CN103937833B - A kind of restructuring of the siRNA based on J subgroup avian leucosis virus LTR gene conserved sequence interference carrier and its preparation method and application - Google Patents
A kind of restructuring of the siRNA based on J subgroup avian leucosis virus LTR gene conserved sequence interference carrier and its preparation method and application Download PDFInfo
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Abstract
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, this carrier utilizes LTR gene this situation very conservative, this gene design is utilized to synthesize siRNA, build the hairpin structure forming siRNA, after further acquisition annealing double-stranded DNA, it is connected with carrier and builds interference carrier of recombinating, by this interference carrier and viral cotransfection cell, final discovery this interference carrier provided by the invention can the Transcription and replication of J subgroup avian leucosis virus in interference in vitro cell and live chickens body effectively, control for J subgroup avian leucosis provides scientific basic and technical support, reduce aviculture and infect the financial loss caused because of ALV-J.
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 and to recombinate the application of interference carrier and preparation method thereof and its Transcription and replication in interference J subgroup avian leucosis virus in vitro cell and live chickens body.
Background technology
J subgroup avian leucosis is a kind of neoplastic disease 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, causes crushing blow to world's Breeder hens industry.ALV-J infects except causing myelocytome, and erythroblastoma, vascular tumor, nephroncus and sarcoma are everlasting and are infected the later stage with occurring, and usual mortality ratio is 1% ~ 5%, and peak period can reach 50%.Research confirms, ALV-J has lasting detrimental effect to immunne response, and make productivity low, secondary infection and polyinfection take place frequently, the sound development of J subgroup avian leucosis serious harm aquaculture.The external generation being controlled this disease by purification, because purification cycle is long, cost is high, also fail at present effectively to implement in China, jumpbogroup purification can only be carried out by eliminating infection chicken, this method due to the infection of not thorough and uncontrollable ALV-J not in time, meanwhile, not science, irrational control techniques, also can accelerate sudden change and the evolution of virus, make disease more sophisticated;
ALV is birnavirus, and its full-length genome is about 7.6-7.8kb.The provirus genome of ALV-J mainly contains 3 structure genes, the archaeal dna polymerase (ThermoScript II) of encoding viral structural albumen, RNA dependence respectively and membrane glycoprotein.Hold it to put in order from 5 ' end to 3 ' and be followed successively by LTR-leader-gag-pol-env-leader-LTR.LTR (longterminalrepeats) is long terminal repeat, comprising virus replication, translate, RNA processing and viral integrase in important sequence component such as host genome.LTR is respectively by 3, and special zone U3, short iteron R and 5, distinct zones U5 tri-part form.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 determines the antigenicity of virus, 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 the prevention and control of current J subgroup avian leucosis, and how this gene being used for the prevention and control of J subgroup avian leucosis becomes problem demanding prompt solution.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, particularly LTR gene is very conservative simultaneously for this situation of significance of ALV, this gene design is utilized to synthesize siRNA interference carrier, after further acquisition annealing double-stranded DNA, by itself and viral cotransfection cell, final discovery this interference carrier provided by the invention can J subgroup avian leucosis virus Transcription and replication in interference in vitro cell and live chickens body effectively, control for J subgroup avian leucosis provides scientific basic and technical support, reduces aviculture and infects the financial loss caused because of ALV-J.
Contriver is the siRNA based on LTR gene conserved sequence design and synthesis first, and base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ IDNO:1;
This section of sequence is identical with one section of sequence of target gene, is utilize siRNA Photographing On-line software screening method to obtain;
Design can form the hairpin structure of siRNA, and reverse transcription obtains dsoligoDNA, connect into interference carrier, after cell is entered in carrier transfection, coding strand wherein can be transcribed out, and in cell, form hairpin structure (in chain, italicized item base complementrity, can form hairpin structure), cut at desmo enzyme and obtain siRNA, siRNA untwists, its antisense strand and target gene complementation, thus be combined with target gene, target gene is degraded.Its mechanism as shown in Figure 1;
And then contriver utilizes aforesaid method to be connected to by dsoligoDNA in pcDNA6.2-GW/EmGFP-miR interference carrier, final acquisition 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 LTR gene conserved regions design and synthesis siRNA;
Its concrete operations are: carry out homology analysis to the different subgroup of ALV and the different strain of J subgroup, compared with the sequence fragment of conservative region fragment as screening siRNA in the LTR gene that its homology is the highest, utilize siRNA Photographing On-line software, obtain siRNA, its base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ IDNO:1;
Strain wherein selected by contriver comprises: NX0101 (ALV-J, DQ115805), MQNCSU (ALV-A, DQ365814), SDAU09E3 (ALV-BJF826241), RSV-Prague (ALV-C, J02342), RSV-Schmidt-RuppinD (ALV-D, D10652), SD0501 (ALV-E, EM467236), HPRS103 (ALV-J, Z46390) andADOL-7501 (ALV-J, AY027920).
