CN103695427B - Small interfering RNA (Ribonucleic Acid) and recombinant vector for knocking down VPS11 (Vacuolar Protein Sorting-Associated Protein 11), and application of small interfering RNA and recombinant vector - Google Patents

Abstract

The invention provides a small interfering RNA for knocking down VPS11, shRNA, encoding DNA of shRNA, recombinant vector, recombinant lentivirus and host cell and application thereof, the small interfering RNA has the function of syntaxin and translation The messenger RNA complementary nucleotide sequence of VPS11 has a length of 19-27bp. The recombinant lentiviral vector provided by the present invention can stably and long-term transcribe VPS11-shRNA in the host cell; the VPS11-shRNA provided by the present invention can generate small molecule interfering RNA capable of silencing VPS11 expression after being processed by the host cell; the present invention provides The small molecule interfering RNA can target the mRNA of the syntaxin VPS11, and cause the degradation of the mRNA, and lead to gene silencing effect, and reduce the expression level of VPS11.

Description

Small molecule interfering RNA, recombinant vector and application thereof for knocking down VPS11

technical field

The invention relates to the fields of molecular biology, genetic engineering technology and biomedicine, in particular to a small molecule interfering RNA for knocking down VPS11, a recombinant vector and an application thereof.

Background technique

VPS11 protein plays an extremely important role in the process of cell vesicle transport, and cell vesicle transport is one of the main pathways for intracellular and intercellular material transport, so the study of the signaling pathway of VPS11 protein in cell vesicle transport is of great importance. It is of great significance to study the life activities of various cells, tissues, organs and organisms. To study the role of VPS11 in the signaling pathway, it is necessary to provide a method for knocking down the expression level of VPS11 and a method for stably knocking down the expression level of VPS11. cell line.

Gene silencing induced by RNA interference (RNAi). When a double-stranded RNA homologous to the endogenous mRNA coding region is introduced into the cell, the mRNA will be degraded to silence gene expression, which is an effective way to reduce the expression level of the target protein.

Therefore, it is necessary to provide a method for gene silencing VPS11 using RNA interference technology and a cell line for stably knocking down the expression level of VPS11.

Contents of the invention

In order to solve the above problems, the present invention provides a small interfering RNA, shRNA, shRNA coding DNA, recombinant vector, recombinant lentivirus, host cell and application for knocking down VPS11.

The small interfering RNA provided by the present invention can directly specifically target the mRNA of VPS11 and cause mRNA degradation, resulting in gene silencing effect; the shRNA provided by the present invention, the encoding DNA of shRNA, the recombinant vector, and the recombinant lentivirus can promote The mRNA degradation of VPS11 indirectly silences the expression of VPS11 and reduces the expression level of VPS11.

English abbreviations and their Chinese interpretations involved in the present invention are as follows:

VPS11: the Genebank accession number of the VPS11 protein is NM_021729.4;

mRNA: messenger RNA; siRNA: small interfering RNA; shRNA: short hairpin RNA;

VPS11-shRNA: short hairpin RNA with messenger RNA targeting VPS11 protein;

In a first aspect, the present invention provides a small molecular interference RNA, the small molecular interference RNA has a nucleotide sequence complementary to the messenger RNA for translating the syntaxin VPS11, and the length is 19-27 bp.

Preferably, the length of the small interfering RNA is 21-27bp.

Preferably, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO:1 or SEQ ID NO:2.

In a second aspect, the present invention provides a shRNA, which has the nucleotide sequence of the sense strand or the antisense strand of the small interfering RNA as described in the first aspect.

Preferably, the shRNA provided by the second aspect of the present invention is a single-stranded RNA with a stem-loop structure, and the shRNA has a nucleotide sequence of a sense strand or an antisense strand of a small interfering RNA, wherein the The small molecular interference RNA has a nucleotide sequence complementary to the messenger RNA for translating the syntaxin VPS11, and the length is 19-27 bp.

