CN101993894A - Construction method of siRNA adenovirus carrier for restraining rat IL-1beta gene expression - Google Patents

Abstract

The invention relates to a siRNA and discloses a siRNA expression carrier for restraining rat IL-1beta gene expression. The construction method designs and synthesizes three pairs of rat IL-1beta gene targeted siRNA sequences and successfully constructs a recombinant adenovirus carrier for expressing the siRNA. The carrier can continuously, effectively and specifically silence the expression of IL-1beta gene and can be used for researching IL-1beta function and treating neurodegenerative diseases.

Description

The siRNA construction of recombinant adenovirus containing method that suppresses rat IL-1 beta gene expression

Technical field

The present invention relates to a kind of siRNA expression vector, more particularly, relate to a kind of construction process that suppresses the siRNA recombinant adenoviral vector of rat IL-1 beta gene expression, belong to gene engineering technology field.

Background technology

Interleukin-(Interleukine), activates and the adjusting immunocyte in the information transmission as the cytokine of immunocyte interphase interaction, plays important effect in mediation T, B cell activation, propagation and differentiation and the inflammatory reaction.Wherein, interleukin 1 (IL-1) is as a kind of important pro-inflammatory cytokine, and its biological action is very extensive.In recent years, studies have shown that more and more interleukin 1 is the maincenter regulon in a kind of stress reaction, neuroendocrine and and the behavior stress reaction aspect bringing into play important effect.Interleukin 1 is that first is proved to be and neuroendocrine system (hypothalmus-pituitary-adrenal axis, HPA) regulate relevant cytokine, now tentatively disclose interleukin 1 and brought into play important effect aspect the activity of stress-induced HPA maincenter, action process and the neuron plasticity, under pathological conditions, interleukin 1 gene expression amount and protein output all can raise, and interleukin 1 is responsible for the inflammatory reaction that the panimmunity stimulation causes in this course.Therefore, interleukin 1 is not only the maincenter medium of interior nerve immunity adjusting of body and inflammatory reaction, and plays an important role in the pathomechanism of many nerve degenerative diseases.

Nerve degenerative diseases (Neurodegenerative disease) is the morbid state of the cellular neural unit forfeiture of a class brain and spinal cord.Along with the quickening of aging population process, the sickness rate of nerve degenerative diseases constantly rises in recent years, becomes the social concern that the whole world faces jointly.State's troubles Parkinson's disease total number of persons has reached 1,720,000 people, crowd's Parkinson's disease morbidity nearly 1% more than 55 years old at present; And the number U.S. in 2000 of suffering from alzheimer's disease has just reached 4,500,000 people, if methods of treatment does not obtain effective progress, the prediction U.S. is when the year two thousand fifty, and Symptomatic case load will reach 13,200,000 people.The pathogeny and the cause of disease of this class disease are intricate, and its pathomechanism is still not fully aware of so far, still lacks effective treatment means in the clinical position.But in clinical, find to find that high-caliber short inflammatory factor is arranged in Kieren Perkins patient's cerebrospinal fluid and the striatum,, in A Zihaimo patient's cerebrospinal fluid, serum, also found high-caliber interleukin 1 β as interleukin 1 β.Interleukin 1 β with high density in experimentation on animals is expelled in the black substance of rat, rat has shown the corresponding symptom of Pa Jinsishi disease after for some time, the morbidity of above-mentioned data prompting interleukin 1 β and nerve degenerative diseases has certain dependency, and the maincenter activity that suppresses pro-inflammatory cytokine is expected to become the potential target spot of treatment nerve degenerative diseases.And the RNA perturbation technique that development in recent years is got up provides very useful instrument for this reason.

RNA disturbs (RNAi) but is meant that specificity in vivo suppresses the expression of goal gene, and this phenomenon occurs in post-transcriptional level, therefore be called again PTGS (post-transcriptional gene silencing, PTGS).RNAi is a kind of conservative defense mechanism that has from the unicellular lower eukaryote to the Mammals.Endogenous or exogenous double-stranded RNA (dsRNA) are cut into a plurality of small fragment RNAs (21-23bp) with length-specific and structure by endonuclease Dicer in cell be siRNA, the reticent mixture of some specific enzymes formation RNA inductive in siRNA and the body (RNA Interference SilencingComplex, RISC).The RISC mixture is by pairing identification and cut purpose mRNA, thereby makes the goal gene silence, i.e. the RNA interference phenomenon.RNAi is by closing the expression of specific gene, and important use is worth demonstrating very aspect the treatment of expression of gene regulation and control, malignant tumour and heredopathia.

