CN109722448A - Target the construction method of wls gene RNA interference recombinant lentivirus vector - Google Patents
Target the construction method of wls gene RNA interference recombinant lentivirus vector Download PDFInfo
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- 238000010276 construction Methods 0.000 title claims description 18
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Abstract
The present invention relates to the buildings of targeting wls gene RNA interference recombinant lentivirus vector, screening and application thereof.The present invention utilizes RNA perturbation technique, for wls gene, the slow virus carrier for expression of eukaryon that shRNA can be expressed in relevant cell is constructed, interference slow virus is obtained by V#wls-shRNA transfer vector plasmid, virus packaging helper plasmid (Helper 1.0, Helper 2.0) three plasmid co-transfection 293T cell culture.The present invention, which targets wls gene RNA interference recombinant lentivirus vector, can efficiently, specifically inhibit cell wls gene expression, can provide a kind of effective tool to the regulating and controlling effect of the differentiation of relevant cell and function, the growth of respective organization and development etc. for research wls.Invention further discloses application of the targeting wls gene RNA interference recombinant lentivirus vector in research bone growth growth course in terms of wnt ligand origins, and to realize that application of the wnt signal path in terms of bone tissue regeneration provides certain theoretical basis.
Description
Technical field
The present invention relates to a kind of construction methods and biologic applications for targeting wls gene RNA interference recombinant lentivirus vector, belong to
In field of biotechnology.
Background technique
The research of the effect of vital wnt secretory protein is concentrated mainly on certain wnt egg in research wnt access at present
On white or b-catenin molecule, however have now been found that the express spectra of 19 kinds of wnt ligandins in mammal is not quite similar,
The function that its position plays also is not quite similar and redundancy is compensatory.Wnt access can pass through the hair such as classical and non-classical access
The effect of waving can not block wnt access to technologies such as the targeting knockouts of b-catenin completely.However targeting knockout wls gene
Advantage is: 1. knockout wls can block the secretion of nearly all wnt albumen, can avoid the functional compensation of numerous wnt albumen.
2. classical and non-classical wnt signal path overall function can be studied by knocking out technology using wls.3. knocking out wls technology can
To study the autocrine and paracrine action of wnt albumen.
Summary of the invention
For the defects in the prior art, the object of the present invention is to provide a kind of targeting wls gene RNA interference recombinant lentiviral diseases
The construction method of poisonous carrier；And it is further disclosed in the research specific source of bone tissue wnt ligand, and its to bone development, bone mine
Change, the application of bone metabolism regulation aspect.
The present invention is achieved by the following technical solutions:
The present invention provides a kind of construction methods for targeting wls gene RNA interference recombinant lentivirus vector comprising as follows
Step:
V#wls-shRNA transfer vector plasmid, virus packaging helper plasmid Helper 1.0 and virus packaging are constructed respectively
Helper plasmid Helper 2.0；
The V#wls-shRNA transfer vector plasmid, virus packaging helper plasmid Helper 1.0 and virus packaging is auxiliary
It helps plasmid Helper 2.0 to transfect jointly to 293T cell, obtains the targeting wls gene RNA interference recombinant lentivirus and carry
Body.
Preferably, the construction method of the building V#wls-shRNA transfer vector plasmid is in V#shRNA carrier
Multiple cloning sites in connect double chain DNA fragment, element orders U6-MCS-Ubiquitin-Cherry-IRES-
Puromycin, restriction enzyme site are cloning site: Age I, EcoR I.