Design and synthesis is containing the hairpin structure of above-mentioned purpose fragment, as topstrandDNA in Fig. 3, its gene order is as shown in sequence table SEQ IDNO:2, bottomstrandDNA corresponding with it, its gene order, as shown in sequence table SEQ IDNO:3, is annealed and is connected in pcDNA6.2-GW/EmGFP-miR interference carrier after forming double-strand, connects product conversion to competent cell DH5 α, after being coated with LB flat board (containing 50 μ g/ml spectinomycins), hatch for 37 DEG C;
Transformation plate is picking 3 clone respectively, checks order after shaking bacterium extracting plasmid, to verify that in recombinant clone, whether Insert Fragment sequence is consistent with oligomerization single stranded DNA (namely topstrandDNA and the bottomstrandDNA corresponding with it) sequence of design;
Find that the present invention does acquisition recombinant vectors by the checking of ordinary method, 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 that this restructuring interference carrier can J subgroup avian leucosis virus Transcription and replication in interference in vitro cell and live chickens body effectively, general jamming effectiveness reaches more than 80%, have great progress than existing gimmick of preventing and treating, wherein adopted external detection method is as follows:
Cultivate DF-1 cell, after 12h, connect poison (ALV-JNX0101 strain), treat that cell state is good, when Cell abundance reaches 70%-80%, DF-1 cell is passed on 24 orifice plates;
(about 8h-12h when Cell abundance reaches 70%,), carry out the transfection of recombinant plasmid, detect the expression level of virogene by Real-timePCR after 72h, the expression level of indirect immunofluorescene assay ALV-J live virus envelope protein, Westernblot detect the expression level of viral envelope proteins after Transfected Recombinant Plasmid DF-1 cell to verify interference effect, protein expression result shows, after interference, 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 viral Simultaneous vaccination instar chicken embryo on the 6th, establish ALV-J to infect positive controls and negative control group simultaneously.
After chick goes out shell, raise to 7 week age, all cut open and kill, period carries out weekly hematology, pathological study and virusology and detects antiviral effect;
The growth of result display recombinant plasmid interference group chicken is normal, and all kinds of white corpuscle of blood has no considerable change, and ALV-J antigen/antibody is negative, and without toxin expelling phenomenon, histopathology detects and has no tissue injury and inflammatory reaction.And serious viremia appears in ALV-J positive controls, toxin expelling, there is significantly damage and inflammation in the vitals such as liver and heart.Experiment proves that RNA disturbs recombinant vectors can reach significant antiviral effect.
In sum, the present invention utilizes LTR gene this situation very conservative, this gene design is utilized to synthesize siRNA interference carrier, and by this interference carrier and viral cotransfection cell, final discovery this interference carrier provided by the invention can J subgroup avian leucosis virus Transcription and replication in interference in vitro cell and live chickens body effectively, control for J subgroup avian leucosis provides scientific basic and technical support, reduces aviculture and infects the financial loss caused because of ALV-J.
Accompanying drawing explanation
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;
The schematic diagram of topstrandDNA and bottomstrandDNA of Fig. 3 designed by the present invention;
Shown in italic font in figure in visible topstrand, sequence is identical with section sequence of on target gene, the other end italic font sequence and its complementation, identical with its mentality of designing in bottomstrand;
Fig. 4 is carrier construction plasmid electrophoresis result schematic diagram of the present invention,
Be wherein DNAmarker on the left of electrophorogram, right side is the band of plasmid;
The spirane structure existed due to plasmid is different, can form many band during electrophoresis, is followed successively by the OC configuration of lax open loop, lax linear L configuration (5700bp) and supercoiled SC configuration from top to bottom;
Fig. 5 is the interference effect figure of gained interference carrier FLuorescent of the present invention,
In figure, p-si-LTR is interference group, Null-vectorcontrol is empty vector control group, ALV-Jinfectedcontrol is that virus infection does not disturb control group, normalcellscontrol to be that normal cell does not connect poison and not disturb in contrast picture group interference group compared with positive controls, fluorocyte number greatly reduces, and proves that this is tested interference plasmid used and has 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
The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art, the reagent adopted also is available reagent;
The preparation of embodiment 1 double-stranded DNA
Target LTR gene conserved regions, initiation site is 7497, design and synthesis siRNA, and its base sequence is
AGGCAACACGGGTCTTATA, its gene order is as shown in sequence table SEQ IDNO:1;
Design and synthesis contains the hairpin structure of above-mentioned purpose fragment, wherein topstrandDNA, and its gene order is as shown in sequence table SEQ IDNO:2; Its gene order of bottomstrandDNA corresponding is with it as shown in sequence table SEQ IDNO:3; Use ddH
2o is dissolved into 100 μMs, and complementary strand is respectively got 5-10 μ l and mixed between two, provides system anneal by table one.By mixture 95 DEG C of heating 5 ~ 10 minutes, then place room temperature 20 minutes, form double-stranded DNA.