Obtain siRNA at present mainly by following two kinds of methods: chemical synthesis and siRNA expression vector.But because the synthetic cost height of RNA, and easily degraded by RNase, complicated operation makes this technology have certain limitation.S iRNA expression vector is low because of cost, and the long-acting rate of acting duration is high and be widely adopted.It is good that recombinant adenoviral vector has a biological safety, expresses longer duration, and host range is wide, but can not only the transfection proliferative cell the also non-proliferative cell of transfection, pathogenic advantage such as low becomes one of s iRNA expression vector of everybody widespread use.We utilize adenovirus AdEasy system, structure can suppress the s iRNA of IL-1 beta gene expression, thereby inquire into the effect of IL-1 β in nerve degenerative diseases, this will help to illustrate the mechanism of causing a disease of IL-1 β nervous system disorders from molecular level, and provide useful means for nerve degenerative diseases treatment.

Summary of the invention

Purpose one of the present invention is to provide a kind of siRNA that suppresses rat IL-1 beta gene expression.

Purpose two of the present invention is to provide the expression vector of above-mentioned siRNA.

Above-mentioned purpose of the present invention can realize by following measure.

Design suppresses the siRNA fragment of rat IL-1 beta gene expression: according to the target sequence of the online searching IL-1 β-siRNA of siRNA design aids in the Ambion company's site (http://www.ambion.com), use BLAST (http://www.ncbi.nlm.nih.gov/biast/blast.cgi) that the target sequence of selecting is carried out homology analysis subsequently, get rid of the possibility that siRNA suppresses other gene fragments non-specificly.

The insertion sequence structure of design IL-1 β-siRNA expression vector: according to adenovirus AdEasy system, the synthetic principle of insertion sequence template is: restriction enzyme site+siRNA sequence+ring texture+terminator sequence+restriction enzyme site, easy to detect for the subsequent recombination shuttle plasmid, when designing and synthesizing the restriction enzyme site at insertion sequence template two ends, only keep four bases, when being connected to insertion sequence on the shuttle plasmid, this restriction enzyme site because base of disappearance so destroyedly fall.(TTTTTT is to guarantee to transcribe accurate termination polyT), and at new Stu I restriction enzyme site of tip designs of synthetic DNA to add transcription termination sequence.

The generation of adenovirus carrier: according to the AdEasy system of foundation such as He in 1998, shuttle plasmid pAdTrack-CMV contains scavenger cell viral promotors (CMV) and green fluorescent protein (GFP), CMV belongs to rna plymerase ii type promotor, there are some researches show that recently rna plymerase ii also can high level expression siRNA, and CMV is stronger than other RNA polymerase 11 promotors (as SV40, RSV etc.) startabilities, plasmid changes cell over to, transcribes under promotor is handled and generates siRNA.And the green fluorescence protein gene that carries can help us to detect the location and the spread condition of adenovirus by fluorescence.After synthetic siRNA sequence, reorganization is connected on the shuttle plasmid, the homologous recombination in bacterium with linearizing recombinant shuttle plasmid and skeleton plasmid again, the gained recombinant adenovirus plasmid can filter out by kantlex, subsequently, recombinant adenovirus plasmid rotaring redyeing 293 cell packing after the PacI enzyme is cut produces recombinant adenovirus, is further used for the research of IL-1 β gene function and the treatment of related neural degenerative disease.

Description of drawings

Fig. 1 is a siRNA hairpin structure synoptic diagram;

Fig. 2 adenovirus vector construct synoptic diagram;

Fig. 3 oligonucleotide fragment annealing gel electrophoresis figure;

Fig. 4 recombinant shuttle plasmid enzyme is cut evaluation figure;

The order-checking collection of illustrative plates of Fig. 5 recombinant shuttle plasmid;

The enzyme of Fig. 6 recombinant adenovirus plasmid is cut evaluation figure;

GFP expresses behind Fig. 7 Adenovirus Transfection HEK293 cell 24h;

Embodiment

Describe the present invention below in conjunction with specific embodiment, should be noted that, these embodiment only are used to illustrate that the present invention should not be construed as limitation of the present invention.

The design of embodiment 1 rat IL-1 β gene siRNA, synthetic and annealing

According to three siRNA of the online design efficient targeting IL-1 of siRNA design aids (http://www.ambion.com) β gene, the siRNA of a control group.The selection principle of siRNA is as follows:

1. with the long sequence of the 19--21nt of AA beginning;

2. the target sequencing is listed as 75--100 nt beginning from the initiator codon downstream, finishes to 75--100nt place, terminator codon upstream;

3. GC content is preferably in about 50% in the sequence of Xuan Zeing; Avoid occurring 3 above G in the sequence;

4. the dna fragmentation of Xuan Zeing is inquired about through BLAST, should not have homology with other Human genome;

The insertion sequence structural order that is cloned in the expression vector is followed successively by: 5 ' end HindIII restriction enzyme site, just sequence, the Loop ring texture, antisense sequences, transcription terminator, 3 ' hold Sal I restriction enzyme site (Fig. 1).Sequence is synthetic by the handsome biotech firm in Shanghai, as following table.