Preferably, the double chain DNA fragment is following sequence:
Wls-RNAi-V oligonucleotide sequence:
The sequence of positive-sense strand is as shown in SEQ ID NO.1:
CCGGTCCAAGGGAAATTGAAGCAAACTCGAGTTTGCTTCAATTTCCCTTGGATTTT TG,
The sequence of positive-sense strand is as shown in SEQ ID NO.2:
GATCCAAAAATCCAAGGGAAATTGAAGCAAACTCGAGTTTGCTTCAATTTCCCTTGGA；
Preferably, the construction method of the virus packaging helper plasmid Helper 1.0 are as follows: Helper 1.0 is carried
Constitution grain overall length 10703bp, the gag gene containing inhibition of HIV, the main structural proteins of coding virus；Pol gene, coding disease
The enzyme of malicious specificity；Rev gene, coding adjust the regulatory factor of gag and pol gene expression.Cag promoter:79-
1670.Gag:1780-3282.Pol:3348-6086.Tat:6180-6398.RRE:6660- 6893.Rev:7272-
7544.beta-globin polyA:7724-8164.pUC ori:8920-9563.Ampicillin:10572-9712.
Preferably, the construction method of the virus packaging helper plasmid Helper 2.0 are as follows: Helper 2.0 is carried
Constitution grain overall length 6503bp, the VSV-G gene containing herpes simplex virus source provide coating egg required for virus is packed
It is white.CMV promoter:114-701. β-globin intron:812-1386.VSV-G:1472-3007.beta-globin
PolyA:3151-3673.Ampicillin:5727-4867.
Preferably, the operating method of the transfection are as follows: after inoculating cell, according to the suitable MOI value of preliminary experiment=
50 carry out purpose virus transfections, and Nostoc commune Vanch liquid is gained after 8-12 hours and continues to cultivate, and are taken pictures sight after 48h using fluorescence microscope
Transfection efficiency is examined, extracts RNA and Protein Detection target gene wls jamming effectiveness after 72h.
Preferably, a kind of targeting wls gene RNA interference recombinant lentivirus vector is in research bone growth development
Wls gene expression in osteocyte system MLO-y4 is interfered in application in terms of wnt ligand origins in the process, targeting, interference wnt ligand
Secretion, row alkaline phosphatase staining and Alizarin red staining detect its mineralization ability respectively after mineralising induces 4 days, 7 days.
Compared with prior art, the present invention have it is following the utility model has the advantages that
Present invention targeting wls gene RNA interference recombinant lentivirus vector can be more efficient and specifically inhibits wls gene table
It reaches, can be the specific source of wnt ligand of research linked groups, wls influence active on wnt signal path, wls and correlation wnt
Regulation of the signal path to cell behaviors and developing tissue growth, wnt signal path answering in terms of regeneration
With etc., a kind of effective tool is provided.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is V#wls-shRNA transfer vector plasmid figure；
Fig. 2 is virus packaging 1.0 figure of helper plasmid Helper；
Fig. 3 is virus packaging 2.0 figure of helper plasmid Helper；
Common light microscopic and fluorescence microscope take pictures and show transfection efficiency figure after Fig. 4 is transfection 48h；
Fig. 5 is q-PCR verifying purpose gene interference effect figure；
Fig. 6 is Westernblot verifying purpose gene jamming effectiveness figure.
Fig. 7 is cell wnt signal path activity reduction figure after transfection purpose virus.
Fig. 8 is osteocyte mineralization ability reduction figure after transfection purpose virus.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1
The present embodiment is related to targeting the specific experiment behaviour of the construction and screening of wls gene RNA interference recombinant lentivirus vector
Make.
1, the preparation of targeting wls gene RNA interference recombinant lentivirus clone
1,1 digests tool carrier using restriction enzyme.
1,2 multiple RNAi target sequences are designed according to RNAi sequence design principle for wls gene order,
Synthesizing single-stranded primer, annealing forms double-stranded DNA, and is attached rear transformed competence colibacillus cell with carrier, through bacterium colony
It is expanded culture after PCR identification, plasmid extraction.
2, targeting wls gene RNA interference recombinant lentivirus packaging and detection
2,1 it will carry the tool carrier plasmid of target gene and assist packaging plasmid helper1.0, helper2.0 corotation
293T cell is contaminated, harvest purifying is carried out after 48h and obtains carrying out later period transfection experiment after high titre saves liquid completion quality testing.
