CN103937833A - siRNA recombinant interference vector based on subgroup J avian leukosis virus LTR gene conserved sequence, preparation method and application thereof - Google Patents
siRNA recombinant interference vector based on subgroup J avian leukosis virus LTR gene conserved sequence, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of gene engineering, and provides a siRNA recombinant interference vector based on a subgroup J avian leukosis virus LTR gene conserved sequence. According to the present invention, the vector adopts the characteristic of the conservation property of LTR gene, and adopts the LTR gene to design and synthesize siRNA and construct a siRNA hairpin structure, the annealed double-stranded DNA is further obtained and is linked to the vector to construct a recombinant interference vector, the interference vector and virus are adopted to co-transfect cells, and finally results show that the interference vector can effectively interfere transcription and replication of subgroup J avian leukosis viruses in vitro and in vivo in chickens so as to provide scientific basis and technical support for prevention and treatment of subgroup J avian leukosis, and reduce economic losses caused by ALV-J infection in the avian breeding industry.
Description
Technical field
The present invention relates to gene engineering technology field, provide a kind of siRNA based on J subgroup avian leucosis virus LTR gene conserved sequence restructuring interference carrier and preparation method thereof with it disturbing the application of Transcription and replication in cell and live chickens body in vitro of J subgroup avian leucosis virus.
Background technology
J subgroup avian leucosis is a kind of neoplastic disease being caused by J subgroup avian leucosis virus (ALV-J).ALV-J is the retrovirus with cyst membrane, and fowl clinical infection main manifestations is myelocytome, immunological tolerance, high mortality and growth retardation.Between 1997～1998 years, ALV-J is global outburst, and world's meat kind chicken industry has been caused to crushing blow.ALV-J infects except causing myelocytome, and erythroblastoma, vascular tumor, nephroncus and sarcoma are everlasting and are infected the later stage and follow generation, and conventionally mortality ratio is 1%～5%, and peak period can reach 50%.Studies confirm that, ALV-J has lasting detrimental effect to immunne response, makes productivity low, and secondary infection and polyinfection take place frequently, the sound development of J subgroup avian leucosis serious harm aquaculture.Control the generation of this disease abroad by purification, because purification cycle is long, cost is high, also fail at present effectively to implement in China, can only carry out jumpbogroup purification by eliminating infection chicken, this method is due to the infection of not thorough and uncontrollable ALV-J not in time, meanwhile, and not science, irrational control techniques, also can accelerate viral sudden change and evolution, make disease more sophisticated;
ALV is birnavirus, the about 7.6-7.8kb of its genome total length.The provirus genome of ALV-J mainly contains 3 structure genes, archaeal dna polymerase (ThermoScript II) and membrane glycoprotein that the virus structural protein of encoding respectively, RNA rely on.Hold it to put in order from 5 ' end to 3 ' and be followed successively by LTR-leader-gag-pol-env-leader-LTR.LTR (long terminal repeats) is long terminal repeat, comprising virus replication, translate, RNA processing and viral integrase be in important sequence compositions such as host genome.LTR is respectively by 3, special zone U3, short iteron R and 5, distinct zones U5 tri-parts compositions.Wherein promotor and transcriptional enhancer are contained in U3 district, with virus replication, translate, tumorigenesis etc. is closely related, has vital role in transcribing;
ALV-J is togavirus simultaneously, and virus envelope has determined viral antigenicity, and antigenicity constantly changes, and this becomes the bottleneck that the development of avian leukosis conventional vaccine cannot be gone beyond.Based on this, development J substock lymphoid leuoosis-resistant biotechnological formulation becomes focus and the focus of current J subgroup avian leucosis prevention and control, how this gene is become to problem demanding prompt solution for the prevention and control of J subgroup avian leucosis.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, particularly the very conservative while of LTR gene is for this situation of significance of ALV, utilize this gene design to synthesize siRNA interference carrier, further obtain after annealing double-stranded DNA, by itself and viral cotransfection cell, final this interference carrier provided by the invention interference in vitro cell and the interior J subgroup avian leucosis virus Transcription and replication of live chickens body effectively of finding, for the control of J subgroup avian leucosis provides scientific basic and technical support, reduce aviculture and infect the financial loss causing because of ALV-J.