Preferably, the length of the small interfering RNA is 21-27bp.

Preferably, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO:1 or SEQ ID NO:2.

In a third aspect, the present invention provides a shRNA encoding DNA, the shRNA has the nucleotide sequence of the sense strand or the antisense strand of the small interfering RNA as described in the first aspect.

Preferably, the third aspect of the present invention provides a shRNA encoding DNA, the shRNA has the nucleotide sequence of the sense strand or the antisense strand of the small interfering RNA, wherein the small interfering RNA has The messenger RNA complementary nucleotide sequence of the syntaxin VPS11 has a length of 19-27 bp.

Preferably, the length of the small interfering RNA is 21-27bp.

Preferably, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO:1 or SEQ ID NO:2.

Preferably, the DNA encoding shRNA contains the nucleotide sequence shown in SEQ ID NO:3 or 4.

Specifically, under this preferred condition, said SEQ ID NO:3 (5'-3') is:

GCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAACAGC TTTTT;

Said SEQ ID NO:4 (5'-3') is:

AAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTTAA ACAGC.

Further preferably, the 5' end or 3' end of the nucleotide sequence shown in SEQ ID NO:3 and 4 has an enzyme cleavage site.

Still further preferably, the cleavage sites at the 5' ends of the nucleotide sequences shown in SEQ ID NO: 3 and 4 are AgeI and EcoRI, respectively.

Under this preferred condition, the DNA encoding shRNA has a nucleotide sequence as shown in SEQ ID NO:5 or 6.

Specifically, the SEQ ID NO:5 (5'-3') is:

GCTGTTTAAGAAGAACCTGTT CTCGAG AACAGGTTTCTTCTTAAACAG C TTTTT

Said SEQ ID NO:6 (5'-3') is:

AATTCAAAAA GCTGTTTAAGAAGAACCTGTT CTCGAG AACAGGTTCTTCTT AAACAGC .

Among them, the bases marked by double dashes are restriction sites, the sequences represented by wavy lines are the sense strands of siRNA, the sequences marked by single dashes are the antisense strands of siRNAs, and the sequences marked in italics are the siRNA hairpins. loop sequence.

Preferably, the DNA encoding shRNA also includes an RNA polymerase III promoter.

Further preferably, the RNA polymerase III promoter is a human U6 promoter or a human H1 promoter.

Further preferably, the RNA polymerase III promoter is a mouse-derived U6 promoter or a mouse-derived H1 promoter.

In a fourth aspect, the present invention provides a recombinant vector, wherein the recombinant vector is the multiple cloning site of the pLKO.1 plasmid, the multiple cloning site of the pEN-hH1c plasmid or the pDSL-hpUGIP plasmid The recombination vector obtained by inserting the recombination site into the DNA encoding shRNA as described in the third aspect.

Preferably, the fourth aspect of the present invention provides a recombinant vector, wherein the recombinant vector is the multiple cloning site of the pLKO.1 plasmid, the multiple cloning site of the pEN-hH1c plasmid or the pDSL- The recombinant vector obtained by inserting the recombination site of the hpUGIP plasmid into the DNA encoding the shRNA, wherein the shRNA has the nucleotide sequence of the sense strand or the antisense strand of the small interfering RNA, wherein the small interfering RNA has a The messenger RNA complementary nucleotide sequence of the syntaxin VPS11 has a length of 19-27 bp.

Preferably, the multiple cloning sites of the pLKO.1 plasmid are AgeI and EcoRI restriction sites.

Preferably, the multiple cloning sites of the pEN-hH1c plasmid are BamHI and XhoI restriction sites.

Preferably, the recombination sites of the pDSL-hpUGIP plasmid are attR1 and attR2, wherein attR1 is located at the 2614-2738 bp site of the pDSL-hpUGIP plasmid, and attR2 is located at the 4194-4318 bp site of the pDSL-hpUGIP plasmid.

