATATCAGCTTCATTATCTAAATCAGCACCACGAGAAGCAAA |
| 11 | 60 | ATGTATTTTTCTACTGAAGGTCCTTCTTCTGGTGGTAAAAAACGTATTAATTGGTTTGAA |
| 12 | 60 | TTCAGGATCTTGAACTTCAATATCAATAATTTCACGATTTTCAAACCAATTAATACGTTT |
| 13 | 60 | ATTGAAGTTCAAGATCCTGAAGAATTTAATATGTTATTAGCTTCTATTTTAGCTCAAGTT |
| 14 | 60 | AGTATCAGGAGCAGTAACAGCTTTAGCTAATAAAATCCAAACTTGAGCTAAAATAGAAGC |
| 15 | 60 | GCTGTTACTGCTCCTGATACTGCTGCTGATTCTGAATTACGTCGTTGGGTTAAATATACT |
| 16 | 60 | TTTATCTAAACGAAATTCACCAATAACACGACGTTGTTGAGTATATTTAACCCAACGACG |
| 17 | 60 | GGTGAATTTCGTTTAGATAAAGGTTGGTTAGATGCTGTTCGTAATCGTATTGCTGAAGAT |
| 18 | 60 | ATCTAAAATTAAAGAAACCATAAAACGACGTAAAGATAAATCTTCAGCAATACGATTACG |
| 19 | 60 | ATGGTTTCTTTAATTTTAGATATTAAACGTACTCCTGGTAATAAACCTCGTATTGCTGAA |
| 20 | 60 | ACCAGCTTCAACAATATAATTATCAATATCACAAATCATTTCAGCAATACGAGGTTTATT |
| 21 | 60 | AATTATATTGTTGAAGCTGGTTTAGCTTCTTTTATTTTAACTATTAAATTTGGTATTGAA |
| 22 | 60 | ACCAGCAAATTCATGTAAACCTAAAGCAGGATACATAGTTTCAATACCAAATTTAATAGT |
| 23 | 60 | GGTTTACATGAATTTGCTGGTGAATTATCTACTATTGAATCTTTAATGAATTTATATCAA |
| 24 | 60 | TTCTAAAATAACCATATAAGGAGCAACTTCACCTAATTGTTGATATAAATTCATTAAAGA |
| 25 | 60 | CCTTATATGGTTATTTTAGAAAATTCTATTCAAAATAAATTTTCTGCTGGTGCTTATCCT |
| 26 | 60 | TAATTCAACACCAACACCCATAGCATAAGACCATAATAAAGGATAAGCACCAGCAGAAAA |
| 27 | 60 | ATGGGTGTTGGTGTTGAATTAGAAAATTCTATGGGTGGTTTAAATTTTGGTCGTTCTTAT |
| 28 | 60 | AACCATTTCTTGACCTAAACGAAAATAAGCAGGATCAAAATAAGAACGACCAAAATTTAA |
| 29 | 60 | CGTTTAGGTCAAGAAATGGTTCGTCGTTCTGCTGGTAAAGTTTCTTCTGTTATTGCTGCT |
| 30 | 60 | AGAAACTAATTTAGCTTCTTCAGCAGTAATACCTAATTCAGCAGCAATAACAGAAGAAAC |
| 31 | 60 | GAAGAAGCTAAATTAGTTTCTGAAATTGCTTCTCAAGCTGGTGATGAACGTACTGCTCGT |
| 32 | 60 | TTGTAAAAAAGAAACTTGAGCTTGACGAGGACCAGTACCACGAGCAGTACGTTCATCACC |
| 33 | 60 | GCTCAAGTTTCTTTTTTACAACATCAAACTGGTGGTGGTGAATCTTCTGCTCCTGCTACT |
| 34 | 60 | TTCAGAACCATTAGGAATAGCAGCTTTAACACCTTCACGAGTAGCAGGAGCAGAAGATTC |
| 35 | 60 | GCTATTCCTAATGGTTCTGAAGAACGTGATCGTAAACAAACTCGTCCTGGTCGTCCTCGT |
| 36 | 60 | AGGCATAATTTCTAATAATAATTGACCAGGAGTTTCACCACGAGGACGACCAGGACGAGT |
| 37 | 60 | TTATTATTAGAAATTATGCCTGAAGATGAAGTTTCTCGTGAATCTGGTCAAAATCCTCGT |
| 38 | 60 | TTGTAAACGAAATAAAGCTTCAGCAGAACGTTGAGCTTCACGAGGATTTTGACCAGATTC |
| 39 | 60 | GAAGCTTTATTTCGTTTACAAGCTATGGCTAAAATTTTAGAAGATCAAGAAGAAGGTGAA |
| 40 | 63 | TTAACCTAATAAATCTTTATCATTATAAACTTGATTATTATCTTCACCTTCTTCTTGATCT TC |
2.PCR amplification gene fragment:
With totally 40 of above-mentioned primers, dividing equally by number order is 4 groups, and 10 every group, per 10 are carried out a PCR reaction.Wherein article one of every group and the last item are the outside primer of reaction, all the other 8 primers for reaction.