First contriver has synthesized siRNA based on the design of LTR gene conserved sequence, and base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ ID NO:1;
This section of sequence is identical with one section of sequence of target gene, is to utilize the online design software screening of siRNA to obtain;
Design can form the hairpin structure of siRNA, and reverse transcription obtains ds oligo DNA, connect into interference carrier, carrier transfection is entered after cell, can transcribe out coding strand wherein, and in cell, form hairpin structure (italicized item base complementrity in chain, can form hairpin structure), cut and obtain siRNA at desmo enzyme, siRNA untwists, its antisense strand and target gene complementation, thus be combined with target gene, make target gene degraded.Its mechanism as shown in Figure 1;
And then contriver utilizes aforesaid method that ds oligo DNA is connected in pcDNA6.2-GW/EmGFP-miR interference carrier, finally obtain this interference carrier of the present invention,
Wherein said interference carrier is purchased from invitrogen company;
Contriver also discloses the concrete preparation method of above-mentioned interference carrier simultaneously, as follows:
Based on the synthetic siRNA of LTR gene conservative region design;
Its concrete operations are: the different subgroups of ALV and the different strain of J subgroup are carried out to homology analysis, in the highest LTR gene of its homology compared with conservative region fragment the sequence fragment as screening siRNA, utilize the online design software of siRNA, obtain siRNA, its base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ ID NO:1;
Wherein the selected strain of contriver comprises: NX0101 (ALV-J, DQ115805), MQNCSU (ALV-A, DQ365814), SDAU09E3 (ALV-B JF826241), RSV-Prague (ALV-C, J02342), RSV-Schmidt-Ruppin D (ALV-D, D10652), SD0501 (ALV-E, EM467236), HPRS103 (ALV-J, Z46390) and ADOL-7501 (ALV-J, AY027920).
The synthetic hairpin structure containing above-mentioned purpose fragment of design, as top strand DNA in Fig. 3, its gene order is as shown in sequence table SEQ ID NO:2, corresponding bottom strand DNA with it, its gene order, as shown in sequence table SEQ ID NO:3, is connected in pcDNA6.2-GW/EmGFP-miR interference carrier after the formation two strands of being annealed, and connects product and is transformed into competent cell DH5 α, be coated with after LB flat board (containing 50 μ g/ml spectinomycins), hatch for 37 DEG C;
Transform dull and stereotyped 3 clones of picking respectively, after shaking bacterium extracting plasmid, check order, to verify that Insert Fragment sequence in recombinant clone is whether consistent with the oligomerization single stranded DNA of design (namely top strand DNA and with it corresponding bottom strand DNA) sequence;
Find that by the checking of ordinary method the present invention does acquisition recombinant vectors, in recombinant clone, Insert Fragment sequence is consistent with the oligomerization single stranded DNA sequence of design,
Further contriver passes through in body and the mode of vitro detection, confirm this restructuring interference carrier interference in vitro cell and interior J subgroup avian leucosis virus Transcription and replication of live chickens body effectively, general jamming effectiveness reaches more than 80%, have great progress than the existing gimmick of preventing and treating, the external detection method that wherein adopted is as follows:
Cultivate DF-1 cell, after 12h, connect poison (ALV-J NX0101 strain), treat that cell state is good, when Cell abundance reaches 70%-80%, DF-1 cell is passed on 24 orifice plates;
(approximately 8h-12h in the time that Cell abundance reaches 70%,), carry out the transfection of recombinant plasmid, expression level, the Westernblot that detects expression level, the indirect immunofluorescene assay ALV-J live virus envelope protein of virogene by Real-time PCR after 72h detects the expression level of virus envelope albumen after Transfected Recombinant Plasmid DF-1 cell with checking interference effect, protein expression result shows, after disturbing, ALV-J protein expression and mRNA transcriptional level obviously reduce, and jamming effectiveness reaches 83.6%;
Same, in vivo test detects:
Restructuring interference plasmid and virus are inoculated instar chicken embryo on the 6th simultaneously, establish ALV-J simultaneously and infect positive controls and negative control group.
Chick goes out after shell, raises to 7 week age, all cuts open and kills, and carries out weekly during this time hematology, pathological study and virusology and detects antiviral effect;
Result shows that the growth of recombinant plasmid interference group chicken is normal, and all kinds of white corpuscles of blood have no considerable change, ALV-J antigen/antibody feminine gender, and without toxin expelling phenomenon, histopathology detects and has no tissue injury and inflammatory reaction.And there is serious viremia in ALV-J positive controls, toxin expelling, there is significantly damage and inflammation in the vitals such as liver and heart.Experimental results show that RNA disturbs recombinant vectors can reach significant antiviral effect.