Preferably, the length of the small interfering RNA is 21-27bp.

Preferably, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO:1 or SEQ ID NO:2.

Preferably, the DNA encoding shRNA contains the nucleotide sequence shown in SEQ ID NO:3 or 4.

Specifically, under this preferred condition, said SEQ ID NO:3 (5'-3') is:

GCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAACAGC TTTTT;

Said SEQ ID NO:4 (5'-3') is:

AAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTTAA ACAGC.

Further preferably, the 5' end or 3' end of the nucleotide sequence shown in SEQ ID NO:3 and 4 has an enzyme cleavage site.

Still further preferably, the cleavage sites at the 5' ends of the nucleotide sequences shown in SEQ ID NO: 3 and 4 are AgeI and EcoRI, respectively.

Under this preferred condition, the DNA encoding shRNA has a nucleotide sequence as shown in SEQ ID NO:5 or 6.

Specifically, the SEQ ID NO:5 (5'-3') is:

CCGGGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAA CAGCTTTTTG;

Said SEQ ID NO:6 (5'-3') is:

AATTCAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTTAAACAGC.

Under this optimal condition, insert the target gene VPS11-shRNA into the AgeI and EcoRI sites of the lentiviral pLKO.1 plasmid to obtain the pLKO.1-VPS11-shRNA vector.

Preferably, the DNA encoding shRNA also includes an RNA polymerase III promoter.

Further preferably, the RNA polymerase III promoter is a human U6 promoter or a human H1 promoter.

Further preferably, the RNA polymerase III promoter is a mouse-derived U6 promoter or a mouse-derived H1 promoter.

The pLKO.1-VPS11-shRNA lentiviral vector constructed by the present invention has high infection efficiency and transcription efficiency, and the DNA gene fragment encoding shRNA on the vector can be inserted into the host genome through recombination, thereby continuously and stably silencing the expression of VPS11.

In the fifth aspect, the present invention provides a recombinant lentivirus, which is a recombinant lentivirus obtained by co-transfecting mammalian cells with the recombinant vector as described in the fourth aspect, the envelope vector psPAX2 and the packaging vector pMD2.G.

Preferably, the recombinant lentivirus provided by the fifth aspect of the present invention is a recombinant lentivirus obtained by co-transfecting mammalian cells with the recombinant vector, the envelope vector psPAX2 and the packaging vector pMD2.G, wherein the recombinant vector is The recombinant vector obtained by inserting the DNA encoding shRNA into the multiple cloning site of the pLKO.1 plasmid, wherein the shRNA has the nucleotide sequence of the sense strand or the antisense strand of the small molecule interfering RNA, and the small molecule interfering RNA has The nucleotide sequence complementary to the messenger RNA of the translated syntaxin VPS11 has a length of 19-27 bp.

Preferably, the multiple cloning sites of the pLKO.1 plasmid are AgeI and EcoRI restriction sites.

Preferably, the multiple cloning sites of the pEN-hH1c plasmid are BamHI and XhoI restriction sites.

Preferably, the recombination sites of the pDSL-hpUGIP plasmid are attR1 and attR2, wherein attR1 is located at the 2614-2738bp site of the pDSL-hpUGIP plasmid, and attR2 is located at the 4194-4318bp site of the pDSL-hpUGIP plasmid.

Preferably, the length of the small interfering RNA is 21-27bp.

Preferably, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO:1 or SEQ ID NO:2.

Preferably, the DNA encoding shRNA contains the nucleotide sequence shown in SEQ ID NO:3 or 4.

Specifically, under this preferred condition, said SEQ ID NO:3 (5'-3') is:

GCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAACAGC TTTTT;

Said SEQ ID NO:4 (5'-3') is:

AAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTTAA ACAGC.

Further preferably, the 5' end or 3' end of the nucleotide sequence shown in SEQ ID NO:3 and 4 has an enzyme cleavage site.