A) the part fragment gene is synthetic
System: 5 * damping fluid, 10 μ l, dNTP 4 μ l, inboard primer 12 μ l, outside primer 3 μ l, taq1 μ l, ddH2O 17 μ l.
Program: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 50s, 25 circulations, extend 72 ℃ of 5min.
Obtain 4 part fragment Nucleotide.
B) total fragment gene is synthetic
System: 5 * damping fluid, 10 μ l, dNTP 4 μ l, each 100ng of part fragment, the outside 2 μ l, taq 1 μ l, ddH2O 17 μ l.
Program: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 2min, 25 circulations, extend 72 ℃ of 5min.
Use 1% agarose electrophoresis, the result of acquisition as shown in Figure 1.Can be clearly seen that, formed the fragment of 4 500bp, and obtain total N protein nucleic acid fragment of a 2000bp.
Downcut the 2000bp band from agarose, it is cloned into the simple carrier of pMD19-T and serves the Hai Shenggong order-checking, result and expection fragment fit like a glove.
3. the expressed proteic detection of gene
Institute's synthetic gene is cloned into expression vector pET-32a, and transformed into escherichia coli Bl21 (DE3) Plyss, 1mM isopropyl ss-D-1-sulfo-gala pyranoside (IPTG) is induced its expression, expression product carries out Westernblotting, wherein one anti-is the proteic monoclonal antibody of N, and two anti-ly are HRP goat anti-mouse IgG antibody.Experimental result is seen Fig. 2.
In sum, contriver's this method is disposable, and to finish nearly 500bp segmental synthetic, and the two-step pcr reaction just can finish the segmental of nearly 1578bp and synthesize, and makes pcr amplification simplify greatly.In addition, improved tibet strain PPR N gene order does not contain complicated secondary structure and high GC content, and do not contain too much restriction enzyme enzyme sequence, successfully express at last by prokaryotic system, expressed product has carried out immunologic detection, and detected result shows by chemical process synthetic gene and provirus gene the common immunological role.
Therefore, the PPR virus N albumen of contriver's genetic modification can obtain by prokaryotic expression system easily, thereby has avoided cultivating the danger of highly pathogenic PPR virus.