Embodiment 2
The present embodiment is related to a kind of targeting wls gene RNA interference experiment of specific bone and its cells MLO-Y4 cell line
1. inoculating cell complete medium diluting cells MLO-Y4 to 5x 104~8x 104A/ml, is separately added into 4ml
The corresponding cell of cell suspension inoculation is counted in the culture dish of two 6cm diameters.
2. each capsule of virus infection is changed to containing 5 μ g/ml Polybrene 2ml complete culture solution of final concentration, according to
Preliminary result (MOI=40-60 is suitable), or be calculated by formula: viral volume=(MOI x cell number)/virus titer
Suitable virus is added in i.e. every capsule.12h changes the liquid time after infecting according to preliminary experiment, is changed to conventional medium, continues to train
It supports.Appropriate selection screening technique: Nostoc commune Vanch liquid culture is changed into after the puromycin medicine sieve 48h of 2ug/ml is added after transfection 72h.
3. observing efficiency of infection
It takes pictures after transfected virus 48h, as shown in figure 4, control virus reaches 70%-90% with purpose virus transfection efficiency
Quantitative fluorescent PCR verifying is distinguished as shown in Figure 5 and Figure 6 with Westernblot verification result after transfected virus 72h:
Fig. 5 showed transfection control virus and purpose virus after 72 hours, and reverse transcription obtains cDNA after carrying out the extraction of total serum IgE,
After verifying to obtain transfection purpose virus by quantitative fluorescent PCR again, the expression of target gene reduces by 69.14 compared to control group ±
3.03%.
Fig. 6 shows that transfection control virus and purpose virus after 72 hours, carry out electricity after carrying out the extraction and determination concentration of total protein
It swims transferring film, immune response colour developing, after verifying obtains transfection purpose virus, the expression of target gene is reduced compared to control group
75.36 ± 4.20%.
4. comparing cell wnt signal path activity after transfection purpose virus.
After 72 hours, reverse transcription obtains cDNA after carrying out the extraction of total serum IgE, then passes through for transfection control virus and purpose virus
Quantitative fluorescent PCR is verified after obtaining transfection purpose virus, and such as Fig. 7, the transcription factor of wnt access and the gene expression of target gene are living
Property is substantially reduced (* * p < 0.01, * p < 0.05).
4. comparing the change of mineralization ability after transfection purpose virus
Row alkaline phosphatase staining and Alizarin red staining detect its mineralization ability respectively after mineralising induces 4 days, 7 days.
Fig. 8 shows that row alkaline phosphatase contaminates respectively after progress mineralising induction 4 days, 7 days after transfection control virus and purpose virus
Color and Alizarin red staining result.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (7)
1. a kind of construction method for targeting wls gene RNA interference recombinant lentivirus vector, which comprises the steps of:
V#wls-shRNA transfer vector plasmid, virus packaging helper plasmid Helper 1.0 and virus packaging auxiliary are constructed respectively
Plasmid Helper 2.0；
By the V#wls-shRNA transfer vector plasmid, virus packaging helper plasmid Helper 1.0 and virus packaging auxiliary matter
Grain Helper 2.0 jointly transfects 293T cell, obtains the targeting wls gene RNA interference recombinant lentivirus vector.
2. the construction method of targeting wls gene RNA interference recombinant lentivirus vector as described in claim 1, which is characterized in that
The construction method of the building V#wls-shRNA transfer vector plasmid is that connection is double in the multiple cloning sites of V#shRNA carrier
Chain DNA segment, element orders U6-MCS-Ubiquitin-Cherry-IRES-puromycin, restriction enzyme site are clone position
Point: Age I, EcoR I.