Still further preferably, the cleavage sites at the 5' ends of the nucleotide sequences shown in SEQ ID NO: 3 and 4 are AgeI and EcoRI, respectively.

Under this preferred condition, the DNA encoding shRNA has a nucleotide sequence as shown in SEQ ID NO:5 or 6.

Specifically, the SEQ ID NO:5 (5'-3') is:

CCGGGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAA CAGCTTTTTG;

Said SEQ ID NO:6 (5'-3') is:

AATTCAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTTAAACAGC.

Under this optimal condition, insert the target gene VPS11-shRNA into the AgeI and EcoRI sites of the lentiviral pLKO.1 plasmid to obtain the pLKO.1-VPS11-shRNA vector.

Preferably, the DNA encoding shRNA also includes an RNA polymerase III promoter.

Further preferably, the RNA polymerase III promoter is a human U6 promoter or a human H1 promoter.

Further preferably, the RNA polymerase III promoter is a mouse-derived U6 promoter or a mouse-derived H1 promoter.

Preferably, the recombinant lentivirus can be obtained by transfecting 293T cells with the pLKO.1-VPS11-shRNA lentiviral expression vector, packaging vector psPAX2 and envelope vector pMD2.G provided by the present invention for lentiviral packaging.

In the present invention, by constructing the DNA encoding VPS11-shRNA between the multiple cloning sites or the recombination sites of the lentiviral vector, after obtaining the recombinant lentiviral expression vector, the recombinant lentiviral expression vector can be constructed by transfecting cells and screening stable expression cell lines. The cell line with stable expression can achieve stable and long-term expression of the target gene, which is very suitable for the research of RNA interference on VPS11.

In addition, the lentiviral vector provided by the present invention can also infect host cells together with the lentiviral packaging plasmid to obtain an active recombinant lentivirus capable of transcribing VPS11-shRNA. After the recombinant lentivirus infects the host cell, it can integrate the gene encoding VPS11-shRNA into the genome of the host cell, which helps the gene encoding VPS11-shRNA to be transcribed in the host cell stably and for a long time, thereby obtaining small molecule interference RNA, degrades the mRNA of VPS11, and leads to gene silencing effect, reducing the expression level of VPS11.

The recombinant lentivirus provided by the present invention not only has high infection efficiency, but also has a wider host range, and can infect various types of cells such as neurons, muscle cells, liver cells, tumor cells, endothelial cells, etc., and rarely Trigger the body's immune response, the scope of application is very wide.

In the sixth aspect, the present invention provides a host cell, comprising the small interfering RNA as described in the first aspect, the shRNA as described in the second aspect, the DNA encoding shRNA as described in the third aspect, and the shRNA as described in the fourth aspect. At least one of the recombinant vector described in the aspect and the recombinant lentivirus described in the fifth aspect.

Preferably, the host cells are 293T cells or Hela cells.

Preferably, the host cell is a cell line stably knocking down the expression level of VPS11.

Further preferably, the host cell is a Hela cell line stably knocking down the expression level of VPS11.

In the seventh aspect, the present invention provides a small interfering RNA as described in the first aspect, the shRNA as described in the second aspect, the DNA encoding shRNA as described in the third aspect, the shRNA as described in the fourth aspect The application of the recombinant vector or the recombinant lentivirus as described in the fifth aspect in the preparation of a drug for inhibiting the expression of the syntaxin VPS11 gene.

The small molecule interfering RNA, shRNA, shRNA coding DNA, recombinant vector, recombinant lentivirus or host cell and application thereof provided by the present invention have the following beneficial effects:

The DNA encoding shRNA provided by the present invention is constructed between the multiple cloning sequence sites of the lentiviral vector or between the recombination sites to obtain a recombinant lentiviral vector. After the recombinant lentiviral vector is transfected into the host cell, or together with the lentiviral packaging plasmid After infecting host cells together, the gene encoding VPS11-shRNA can be integrated into the host cell genome, which helps the gene encoding VPS11-shRNA to be stably and long-term transcribed in the host cell to obtain VPS11-shRN;

The VPS11-shRNA provided by the present invention can produce small molecule interfering RNA capable of silencing the expression of VPS11 after being processed by host cells;

The small molecule interfering RNA provided by the present invention can target the mRNA of the syntaxin VPS11, and cause the degradation of the mRNA, and cause gene silencing effect, and reduce the expression level of VPS11.

