CN103804485B - Red-crowned crane interferon-alpha, its encoding gene and the application in antiviral - Google Patents
Red-crowned crane interferon-alpha, its encoding gene and the application in antiviral Download PDFInfo
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Abstract
The invention discloses a kind of red-crowned crane interferon-alpha, its encoding gene and the application in antiviral.A kind of red-crowned crane interferon-alpha (IFN-α) of the present invention, is characterized in that its aminoacid sequence is as SEQ? ID? shown in NO.3 or SEQ? ID? shown in NO.5.One aspect of the present invention discloses the nucleotide sequence of red-crowned crane IFN-α and the protein sequence of coding thereof, also demonstrate on the other hand the red-crowned crane IFN-α that clones and obtain antiviral, the application particularly in anti-vesicular stomatitis virus and anti-H9N2 avian influenza virus.Proposition of the present invention is that the further investigation of red-crowned crane Interferon, rabbit to other virus diseases is laid a good foundation, simultaneously also for the appearance of red-crowned crane Interferon, rabbit product provides theoretical basis.
Description
Technical field
The present invention relates to a kind of Interferon, rabbit and application thereof, particularly a kind of red-crowned crane interferon-alpha and the application in antiviral, the invention belongs to biological technical field.
Background technology
China is the red-crowned crane major country of perching in the world, and the State Administration of Forestry reports to the State Council red-crowned crane as unique national bird candidate bird, be animals under first-class state protection.In recent years, the transmissible disease of the harm such as red-crowned crane bird flu, newcastle disease, bleb, Marek’s disease red-crowned crane existence occurs often, causes serious loss, is therefore very important to the epidemic prevention of these diseases and treatment.
Interferon, rabbit (interferon, IFN) is that (or many) being controlled to produce by cellular genome under the effect of specific inducer plant protein, has the biological functions such as antiviral breeding and immunomodulatory.Interferon, rabbit can be divided into I type, II type and type III.I type Interferon, rabbit is also known as viral interferon or LeIF, and comprise IFN-α, β, ω, κ, τ, δ and ζ etc., major function is virus replication.II type Interferon, rabbit, also known as type II interferon, only has IFN-γ member, mainly plays immunoregulatory effect.Type iii interferon comprises IFN-λ (IL-28/29).
Up to now, the research (especially the research of people and mouse) of mammiferous IFN-α has obtained a lot of achievement, but the research of the IFN-α of bird is started late, only some progress in the species such as chicken, duck, goose, parrot, francolin.Not yet there is the report of the aminoacid sequence that the nucleotide sequence of red-crowned crane IFN-α gene is corresponding with it.Owing to also not having the product of red-crowned crane Interferon, rabbit to come out, be use chicken interferon to replace red-crowned crane Interferon, rabbit mostly.But there is species variation between chicken and red-crowned crane, the result for the treatment of of its Interferon, rabbit to the virus disease of red-crowned crane is not very well, thus studies red-crowned crane Interferon, rabbit and is necessary.
Summary of the invention
One of technical problem to be solved by this invention is to provide the Nucleotide of red-crowned crane interferon-alpha (IFN-α) gene and corresponding aminoacid sequence;
Two of technical problem to be solved by this invention is by carrying out recombinant expressed in prokaryotic cell prokaryocyte by red-crowned crane interferon-alpha (IFN-α) gene, thus verifies the restraining effect of this albumen to viral growth.
In order to achieve the above object, the technique means that the present invention adopts is:
The embodiment of clone red-crowned crane IFN-α complete sequence:
(1) extraction of red-crowned crane genomic dna;
(2) two couples of degenerated primer AIF-TY1a and AIF-TY2b are utilized, POAU and AIF-TY2b carries out pcr amplification, PCR primer is connected on suitable cloning vector, be transformed in competent escherichia coli cell, order-checking obtains the partial sequence of red-crowned crane IFN-α gene, wherein the nucleotides sequence of primer AIF-TY1a is classified as: 5 '-CASCRSYACRBCCASCASCTCSAGC-3 ', the nucleotides sequence of primer POAU is classified as: 5 '-CYRSYSCTCNYGCTCCTYCT-3 ', the nucleotides sequence of its common downstream degenerated primer AIF-TY2b is classified as: 5 '-AGGMGGACGRGKTCCCASGCGCAG-3 ', wherein R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, B=C/G/T, N=A/C/G/T,
(3) utilize chromosome walking technology, obtain the unknown nucleotide sequence of flank;
(4) by obtaining the complete sequence of red-crowned crane IFN-α to the splicing of extension increasing sequence.