Sequence table
＜110〉Academy of Agricultural Sciences, Shanghai City
＜120〉through the PPR N of genetic modification gene, its remodeling method and chemosynthesis
<130>096436
<160>41
<170>PatentIn?version?3.3
<210>1
<211>54
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
atggctactt?tattaaaatc?tttagcttta?tttaaacgta?ataaagataa?agct 54
<210>2
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
aataccacga?atagcaccac?cagaaccaga?agcagtagga?gctttatctt?tattacgttt 60
<210>3
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
ggtggtgcta?ttcgtggtat?taaaaatgtt?attattgttc?ctattcctgg?tgattcttct 60
<210>4
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
acgaactaaa?cgatctaata?aacgagaacg?agtagtaata?gaagaatcac?caggaatagg 60
<210>5
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>5
ttattagatc?gtttagttcg?tttagctggt?gatcctgata?ttaatggttc?taaattaact 60
<210>6
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>6
agattcaaca?aataaagata?acatagaaat?cataacacca?gttaatttag?aaccattaat 60
<210>7
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>7
ttatctttat?ttgttgaatc?tcctggtcaa?ttaattcaac?gtattactga?tgatcctgat 60
<210>8
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>8
acgagtagat?tgaacaactt?caactaaacg?aatagaaaca?tcaggatcat?cagtaatacg 60
<210>9
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>9
gaagttgttc?aatctactcg?ttctcaatct?ggtttaactt?ttgcttctcg?tggtgctgat 60
<210>10
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>10
accttcagta?gaaaaataca?tatcagcttc?attatctaaa?tcagcaccac?gagaagcaaa 60
<210>11
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>11
atgtattttt?ctactgaagg?tccttcttct?ggtggtaaaa?aacgtattaa?ttggtttgaa 60
<210>12
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>12
ttcaggatct?tgaacttcaa?tatcaataat?ttcacgattt?tcaaaccaat?taatacgttt 60
<210>13
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>13
attgaagttc?aagatcctga?agaatttaat?atgttattag?cttctatttt?agctcaagtt 60
<210>14
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>14
agtatcagga?gcagtaacag?ctttagctaa?taaaatccaa?acttgagcta?aaatagaagc 60
<210>15
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>15
gctgttactg?ctcctgatac?tgctgctgat?tctgaattac?gtcgttgggt?taaatatact 60
<210>16
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>16
tttatctaaa?cgaaattcac?caataacacg?acgttgttga?gtatatttaa?cccaacgacg 60
<210>17
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>17
ggtgaatttc?gtttagataa?aggttggtta?gatgctgttc?gtaatcgtat?tgctgaagat 60
<210>18
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>18
atctaaaatt?aaagaaacca?taaaacgacg?taaagataaa?tcttcagcaa?tacgattacg 60
<210>19
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>19
atggtttctt?taattttaga?tattaaacgt?actcctggta?ataaacctcg?tattgctgaa 60
<210>20
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>20
accagcttca?acaatataat?tatcaatatc?acaaatcatt?tcagcaatac?gaggtttatt 60
<210>21
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>21
aattatattg?ttgaagctgg?tttagcttct?tttattttaa?ctattaaatt?tggtattgaa 60
<210>22
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>22
accagcaaat?tcatgtaaac?ctaaagcagg?atacatagtt?tcaataccaa?atttaatagt 60
<210>23
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>23
ggtttacatg?aatttgctgg?tgaattatct?actattgaat?ctttaatgaa?tttatatcaa 60
<210>24
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>24
ttctaaaata?accatataag?gagcaacttc?acctaattgt?tgatataaat?tcattaaaga 60
<210>25
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>25
ccttatatgg?ttattttaga?aaattctatt?caaaataaat?tttctgctgg?tgcttatcct 60
<210>26
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>26
taattcaaca?ccaacaccca?tagcataaga?ccataataaa?ggataagcac?cagcagaaaa 60
<210>27
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>27
atgggtgttg?gtgttgaatt?agaaaattct?atgggtggtt?taaattttgg?tcgttcttat 60