In summary, the present invention provides an effective RNA interference solution for reducing the expression level of VPS11. The solution is preferably the small interfering RNA, shRNA, shRNA-encoding DNA, recombinant vector, recombinant lentivirus or host provided by the present invention. Cells undergo RNA interference.

Description of drawings

Fig. 1 is the plasmid map of the pLKO.1 vector used in the embodiment of the present invention;

Fig. 2 is the RT-PCR detection result of the mRNA of Hela cell VPS11 that the embodiment of the present invention 4 provides;

Fig. 3 is the western blot detection result of the expression level of VPS11 in Hela cells provided in Example 4 of the present invention.

detailed description

The following description is a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered Be the protection scope of the present invention.

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3 ^rd edition, 2001, NY, Cold Spring Harbor). The primers and DNA sequences used were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

The pLKO.1 vector used in the present invention was purchased from Open Biosystems, and the plasmid maps of the pLKO.1 vector were shown in Figure 1; oligo dT, various restriction enzymes and Taq enzymes were purchased from TaKaRa, and T4DNA ligase Purchased from NEB Company, Gel Recovery Kit and Small Plasmid Extraction Kit were purchased from OMEGA Company, Large Plasmid Extraction Kit was purchased from Nucleo Bond Company; DMEM High Glucose Medium and Fetal Calf Serum were purchased from HyClone Company; transfection Reagent Magetran was purchased from Origene; the inverted microscope was Olympus; the fluorescent inverted microscope was Nikon.

The Hela cell culture medium of the embodiment of the present invention is DMEM high-glucose medium containing 10% fetal bovine serum.

Unless otherwise specified, other reagents used in the present invention are commercially available.

Example 1

A method for preparing DNA encoding VPS11-shRNA, comprising the steps of:

According to the existing human VPS11 sequence and shRNA design principles in the GeneBank sequence library, the siRNA online design tool (http://www.sirnawizard.com/index.php) was used to design the coding DNA (interference fragment) of the human VPS11 sequence, And BLAST verification was performed on the designed fragment on the NCBI website to ensure that there was no homologous sequence of this fragment in other genes of the guarantor (except VPS11), and the following sequence was obtained:

VPS11-shRNA (SEQ ID NO: 3) (5'-3'):

GCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAACAGC TTTTT;

VPS11-shRNA (SEQ ID NO: 4) (5'-3'):

AAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTTCTTCTTAAAC AGC;

The restriction endonucleases AgeI and EcoRI were respectively introduced to synthesize the following primer sequences of VPS11-shRNA:

VPS11-shRNA-F (SEQ ID NO:5) (5'-3'):

GCTGTTTAAGAAGAACCTGTT CTCGAG AACAGGTTTCTTCTTAAA CAGC TTTTT

VPS11-shRNA-R (SEQ ID NO:6) (5'-3'):

AAAAAAGCTGTTTAAGAAGAACCTGTTCTCGAGAACAGGTTCTTCTT AAACAGC;

In said SEQ ID NO: 5 and SEQ ID NO: 6, the underlined parts are the restriction sites of AgeI and EcoRI respectively.

Example 2

A method for constructing a lentiviral recombinant vector pLKO.1-VPS11-shRNA, by applying DNA subcloning technology, mixing and annealing the synthesized interference fragments VPS11-shRNA-F and VPS11-shRNA-R, and passing the viscous The ends were connected to the Age Ⅰ and EcoR Ⅰ polyclonal microdots of the vector pLKO.1 to obtain the pLKO.1-VPS11-shRNA lentiviral recombinant vector, which was sequenced and identified.