The embodiment of red-crowned crane IFN-α gene function checking:
(1) the mature peptide gene of pcr amplification red-crowned crane IFN-α;
(2) prokaryotic expression carrier building restructuring is transformed in intestinal bacteria, abduction delivering interferon protein;
(3) if inclusion body is expressed, by inclusion body protein purifying, renaturation;
(4) the IFN-α after measuring renaturation on cell to the suppression situation of virus replication to verify the function of this albumen.
On the basis of above-mentioned research, the present invention proposes a kind of red-crowned crane interferon-alpha (IFN-α), its aminoacid sequence is (propetide, containing signal peptide) or SEQIDNO.5 shown (mature peptide) as shown in SEQIDNO.3.
Further, the invention allows for the nucleotide sequence of the red-crowned crane interferon-alpha described in coding.
Preferably, described nucleotides sequence is classified as complete genome sequence or its coding region sequence of red-crowned crane interferon-alpha, wherein, the complete genome sequence of described red-crowned crane interferon-alpha is as shown in SEQIDNO.1, and described coding region sequence is as shown in SEQIDNO.2 or SEQIDNO.6.
Expression vector containing described nucleotide sequence and the host cell containing described expression vector are also within protection scope of the present invention.
Further, the invention allows for the application of described red-crowned crane interferon-alpha in preparation antiviral.Wherein, preferably, described virus is vesicular stomatitis virus or bird flu H9N2 virus.
Further, the present invention proposes a kind of pharmaceutical composition of anti-vesicular stomatitis virus, it is characterized in that effective constituent is red-crowned crane interferon-alpha of the present invention (IFN-α).And
A pharmaceutical composition for anti-H9N2 avian influenza virus, is characterized in that effective constituent is red-crowned crane interferon-alpha according to claim 1.
To sum up, one aspect of the present invention discloses the nucleotide sequence of red-crowned crane IFN-α and the protein sequence of coding thereof, the simultaneous verification anti-vesicular stomatitis virus (Vesicularstomatitisvirus of this red-crowned crane IFN-α, and the function of anti-H9N2 avian influenza virus VSV), for the further investigation of red-crowned crane Interferon, rabbit to other virus diseases is laid a good foundation, for the appearance of red-crowned crane Interferon, rabbit product provides possibility.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure using degenerated primer AIF-TY1a and AIF-TY2bPCR amplification IFN-α portion gene;
1 road: object band; 2 roads: negative control;
Fig. 2 is the electrophoresis result figure using degenerated primer POAU and AIF-TY2bPCR amplification IFN-α portion gene;
1 road: negative control; 2 roads: object band;
Fig. 3 is the amplification electrophoresis result figure of IFN-α 5 ' and 3 ' flanking sequence;
1 road: 3 ' end first time amplified production; 2 roads: 3 ' end second time amplified production; 3 roads: 3 ' end third time amplified production; 4 roads: 5 ' end third time amplified production; 5 roads: 5 ' end second time amplified production; 6 roads: 5 ' end first time amplified production;
Fig. 4 is the amplification electrophoresis result figure of IFN-α mature peptide sequence;
1 road: object band; 2 roads: negative control;
Fig. 5 is the SDS-PAGE result of albumen;
After 1 road: pET32a zero load induction; 2 roads: before recombinant bacterium induction; 3 roads: recombinant bacterium whole cell; 4 roads: ultrasonic postprecipitation; 5 roads: ultrasonic rear supernatant;
Fig. 6 is that restructuring IFN-α is to the microscopic examination figure of the inhibition test of VSV virus;
A: virus control group; B: experimental group; C: negative control-cell controls group;
Fig. 7 is that the IFN-α of different concns is to the statistical graph of the suppression situation of VSV;
The different concns longitudinal axis of transverse axis: IFN: IFN is to the inhibiting rate of VSV;
Fig. 8 is that restructuring IFN-α is to the microscopic examination figure of the inhibition test of H9N2 virus;
A: virus control group; B: experimental group; C: negative control-cell controls group;
Fig. 9 is that the IFN-α of different concns is to the statistical graph of the suppression situation of H9N2.