In sum, the present invention utilizes very conservative this situation of LTR gene, utilize this gene design to synthesize siRNA interference carrier, and by this interference carrier and viral cotransfection cell, final this interference carrier provided by the invention interference in vitro cell and the interior J subgroup avian leucosis virus Transcription and replication of live chickens body effectively of finding, for the control of J subgroup avian leucosis provides scientific basic and technical support, reduce aviculture and infect the financial loss causing because of ALV-J.
Brief description of the drawings
Fig. 1 is interference principle figure involved in the present invention;
Fig. 2 is interference carrier collection of illustrative plates involved in the present invention;
Fig. 3 is the schematic diagram of the designed top strand DNA of the present invention and bottom strand DNA;
Shown in italic font in figure in visible top strand, sequence is identical with one section of sequence on target gene, and the other end italic font sequence and its complementation are identical with its mentality of designing in bottom strand;
Fig. 4 is carrier construction plasmid electrophoresis result schematic diagram of the present invention,
Wherein electrophorogram left side is DNA marker, the band that right side is plasmid;
Due to the spirane structure difference that plasmid exists, when electrophoresis, can form many bands, be followed successively by from top to bottom the OC configuration of lax open loop, lax linear L configuration (5700bp) and supercoiled SC configuration;
Fig. 5 is the interference effect figure of gained interference carrier FLuorescent of the present invention,
In figure p-si-LTR be interference group, Null-vector control be empty carrier control group, ALV-J infected control be virus infection do not disturb control group, normal cells control be normal cell do not connect poison do not disturb contrast picture group in interference group compared with positive controls, fluorocyte number greatly reduces, and proves that this tests interference plasmid used and have good interference effect;
Fig. 6 is the relative quantification comparative result that RT-PCR of the present invention detects expressing viral,
Scheme known interference group and positive control compare, the expression level of its virogene significantly reduces.
Embodiment
Further definition the present invention in following examples, according to above description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and in the situation that not departing from spirit and scope of the invention, can make various changes and amendment to the present invention, so that its applicable various uses and condition.Except special indicating, the state of the art that is of the present invention, the reagent adopting is also available reagent;
The preparation of embodiment 1 double-stranded DNA
Target LTR gene conservative region, initiation site is 7497, the synthetic siRNA of design, its base sequence is
AGGCAACACGGGTCTTATA, its gene order is as shown in sequence table SEQ ID NO:1;
The synthetic hairpin structure containing above-mentioned purpose fragment of design, wherein top strand DNA, its gene order is as shown in sequence table SEQ ID NO:2; Corresponding its gene order of bottom strand DNA is as shown in sequence table SEQ ID NO:3 with it; Use ddH
2o is dissolved into 100 μ M, and complementary strand is respectively got 5-10 μ l and mixed between two, provides system anneal by table one.Mixture, 95 DEG C of heating 5～10 minutes, is then placed to room temperature 20 minutes, form double-stranded DNA.
Table one, the oligo DNA system of annealing
| 100μM?top?strand?oligo | 5μl |
| 100μM?bottom?strand?oligo | 5μl |
| 10×oligo?annealing?buffer | 2μl |
| ddH 2O | 8μl |
| Total?volume | 20μl |
The recombinate structure of interference carrier of embodiment 2:
By the double-stranded DNA sterilizing ddH of annealing
2o continues to be diluted to 10nM concentration, provides system connect 30-45 minute in room temperature by table three.
Table two, enzyme linked system
| 5×ligation?buffer | 4μl |
| pcDNA6.2-GW/EmGFP-miR | 2μl |
| ds?oligo(10nM) | 4μl |
| T4DNA?ligase（1U/μl） | 1μl |
| ddH2O | 9μl |
| Total?volume | 20μl |
Embodiment 3 transforms test
Get 10 μ l connection products and transform 100 μ l competent cell DH5 α, be coated with after LB flat board (containing 50 μ g/ml spectinomycins), hatch for 37 DEG C.
Transform dull and stereotyped 3 clones of picking respectively, check order after shaking bacterium extracting plasmid, with verify Insert Fragment sequence in recombinant clone whether with the oligomerization single stranded DNA of design, namely top strand DNA and bottom strand consensus dna sequence;
Deliver the order-checking of order-checking company by the recombinant vectors that clone is obtained, found that the present invention does acquisition recombinant vectors, in recombinant clone, Insert Fragment sequence is consistent with the oligomerization single stranded DNA sequence of design, and visual target fragment has successfully been inserted in cloning vector.