Specifically include the following steps:

1. Interference segment annealing

Dissolve the newly synthesized paired interference fragments into a 50uM solution, pipette 10uL into PCR tubes and mix well, put them in a 95°C water bath for 10min, then close the water and cool down to room temperature naturally.

The reaction system is as follows:

overall system 20μl

VPS11-shRNA-F 10μl VPS11-shRNA-R 10μl

2. Double enzyme digestion and gel recovery of the empty vector pLKO.1

1) Double enzyme digestion of the empty vector pLKO.1

The pLKO.1 empty vector was double digested with restriction endonucleases AgeI and EcoRI, and the reaction system was as follows:

overall system 20μl 10xdisgestion Buffer 2μl pLKO.1 empty vector 5μl Age Ⅰ and EcoR Ⅰ 1μl MiliQ-H2O 12μl

After the preparation is fully mixed, it is placed in a 37°C water bath for 3 hours, and then detected by electrophoresis.

2) Gel recovery of the digested product

The digested products were purified using the Column DNA Gel Recovery Kit from Tiangen Bio (Beijing) Co., Ltd. Methods as below:

Column equilibration step: add 500ul of equilibrium solution BL to the adsorption column CA2, centrifuge at 12,000rpm for 1min, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

Cut out a single target DNA band from the agarose gel and put it into a clean centrifuge tube, and weigh it;

Add 3 times the volume of sol solution PN to the gel block;

Place in a water bath at 50°C for 10 minutes, during which time gently turn the centrifuge tube up and down to ensure that the glue block is fully dissolved;

Add the solution obtained in the previous step into an adsorption column CA2, place it at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30-60 sec, pour off the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube;

Add 600 ul of rinse solution PW to the adsorption column CA2, centrifuge at 12,000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube. Repeat step 5;

Put the adsorption column CA2 back into the collection tube, centrifuge at 12,000rpm for 2min, and remove the rinsing solution as much as possible. Place the adsorption column CA2 at room temperature for several minutes and dry it thoroughly;

Put the adsorption column CA2 into a clean centrifuge tube, add an appropriate amount of sterile water dropwise to the middle of the adsorption membrane (the elution buffer EB should be preheated in a 65-70°C water bath), and place at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12,000rpm for 2min.

3. Ligation and transformation of interference fragment and empty vector pLKO.1

Use the annealed fragment obtained in the previous step to ligate the recovered product of the pLKO.1 vector (the ratio of the target fragment to the vector is controlled at 3:1-6:1), and the reaction system is prepared as follows:

overall system 20μl 10×Ligation Buffer 2μl Annealed product 5μl pLKO.1 vector 8μl T4 DNA ligase 1.5μl MiliQ-H2O 3.5

After the preparation is fully mixed, it is placed at 16°C for 12 hours.

The ligation product was transformed. Transformation steps are as follows: ①Take 100 μl of competent cells and melt in ice bath; ②Quickly add the ligation product to competent cells, mix gently and then ice-bath for 30 minutes; ③42°C, heat shock for 30 seconds, and cool in ice bath for 2 minutes; ④ Add 1000 μl of SOC medium, and culture in a 37°C incubator for 1 hour; ⑤Apply an appropriate amount on an LB culture plate (ampicillin-resistant), and place it upside down in a 37°C incubator for overnight cultivation.

4. Extraction and identification of recombinant plasmid pLKO.1-VPS11-shRNA

Pick a single colony for plasmid extraction, and select an appropriate endonuclease for restriction detection to ensure that the inserted fragment of the vector is the target gene. The specific operation steps were carried out according to the instructions of the Tiangen kit.