The different concns longitudinal axis of transverse axis: IFN: IFN is to the inhibiting rate of H9N2.
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The amplification of the partial sequence of embodiment 1 red-crowned crane IFN-α gene
1, according to the simple and fast extraction method of bird poba gene group DNA, from the blood of red-crowned crane, genomic dna is extracted;
2, with red-crowned crane genomic dna for template, degenerated primer AIF-TY1a and AIF-TY2b is primer, pcr amplification IFN-α gene, wherein the nucleotides sequence of primer AIF-TY1a is classified as: 5 '-CASCRSYACRBCCASCASCTCSAGC-3 ', and the nucleotides sequence of primer AIF-TY2b is classified as: 5 '-AGGMGGACGRGKTCCCASGCGCAG-3 ', wherein R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, B=C/G/T; Reaction system and reaction conditions as follows:
PCR reaction system:
Reaction conditions: 94 DEG C of denaturation 8min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C of ends extend 10min, 4 DEG C of termination reactions.PCR primer 1% agarose gel electrophoresis result as shown in Figure 1.
Reclaim object fragment, glue is reclaimed product and be connected into pMD19-T carrier, after 16 DEG C of connection 5h, be transformed in bacillus coli DH 5 alpha competent cell, cultivate 12h for 37 DEG C.
Through bacterium liquid PCR, picking mono-clonal bacterium colony, identifies that (AIF-TY1a and AIF-TY2b is primer) positive Hou Song Beijing Liuhe Huada Genomics Technology Co., Ltd order-checking obtains part (167bp) red-crowned crane IFN-α gene.
The amplification of the partial sequence of embodiment 2 red-crowned crane IFN-α gene
1, with red-crowned crane genomic dna for template, degenerated primer POAU and AIF-TY2b is primer, pcr amplification IFN-α gene, and wherein the nucleotides sequence of primer POAU is classified as: 5 '-CYRSYSCTCNYGCTCCTYCT-3 ', the nucleotides sequence of primer AIF-TY2b is classified as: 5 '-AGGMGGACGRGKTCCCASGCGCAG-3 ', wherein R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, B=C/G/T, N=A/C/G/T;
Reaction system and reaction conditions as follows:
PCR reaction system:
Reaction conditions: 94 DEG C of denaturation 8min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C of ends extend 10min, 4 DEG C of termination reactions.PCR primer 1% agarose gel electrophoresis result as shown in Figure 2.
2, reclaim object fragment, glue is reclaimed product and be connected into pMD19-T carrier, after 16 DEG C of connection 5h, be transformed in bacillus coli DH 5 alpha competent cell, cultivate 12h for 37 DEG C.
3, picking mono-clonal bacterium colony, identify that (AIF-TY1a and AIF-TY2b is primer, POAU and AIF-TY2b is primer) is positive Hou Song Beijing Liuhe Huada Genomics Technology Co., Ltd order-checking acquisition part (480bp) red-crowned crane IFN-α gene through bacterium liquid PCR.
Find through sequence alignment, to increase the on all four sequence of 167bp sequence obtained containing with embodiment 1 in the 480bp sequence of acquisition.
The amplification of 5 ' side wing unknown nucleotide sequence of embodiment 3 red-crowned crane IFN-α gene
On the basis of the 480bp interferon gene of embodiment 2 acquisition, according to the explanation of GenomeWalkingKit test kit, design the Auele Specific Primer SP1 that 3 annealing temperatures are relatively high, SP2, SP3, its nucleotide sequence is respectively:
SP1：5’-TTGCCTTTTGAAGCGCGTCCCGT-3’；
SP2：5’-CCTTGGTTCGTGTGGTTGTCGTG-3’；
SP3：5’-GAGGTTGAGGCTGTCCCAGGTGA-3’。
Illustratively carry out three nest-type PRCs, PCR reaction system is as follows for the first time:
PCR reaction conditions is as follows for the first time:
The PCR reaction system of second time PCR reaction and reaction conditions, as shown in first time, need carry out the part of changing: dilute 100 times afterwards for second time template with first time PCR primer, SP2 is the Auele Specific Primer of second time reaction.