Table one, oligoDNA annealing system
| 100μM top strand oligo | 5μl |
| 100μM bottom strand oligo | 5μl |
| 10×oligo annealing buffer | 2μl |
| ddH 2O | 8μl |
| Total volume | 20μl |
Embodiment 2 is recombinated the structure of interference carrier:
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 and connect product conversion 100 μ l competent cell DH5 α, after being coated with LB flat board (containing 50 μ g/ml spectinomycins), hatch for 37 DEG C.
Transformation plate is picking 3 clone respectively, checks order after shaking bacterium extracting plasmid, with to verify in recombinant clone Insert Fragment sequence whether with the oligomerization single stranded DNA designed, namely topstrandDNA with bottomstrandDNA sequence is consistent;
The order-checking of order-checking company is delivered by the recombinant vectors obtained by clone, 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-JNX0101 strain), treat that cell state is good, when Cell abundance reaches 70%-80%, DF-1 cell is passed on 24 orifice plates;
When Cell abundance reaches 70% (about 8h-12h), carry out the transfection of recombinant plasmid, detailed process is: prepare RNA interference transfection composite with the DMEM without blood nonreactive, specific as follows: transfection reagent Lipofectamine
tM2000(μ l) prepare RNA according to the ratio of 2.5:0.5 disturb transfection composite with interference plasmid (μ g), plasmid final concentration is made to be 1 μ g/ μ l by the DMEM consumption adjusted without blood nonreactive, cell growth medium is changed to without blood nonreactive DMEM, add the transfection composite that above-mentioned plasmid final concentration is 1 μ g/ μ l afterwards, after 8h, be replaced by maintain base;
Detect the expression level of virogene by Real-timePCR after 72h, the expression level of indirect immunofluorescene assay ALV-J live virus envelope protein, Westernblot detect the expression level of viral envelope proteins after Transfected Recombinant Plasmid DF-1 cell to verify interference effect, protein expression result shows, after interference, 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 detects:
Restructuring interference plasmid and viral Simultaneous vaccination instar chicken embryo on the 6th, establish ALV-J to infect positive controls and negative control group simultaneously.
After chick goes out shell, raise to 7 week age, all cut open and kill, period carries out weekly hematology, pathological study and virusology and detects antiviral effect;
The growth of result display restructuring material interference group chicken is normal, and all kinds of white corpuscle of blood has no considerable change, and ALV-J antigen/antibody is negative, and without toxin expelling phenomenon, histopathology detects and has no tissue injury and inflammatory reaction.And serious viremia appears in ALV-J positive controls, toxin expelling, there is significantly damage and inflammation in the vitals such as liver and heart.Experiment proves that RNA disturbs recombinant vectors can reach significant antiviral effect.
Claims (2)
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 the siRNA designed by utilization, build its hairpin structure, to be connected to by the double-stranded DNA of formation after annealing and to obtain in pcDNA6.2-GW/EmGFP-miR interference carrier, wherein its gene order of siRNA is as shown in sequence table SEQ IDNO:1.
2. the preparation method of restructuring interference carrier according to claim 1, is characterized in that: concrete steps are as follows:
(1) based on LTR gene conserved regions design and synthesis siRNA; Its concrete operations are: carry out homology analysis to the different subgroup of ALV and the different strain of J subgroup, compared with the sequence fragment of conservative region fragment as screening siRNA in the LTR gene that its homology is the highest, utilize siRNA Photographing On-line software, obtain siRNA, base sequence is AGGCAACACGGGTCTTATA, and its gene order is as shown in sequence table SEQ IDNO:1;
(2) design and synthesis is containing the hairpin structure of above-mentioned purpose fragment, wherein topstrandDNA, its gene order is as shown in sequence table SEQ IDNO:2, bottomstrandDNA, its gene order, as shown in sequence table SEQ IDNO:3, is annealed and is connected in pcDNA6.2-GW/EmGFP-miR interference carrier after forming double-strand and get final product.
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| Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs;Qing-Wen Meng，et al;《Virology Journal》;20111222;第8卷(第556期);1－9 * |
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