<210>28
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>28
aaccatttct?tgacctaaac?gaaaataagc?aggatcaaaa?taagaacgac?caaaatttaa 60
<210>29
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>29
cgtttaggtc?aagaaatggt?tcgtcgttct?gctggtaaag?tttcttctgt?tattgctgct 60
<210>30
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>30
agaaactaat?ttagcttctt?cagcagtaat?acctaattca?gcagcaataa?cagaagaaac 60
<210>31
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>31
gaagaagcta?aattagtttc?tgaaattgct?tctcaagctg?gtgatgaacg?tactgctcgt 60
<210>32
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>32
ttgtaaaaaa?gaaacttgag?cttgacgagg?accagtacca?cgagcagtac?gttcatcacc 60
<210>33
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>33
gctcaagttt?cttttttaca?acatcaaact?ggtggtggtg?aatcttctgc?tcctgctact 60
<210>34
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>34
ttcagaacca?ttaggaatag?cagctttaac?accttcacga?gtagcaggag?cagaagattc 60
<210>35
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>35
gctattccta?atggttctga?agaacgtgat?cgtaaacaaa?ctcgtcctgg?tcgtcctcgt 60
<210>36
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>36
aggcataatt?tctaataata?attgaccagg?agtttcacca?cgaggacgac?caggacgagt 60
<210>37
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>37
ttattattag?aaattatgcc?tgaagatgaa?gtttctcgtg?aatctggtca?aaatcctcgt 60
<210>38
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>38
ttgtaaacga?aataaagctt?cagcagaacg?ttgagcttca?cgaggatttt?gaccagattc 60
<210>39
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>39
gaagctttat?ttcgtttaca?agctatggct?aaaattttag?aagatcaaga?agaaggtgaa 60
<210>40
<211>63
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>40
ttaacctaat?aaatctttat?cattataaac?ttgattatta?tcttcacctt?cttcttgatc 60
ttc 63
<210>41
<211>1578
<212>DNA
＜213〉artificial sequence
<400>41
atggctactt?tattaaaatc?tttagcttta?tttaaacgta?ataaagataa?agctcctact 60
gcttctggtt?ctggtggtgc?tattcgtggt?attaaaaatg?ttattattgt?tcctattcct 120
ggtgattctt?ctattactac?tcgttctcgt?ttattagatc?gtttagttcg?tttagctggt 180
gatcctgata?ttaatggttc?taaattaact?ggtgttatga?tttctatgtt?atctttattt 240
gttgaatctc?ctggtcaatt?aattcaacgt?attactgatg?atcctgatgt?ttctattcgt 300
ttagttgaag?ttgttcaatc?tactcgttct?caatctggtt?taacttttgc?ttctcgtggt 360
gctgatttag?ataatgaagc?tgatatgtat?ttttctactg?aaggtccttc?ttctggtggt 420
aaaaaacgta?ttaattggtt?tgaaaatcgt?gaaattattg?atattgaagt?tcaagatcct 480
gaagaattta?atatgttatt?agcttctatt?ttagctcaag?tttggatttt?attagctaaa 540
gctgttactg?ctcctgatac?tgctgctgat?tctgaattac?gtcgttgggt?taaatatact 600
caacaacgtc?gtgttattgg?tgaatttcgt?ttagataaag?gttggttaga?tgctgttcgt 660
aatcgtattg?ctgaagattt?atctttacgt?cgttttatgg?tttctttaat?tttagatatt 720
aaacgtactc?ctggtaataa?acctcgtatt?gctgaaatga?tttgtgatat?tgataattat 780
attgttgaag?ctggtttagc?ttcttttatt?ttaactatta?aatttggtat?tgaaactatg 840
tatcctgctt?taggtttaca?tgaatttgct?ggtgaattat?ctactattga?atctttaatg 900
aatttatatc?aacaattagg?tgaagttgct?ccttatatgg?ttattttaga?aaattctatt 960
caaaataaat?tttctgctgg?tgcttatcct?ttattatggt?cttatgctat?gggtgttggt?1020
gttgaattag?aaaattctat?gggtggttta?aattttggtc?gttcttattt?tgatcctgct?1080
tattttcgtt?taggtcaaga?aatggttcgt?cgttctgctg?gtaaagtttc?ttctgttatt?1140
gctgctgaat?taggtattac?tgctgaagaa?gctaaattag?tttctgaaat?tgcttctcaa?1200
gctggtgatg?aacgtactgc?tcgtggtact?ggtcctcgtc?aagctcaagt?ttctttttta?1260
caacatcaaa?ctggtggtgg?tgaatcttct?gctcctgcta?ctcgtgaagg?tgttaaagct?1320
gctattccta?atggttctga?agaacgtgat?cgtaaacaaa?ctcgtcctgg?tcgtcctcgt?1380
ggtgaaactc?ctggtcaatt?attattagaa?attatgcctg?aagatgaagt?ttctcgtgaa?1440
tctggtcaaa?atcctcgtga?agctcaacgt?tctgctgaag?ctttatttcg?tttacaagct?1500
atggctaaaa?ttttagaaga?tcaagaagaa?ggtgaagata?ataatcaagt?ttataatgat 1560
aaagatttat?taggttaa 1578
Claims (10)
1. the proteic isolating nucleic acid of coding PPR virus N is characterized in that, is the sequence of SEQ IDNO:41.