PCR reactive moieties for the third time: dilute 100 times afterwards for third time template with second time PCR primer, SP3 is the Auele Specific Primer of third time reaction.Its reaction system and reaction conditions are as shown in first time.
First time PCR, second time PCR, third time PCR tri-nest-type PRCs PCR primer 1% agarose gel electrophoresis result as shown in Figure 3.
2, reclaim third time PCR primer, recovery product is connected into pMD19-T carrier, is transformed in bacillus coli DH 5 alpha competent cell, cultivate 12h for 37 °.
3, picking mono-clonal bacterium colony, cuts qualification (EcoRI, HindIII) for checking order positive Hou Song Beijing Liuhe Huada Genomics Technology Co., Ltd through restriction enzyme, obtains 5 ' end flanking sequence.
The amplification of 3 ' side wing unknown nucleotide sequence of embodiment 4 red-crowned crane IFN-α gene
As described in Example 3, reaction system and reaction conditions are all identical, and the nucleotides sequence of required Auele Specific Primer is classified as the amplification method of 3 ' side wing unknown nucleotide sequence:
P1：5’-ACCCTCACCTGGGACAGCCTCAACC-3’；
P2：5’-CGCCACCTACTCCTCAACAACCTC-3’；
P3：5’-CACCACCACATCCAGCAGCTCCAGC-3’。
First time PCR, second time PCR, third time PCR tri-nest-type PRCs PCR primer 1% agarose gel electrophoresis result as shown in Figure 3.Flanking sequence is held through order-checking acquisition 3 '.
By obtaining the complete sequence of red-crowned crane IFN-α to the splicing of embodiment 1-4 extension increasing sequence, sequence is as shown in SEQIDNO.1, wherein 487-1227 position Nucleotide is IFN-α coding region sequence (open reading frame, ORF), sequence as shown in SEQIDNO.2, the aminoacid sequence of IFN-α of its coding as shown in SEQIDNO.3, comprising signal peptide and mature peptide sequence, the aminoacid sequence of signal peptide is as shown in SEQIDNO.4, and the amino acid sequence of mature peptide is as shown in SEQIDNO.5.
The prokaryotic expression of embodiment 5 red-crowned crane IFN-α gene
In the process of prokaryotic expression, the signal peptide part of red-crowned crane IFN-α will be removed, therefore primer MORF1a and MORF2b of red-crowned crane IFN-alpha expression mature peptide will first be designed, nucleotides sequence is classified as: MORF1a:5 '-GAATTCTGCCACCACCTGCGGCCACGC-3 ' (underscore: EcoRI restriction enzyme site), MORF2b:5 '-AAGCTTCATGCACGTTTCCCCATCGGTT-3 ' (underscore: HindIII restriction enzyme site).
The nucleotide sequence of pcr amplification coding red-crowned crane IFN-α mature peptide, reaction system is as follows:
Reaction conditions: 94 DEG C of denaturation 8min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C of ends extend 10min, 4 DEG C of termination reactions.PCR primer 1% agarose gel electrophoresis result as shown in Figure 4.
3, after object fragment reclaims, be connected into blunt vector pEASY-Blunt-Simple, obtain pEASY-IFN-α, transformation of E. coli DH5 α, cultivate 12h, identify through bacterium liquid PCR for 37 DEG C, the mono-double digestion of EcoRI, HindIII is accredited as positive bacterium liquid and send Hua Da to check order, sequencing result display clones the nucleotide sequence of the coding red-crowned crane IFN-α mature peptide obtained as shown in SEQIDNO.6, consistent with expection.
4, the structure of recombinant expression vector pET32a-IFN-α: pEASY-IFN-α and pET32a by IFN-α and linear expression vector pET32a, connects into pET32a-IFN-α after cutting through EcoRI, HindIII pair under the effect of T4DNA ligase enzyme.
5, in the BL21 intestinal bacteria transformed by recombinant expression vector pET32a-IFN-α, treat that OD value adds IPTG abduction delivering about 0.5.As shown in Figure 7, aminoacid sequence is as shown in SEQIDNO.5 for the SDS-PAGE result of the recombinant protein after expression.
6, the ni-sepharose purification of albumen: guanidine hydrochloride solution recombinant protein being dissolved in 6M, by purifying description operation, 10mM imidazoles, the assorted band of upper prop washing is white respectively for 100mM imidazoles, 250mM eluting interferon albumen.