3. the construction method of targeting wls gene RNA interference recombinant lentivirus vector as claimed in claim 2, which is characterized in that
The double chain DNA fragment is following sequence:
Wls-RNAi oligonucleotide sequence:
The sequence of positive-sense strand is as shown in SEQ ID NO.1:
CCGGTCCAAGGGAAATTGAAGCAAACTCGAGTTTGCTTCAATTTCCCTTGGATTTT TG,
The sequence of positive-sense strand is as shown in SEQ ID NO.2:
GATCCAAAAATCCAAGGGAAATTGAAGCAAACTCGAGTTTGCTTCAATTTCCCTTGGA。
4. the construction method of targeting wls gene RNA interference recombinant lentivirus vector as described in claim 1, which is characterized in that
The construction method of the virus packaging helper plasmid Helper 1.0 are as follows: 1.0 vector plasmid overall length 10703bp of Helper contains
The gag gene of inhibition of HIV, the main structural proteins of coding virus；Pol gene encodes the enzyme of virus-specific；Rev gene is compiled
Code adjusts the regulatory factor of gag and pol gene expression.
5. the construction method of targeting wls gene RNA interference recombinant lentivirus vector as described in claim 1, which is characterized in that
The construction method of the virus packaging helper plasmid Helper 2.0 are as follows: 2.0 vector plasmid overall length 6503bp of Helper contains
The VSV-G gene in herpes simplex virus source provides envelope protein required for virus is packed.
6. the construction method of targeting wls gene RNA interference recombinant lentivirus vector as described in claim 1, which is characterized in that
The operating method of the transfection are as follows: after inoculating cell, carry out purpose virus transfection, 8- according to the suitable MOI value of preliminary experiment=50
Nostoc commune Vanch liquid is gained after 12 hours to continue to cultivate, is taken pictures after 48h using fluorescence microscope and observes transfection efficiency, is extracted after 72h
RNA and Protein Detection target gene wls jamming effectiveness.
7. a kind of targeting wls gene RNA interference recombinant lentivirus vector as described in claim 1 is in research bone growth hair
Application during educating in terms of wnt ligand origins, which comprises the steps of: targeting interference osteocyte system MLO-y4
Middle wls gene expression, row alkaline phosphatase staining and alizarin red respectively after interfering the secretion of wnt ligand, mineralising to induce 4 days, 7 days
Dyeing detects its mineralization ability.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033430A1 (en) * | 2007-02-09 | 2011-02-10 | The General Hospital Corporation | Methods for the induction of a cell to enter the islet 1+ lineage and a method for the expansion thereof |
| WO2013106934A1 (en) * | 2012-01-17 | 2013-07-25 | Université de Montréal | Novel regulators of innate immunity and uses thereof |
| CN104096233A (en) * | 2013-04-03 | 2014-10-15 | 中国科学院上海生命科学研究院 | Prevention and treatment of bone diseases caused by glucocorticoid medicines |
| CN107073117A (en) * | 2014-08-26 | 2017-08-18 | 埃塔根有限公司 | For recognizing the antibody of the specific motif of WLS albumen and pharmaceutical composition containing it |
-
2017
- 2017-10-27 CN CN201711027633.1A patent/CN109722448A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033430A1 (en) * | 2007-02-09 | 2011-02-10 | The General Hospital Corporation | Methods for the induction of a cell to enter the islet 1+ lineage and a method for the expansion thereof |
| WO2013106934A1 (en) * | 2012-01-17 | 2013-07-25 | Université de Montréal | Novel regulators of innate immunity and uses thereof |
| CN104096233A (en) * | 2013-04-03 | 2014-10-15 | 中国科学院上海生命科学研究院 | Prevention and treatment of bone diseases caused by glucocorticoid medicines |
| CN107073117A (en) * | 2014-08-26 | 2017-08-18 | 埃塔根有限公司 | For recognizing the antibody of the specific motif of WLS albumen and pharmaceutical composition containing it |
Non-Patent Citations (1)
| Title |
|---|
| 杜佳慧等: "骨细胞来源的wnt分泌蛋白在骨生长发育中的作用", 《2017全国口腔生物医学学术年会论文汇编》 * |
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