2. the method for the described nucleic acid of claim 1 that increases is characterized in that this method comprises the following steps:
A) be template with the described nucleic acid of claim 1, organize primer: SEQ ID NO:1-10, SEQ IDNO:11-20, SEQ ID NO:21-30, SEQ ID NO:31-40 carry out the PCR reaction respectively with each.Obtain 4 nucleic acid fragments;
B) with the nucleic acid fragment that obtains in the step a) as template, increase with SEQ ID NO:1,10,11,20,21,30,31,40 primer, amplification obtains the described nucleic acid of claim 1.
3. method as claimed in claim 2 is characterized in that, the reaction conditions in the step a) is: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 50s, 25 circulations, extend 72 ℃ of 5min.
4. method as claimed in claim 2 is characterized in that, the reaction system in the step a) is: 5 * damping fluid, 10 μ l, dNTP 4 μ l, inboard primer 12 μ l, outside primer 3 μ l, taq 1 μ l, ddH2O 17 μ l.
5. method as claimed in claim 2 is characterized in that, the reaction conditions in the step b) is: 94 ℃ of 1min, 94 ℃ of 30s, 60 ℃ of 15s, 72 ℃ of 2min, 25 circulations, extend 72 ℃ of 5min.
6. method as claimed in claim 2 is characterized in that, the reaction system in the step b) is: 5 * damping fluid, 10 μ l, dNTP 4 μ l, each 100ng of part fragment, the outside 2 μ l, taq 1 μ l, ddH2O 17 μ l.
7. the test kit of the described nucleic acid of claim 1 that is used to increase is characterized in that contain the primer of SEQ IDNO:1-40, SEQ ID NO:41's is nucleic acid-templated, dNTP, PCR damping fluid, carrier and label.
8. an expression vector is characterized in that, described expression vector contains the described nucleotide sequence of SEQ ID NO:41 and is used to express this expression of nucleic acids box.
9. a host cell is characterized in that, described host cell contains the described expression vector of claim 8.
10. host cell as claimed in claim 9 is characterized in that described host cell is a prokaryotic cell prokaryocyte.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224914A (en) * | 2013-05-17 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein |
| CN105154588A (en) * | 2015-09-09 | 2015-12-16 | 江苏省农业科学院 | Primer pair for detecting caprine parainfluenza virus type 3 (CPIV3) and peste des petits ruminants virus (PPRV) |
| CN107586783A (en) * | 2016-07-06 | 2018-01-16 | 华中农业大学 | Anti- PPR virus N protein monoclonal antibody and its application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131800B (en) * | 2013-03-25 | 2015-02-25 | 中国动物卫生与流行病学中心 | Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof |
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2009
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224914A (en) * | 2013-05-17 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Construction and application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein |
| CN103224914B (en) * | 2013-05-17 | 2016-01-20 | 中国农业科学院哈尔滨兽医研究所 | Express the construction and application of the restructuring PPR virus merging foreign epitope N protein |
| CN105154588A (en) * | 2015-09-09 | 2015-12-16 | 江苏省农业科学院 | Primer pair for detecting caprine parainfluenza virus type 3 (CPIV3) and peste des petits ruminants virus (PPRV) |
| CN107586783A (en) * | 2016-07-06 | 2018-01-16 | 华中农业大学 | Anti- PPR virus N protein monoclonal antibody and its application |
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| Publication number | Publication date |
|---|---|
| CN102108359B (en) | 2012-12-19 |
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