The synthetic oligonucleotide fragment is dissolved with sterilized water, carry out annealing reaction according to following system: positive-sense strand 2ul, antisense strand 2ul, 95 ℃ of 2min behind the abundant mixing of annealing buffer 16ul naturally cool to 37 ℃ of 1h of room temperature afterwards.Product is stored in 4 ℃ at last.Get 2ul annealing product and carry out native polyacrylamide gel electrophoresis, observe annealing result (Fig. 3).The result just in time is the long fragment of needed 63bp after showing annealing.

The structure of embodiment 2 recombinant shuttle plasmid pAdTrack-CMV-siIL-1 β

After HindIII and Sal I enzyme were cut, agarose reclaimed the big fragment of DNA with the AdTrack-CMV carrier.Be connected transformed competence colibacillus bacterium DH5 α with fragment annealing, select the mono-clonal bacterium colony, the plasmid extraction is carried out enzyme and is cut evaluation, sends to order-checking (Fig. 4,5) at last.After 1 and 2 bands are represented to cut with HindIII and SalI enzyme respectively among Fig. 4, plasmid does not have linearized, show that this plasmid is a positive colony, because we are when the design insertion sequence, removed a base in HindIII and the Sal I recognition sequence respectively, when insertion sequence correct be connected on the shuttle plasmid time, so positive colony is going enzyme to cut can not be with plasmid linearization with this enzyme.Newly added the StuI restriction enzyme site that shuttle plasmid itself does not have in the insertion sequence, a linearizing band (No. 3 bands among Fig. 4) has just appearred in positive plasmid after cutting with this enzyme enzyme.

The structure of the Ad-siIL-1 β of embodiment 3 recombinant adenovirus plasmids

With PmeI restriction endonuclease linearizing reorganization pAdTrack-CMV-s i IL-1 β plasmid, change over to behind the purifying among the competence BJ5183 that contains pAdEasy-1, carry out homologous recombination, select little bacterium colony, shake bacterium extracting plasmid, cut evaluation with the PacI enzyme, the big fragment of a 30kb and the fragment (Fig. 6) of a treaty 3-4kb can appear in positive colony.

Embodiment 4 packing recombinant adenovirus

With the PacI linearizing of Ad-siIL-1 β plasmid, ethanol sedimentation, washing adds dH20 and fully dissolves, use the LIP2000 transfection reagent, transfection HEK293 cell is observed the expression (Fig. 7) of green fluorescent protein behind the 24h to specifications, and observation of cell pathological change and collecting cell are to centrifuge tube behind the 7d, behind the multigelation 3 times, the centrifugal supernatant liquor that obtains infects HEK293 with supernatant liquor, reclaims cell when tangible cytopathy occurring, multigelation centrifuging and taking supernatant ,-80 ℃ of preservations are standby.

Claims (6)

1. recombinant adenoviral vector is characterized in that: s iRNA that can expression inhibiting rat IL-1 beta gene expression.

2. carrier that can produce the siRNA that suppresses rat IL-1 beta gene expression is characterized in that having following basic structure:

The circular double stranded DNA molecule that is linked in sequence and forms by promotor, IL-1 β coding region, termination codon, dna sequence dna;

Promotor is for starting the sequence that produces s iRNA, and it is the element that starts its downstream next-door neighbour's expression of gene;

IL-1 β coding region is made up of 19 Nucleotide for expressing the coding region of the described siRNA of claim 1, can adopt the principle of base complementrity to form double-stranded RNA in cell;

Termination codon is 6 successive T;

Dna sequence dna is the dna sequence dna between between the RNA coding region, contains replication origin and the antibiotics resistance gene of plasmid vector DNA.

3. expression vector according to claim 1 and 2 is characterized in that: the described s iRNA gene that is inserted into has two sticky ends difference complementary sticky ends with the siRNA expression vector.

4. recombinant adenoviral vector according to claim 1, its feature also is: adenovirus carrier is the replication-defective virus of E1 district disappearance.

5. recombinant adenoviral vector according to claim 1, its feature also is: packing cell is HEK293.

6. the construction process of recombinant adenoviral vector according to claim 1, it is characterized in that: design and synthesize special interference siRNA at rat IL-1 β, annealing forms among the double-stranded oligonucleotide fragment insertion shuttle plasmid pAdTrack-CMV and has obtained the pAdTrack-CMV-shRNA recombinant shuttle plasmid, and carries out sequencing analysis; Zone of transformation has the competent cell BJ5183 of viral skeleton plasmid pAdeasy-1 behind the PmeI enzyme linearizing recombinant plasmid pAdTrack-CMV-siIL-1 β, obtain virus particle pAd-siIL-1 β, change the HEK293 cell after the linearizing and pack acquisition recombinant adenovirus Ad-siIL-1 β particle.

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