1) Add 500ul of balance solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12000rpm for 1min, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2) Take 1-5ml of overnight cultured bacterial solution, add it to a centrifuge tube, use a conventional desktop centrifuge, centrifuge at 12000rpm for 1min, and remove the supernatant as much as possible.

3) Add 250ul of solution P1 to the centrifuge tube with the bacterial pellet left, and use a pipette or a vortex shaker to thoroughly suspend the bacterial pellet.

4) Add 250ul of solution P2 to the centrifuge tube, gently turn it up and down 6-8 times to fully lyse the bacteria.

5) Add 350ul of solution P3 to the centrifuge tube, turn it up and down gently 6-8 times immediately, and mix well, at this time, a white flocculent precipitate will appear. Centrifuge at 12000rpm for 10min.

6) Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette, and pay attention not to suck out the precipitate as much as possible. Centrifuge at 12000rpm for 30-60sec, pour off the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

7) Add 600 ul of rinse solution PW to the adsorption column CP3, centrifuge at 12000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

8) Repeat step 7.

9) Put the adsorption column CP3 into the collection tube, and centrifuge at 12000rpm for 2min.

10) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100ulMlliQ-H2O dropwise to the middle of the adsorption membrane, place at room temperature for 2min, and centrifuge at 12000rpm for 2min to collect the plasmid solution into the centrifuge tube.

After the plasmid was extracted, Age Ⅰ and EcoR Ⅰ double enzyme digestion was performed for identification.

After electrophoresis detection, the plasmids that were initially identified as positive clones were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the positive clones were selected and named pLKO.1-VPS11-shRNA.

Example 3

A method for packaging and producing pLKO.1-VPS11-shRNA lentivirus, comprising the following steps:

1) Plating of 293T cells (use ordinary DMEM complete medium with pH 7.4 when plating), the 293T cells normally cultured in a 6cm plate with a density of about 85-95% are divided into two new 6cm plates (about 6 cm) *105 cells), ^12-18h after plating is the best transfection time;

2) Transfection: Change the medium with DMEM complete medium with pH 7.4 before transfection. Take two 1.5ml EP tubes, add 300ul DMEM complete medium respectively, dissolve plasmid pLKO.1-VPS11-shRNA4ug, envelope vector psPAX23ug and packaging vector pMD2.G1ug in EP tubes, and dissolve 24ul polyjet transfection reagent in another In an EP tube, after preliminary mixing, transfer to the EP tube containing the plasmid, further gently pipette and mix several times, let it stand for 15 minutes, and then add it dropwise to the plate to be transfected;

3) 13-18 hours after transfection, replace the medium with 3ml of DMEM complete medium with normal pH7.4. 48h, 72h, and 96h after transfection were observed under a fluorescent microscope and photographed. At the same time, the virus stock solution was harvested 3 times. The virus stock solution harvested first could be temporarily stored in a refrigerator at 4°C to obtain pLKO.1-VPS11-shRNA lentivirus;

4) Virus concentration: Collect the viruses harvested three times and filter them with a 0.45um filter membrane to remove cell debris. Take 15ml of the filtered virus stock solution in a 100kD ultrafiltration column collection pool, centrifuge at 4000g for 25-30min at 4°C, and about 200ul remains in the collection pool. Virus concentrate, for virus titer determination, 50ul/tube is dispensed into 1.5ml EP tubes, stored at -80°C for long-term storage.

In order to fully illustrate the beneficial effects of the present invention, the present invention also provides a cell line that stably reduces the expression of VPS11 by using the pLKO.1-VPS11-shRNA lentivirus provided in this example, including the following steps: taking the pLKO.1-VPS11-shRNA lentivirus infected (infect) Hela cells, and puromycin was used to screen cells with stably integrated shRNA sequences in the genome to obtain a cell line that stably knocks down the VPS11 gene, that is, a cell line that can stably reduce the expression of VPS11.