Embodiment 4
The vitro detection of interference carrier effect:
Cultivate DF-1 cell, after 12h, connect poison (ALV-J NX0101 strain), treat that cell state is good, when Cell abundance reaches 70%-80%, DF-1 cell is passed on 24 orifice plates;
In the time that Cell abundance reaches 70%, (approximately 8h-12h), carries out the transfection of recombinant plasmid, and detailed process is: use without the DMEM preparation RNA of blood nonreactive and disturb transfection composite, and specific as follows: transfection reagent Lipofectamine
tMl) (μ g) prepares RNA according to the ratio of 2.5:0.5 and disturbs transfection composite 2000(μ with interference plasmid, making plasmid final concentration by adjustment without the DMEM consumption of blood nonreactive is 1 μ g/ μ l, cell growth medium is changed to without blood nonreactive DMEM, adding afterwards above-mentioned plasmid final concentration is the transfection composite of 1 μ g/ μ l, is replaced by maintain base after 8h;
Expression level, the Western blot that detects expression level, the indirect immunofluorescene assay ALV-J live virus envelope protein of virogene by Real-time PCR after 72h detects the expression level of virus envelope albumen after Transfected Recombinant Plasmid DF-1 cell with checking interference effect, protein expression result shows, after disturbing, ALV-J protein expression and mRNA transcriptional level obviously reduce, and jamming effectiveness reaches 83.6%; (as shown in Figure 6)
Embodiment 4 in vivo test detect:
Restructuring interference plasmid and virus are inoculated instar chicken embryo on the 6th simultaneously, establish ALV-J simultaneously and infect positive controls and negative control group.
Chick goes out after shell, raises to 7 week age, all cuts open and kills, and carries out weekly during this time hematology, pathological study and virusology and detects antiviral effect;
Result shows that the growth of restructuring material interference group chicken is normal, and all kinds of white corpuscles of blood have no considerable change, ALV-J antigen/antibody feminine gender, and without toxin expelling phenomenon, histopathology detects and has no tissue injury and inflammatory reaction.And there is serious viremia in ALV-J positive controls, toxin expelling, there is significantly damage and inflammation in the vitals such as liver and heart.Experimental results show that RNA disturbs recombinant vectors can reach significant antiviral effect.
Claims (3)
1. the restructuring of the siRNA based on a J subgroup avian leucosis virus LTR gene conserved sequence interference carrier, it is characterized in that: this carrier is to utilize designed siRNA, build its hairpin structure, after annealing, the double-stranded DNA of formation is connected to and obtains in pcDNA6.2-GW/EmGFP-miR interference carrier, wherein its gene order of siRNA is as shown in sequence table SEQ ID NO:1.
2. the preparation method of restructuring interference carrier claimed in claim 1, is characterized in that: concrete steps are as follows:
(1) based on the synthetic siRNA of LTR gene conservative region design; Its concrete operations are: the different subgroups of ALV and the different strain of J subgroup are carried out to homology analysis, in the highest LTR gene of its homology compared with conservative region fragment the sequence fragment as screening siRNA, utilize the online design software of siRNA, obtain siRNA, base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ ID NO:1;
(2) the synthetic hairpin structure containing above-mentioned purpose fragment of design, wherein top strand DNA, its gene order is as shown in sequence table SEQ ID NO:2, bottom strand DNA, its gene order, as shown in sequence table SEQ ID NO:3, is connected in pcDNA6.2-GW/EmGFP-miR interference carrier and get final product after the formation two strands of being annealed.
As claimed in claim 1 carrier disturbing the application of Transcription and replication in cell and live chickens body in vitro of J subgroup avian leucosis virus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104262477A (en) * | 2014-09-25 | 2015-01-07 | 山东农业大学 | Avian CCCH type zinc finger protein chZAP and application thereof |
| CN104306995A (en) * | 2014-10-29 | 2015-01-28 | 山东农业大学 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
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| QING-WEN MENG，ET AL: "Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs", 《VIROLOGY JOURNAL》 * |
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| 孟祥凯: "J 亚群禽白血病病毒gp37基因siRNA表达载体的构建及体外干扰效应的初步研究", 《中国优秀硕士学位论文全文数据库.农业科学辑》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262477A (en) * | 2014-09-25 | 2015-01-07 | 山东农业大学 | Avian CCCH type zinc finger protein chZAP and application thereof |
| CN104306995A (en) * | 2014-10-29 | 2015-01-28 | 山东农业大学 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
| CN104306995B (en) * | 2014-10-29 | 2017-04-12 | 山东农业大学 | Epitope vaccine for resisting subgroup J avian leukosis virus infection as well as preparation method and application of epitope vaccine |
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