7, the dialysis renaturation of inclusion body: the present invention attempts the recombinant interferon after with 2M Guanidinium hydrochloride dialysis purifying to renaturing inclusion bodies, finally prepares against its biological function of research with PBS dialysis.
Embodiment 6 is recombinated the anti-VSV virus activity functional verification of red-crowned crane IFN-α
1, chick embryo fibroblast is prepared, 37 DEG C, 5%CO
2cultivate 24h;
2, change cell culture fluid, in chick embryo fibroblast, access restructuring red-crowned crane IFN-α albumen (prepared by embodiment 5) of the different extension rate of 100 μ L simultaneously, cultivate 24h;
3, change cell culture fluid, add 100 μ L vesicular stomatitis virus (VSV) simultaneously, 100pfu hole, cultivate 24h, examine under a microscope, and add up the suppression situation (inhibiting rate) that virus is subject to, result is as shown in Fig. 6 and Fig. 7.
Result shows, the red-crowned crane IFN-α albumen that the present invention prepares has the effect that suppression vesicular stomatitis virus (VSV) is bred, and suppression degree raises along with the increase of red-crowned crane IFN-α protein concentration within the specific limits.
Embodiment 7 is recombinated the anti-H9N2 virus activity functional verification of red-crowned crane IFN-α
1, chick embryo fibroblast is prepared, 37 DEG C, 5%CO
2cultivate 24h;
2, change cell culture fluid, in chick embryo fibroblast, access restructuring red-crowned crane IFN-α albumen (prepared by embodiment 5) of the different extension rate of 100 μ L simultaneously, cultivate 24h;
3, change cell culture fluid, add 100 μ L avian influenza virus H9N2 simultaneously, 100pfu hole, cultivate 24h, examine under a microscope, and add up the suppression situation (inhibiting rate) that virus is subject to, result is as shown in FIG. 8 and 9.
Result shows, the red-crowned crane IFN-α albumen that the present invention prepares has the effect suppressing avian influenza virus H9N2 propagation, and suppression degree raises along with the increase of red-crowned crane IFN-α protein concentration within the specific limits.
Claims (8)
1. red-crowned crane interferon-alpha (IFN-α), is characterized in that its aminoacid sequence is as shown in SEQIDNO.3 or shown in SEQIDNO.5.
2. the nucleic acid molecule of red-crowned crane interferon-alpha according to claim 1 of encoding.
3. nucleic acid molecule as claimed in claim 2, it is characterized in that the sequence of described nucleic acid molecule is complete genome sequence or its coding region sequence of red-crowned crane interferon-alpha, wherein, the complete genome sequence of described red-crowned crane interferon-alpha is as shown in SEQIDNO.1, and described coding region sequence is as shown in SEQIDNO.2 or SEQIDNO.6.
4. an expression vector, is characterized in that containing the nucleic acid molecule described in Claims 2 or 3.
5. a host cell, is characterized in that containing expression vector according to claim 4.
6. the application of red-crowned crane interferon-alpha according to claim 1 in preparation antiviral, described virus is vesicular stomatitis virus or H9N2 avian influenza virus.
7. a pharmaceutical composition for anti-vesicular stomatitis virus, is characterized in that effective constituent is red-crowned crane interferon-alpha according to claim 1.
8. a pharmaceutical composition for anti-H9N2 avian influenza virus, is characterized in that effective constituent is red-crowned crane interferon-alpha according to claim 1.
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| CN116143900A (en) * | 2023-02-10 | 2023-05-23 | 江苏省农业科学院 | Recombinant pigeon interferon alpha, expression engineering strain thereof and preparation method thereof |
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| CN101351218A (en) * | 2005-02-04 | 2009-01-21 | 维拉纳帝弗公司 | Method and use of interferon compositions for the treatment of avian influenza |
| CN101948845A (en) * | 2010-09-01 | 2011-01-19 | 中国科学院微生物研究所 | Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha |
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| CN101351218A (en) * | 2005-02-04 | 2009-01-21 | 维拉纳帝弗公司 | Method and use of interferon compositions for the treatment of avian influenza |
| CN101948845A (en) * | 2010-09-01 | 2011-01-19 | 中国科学院微生物研究所 | Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha |
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