Example 4

A method for preparing a cell line stably reducing VPS11 expression, comprising the steps of:

(1) Determine the appropriate concentration of G418 in the screening medium

Preparation of G418: Prepare 1M HEPES: dissolve 23.8g HEPES powder in 100ml ddH2O, adjust the pH to 7.3 with 10N NaOH, filter and sterilize, store at 4°C, and the final concentration is 1mol/L;

Prepare screening medium: Dilute G418 with medium to 0 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml, 1000 μg/ml ml, 1100μg/ml, 1200μg/ml 24-well plates, 1ml of medium (complete medium containing G418) per well, 4 replicates for each concentration, and 4ml of medium of each concentration is required for each medium change, which is ready-to-use;

(2) Resuscitate the cryopreserved cells, passage 3 to 4 times to make the cells reach a good growth state, spread 24-well plates (20000/well), change the medium for 12 hours, and add screening medium for culture;

Determine the optimal screening concentration of G418: Aspirate the culture medium in the culture wells, wash once with PBS, add different concentrations of screening medium to each well, replace the screening medium every other day, and culture for 10-14 days. Benchmark; the optimal screening concentration that the present embodiment obtains is 1200 μ g/ml;

(3) Magetran reagent transfection adherent cells and screening of resistant cell clones

Inoculate Hela cells into a 6-well plate and culture in DMEM high-glucose medium containing 10% fetal bovine serum until the density is about 70% to 80%, which can be used for transfection;

Equilibrate the Magetran reagent and pLKO.1-VPS11-shRNA plasmid solution to room temperature, prepare the following mixture in a centrifuge tube: 200 μl opti-DMEM, 2 μg pLKO.1-VPS11-shRNA plasmid, 6 μl Magetran transfection reagent, shake and mix well Incubate at room temperature for 15 minutes;

After replacing the cells with new medium, add the mixture into the culture plate, shake well, and then culture in a 37°C, 5% CO2 incubator. After 12 hours of culture, remove the medium and replace it with fresh 10% fetal bovine Continue culturing in DMEM high-glucose medium with serum, add the medium with optimal screening concentration 24 hours after transfection, and change the medium every other day; after changing the medium once or twice (cultivate overnight after changing the medium), the cells reach 50%-80% At this time, suck out all the culture solution, centrifuge at 3000-4000rpm, filter the supernatant at 0.22μm, add 2 times the volume of fresh culture solution with the optimal screening concentration, mix well at 4°C for later use; when the cells die in a large number after about 6 days of culture, you can replace them. Use adaptive medium, or increase the concentration of serum for culture, for example, 10% serum was originally used, and 20% serum can be used at this time; after 10 days of culture, the concentration of G418 is halved to maintain the screening pressure; resistance can be seen after screening for about 14 days When clones appear, single clones are picked, and positive clones are marked under a high-magnification microscope. Positive clones are screened by loop method or scraping method combined with limiting dilution method, and transferred to multi-well plates for culture.

In this example, after transfecting Hela cells with the pLKO.1-VPS11-shRNA recombinant vector, screening was performed using DMEM high-glucose medium (G418 concentration: 1200 μg/ml) containing 10% fetal bovine serum to obtain resistant clones, wherein , in the culture medium containing G418, the cells that were not transfected with the plasmid died; then the obtained resistant cell mixed clones were serially diluted, identified, and selected with G418 to obtain a monoclonal resistant cell line, that is, stably knocking down VPS11 (VPS11knock-down) Hela cell line, the Hela cell line stably knocking down VPS11 was continuously cultured in DMEM high glucose medium with 10% fetal bovine serum.

In order to fully illustrate the beneficial effect of the present invention, this embodiment also identifies the obtained cell line monoclonal stably reducing the expression of VPS11: including RT-PCR detection of the expression of the target gene; and western detection of the expression of the target gene, the steps are as follows:

1. Real-time-PCR detection of target gene expression

The resulting cells with stably reduced VPS11 expression were extracted for total RNA, and at the same time, Hela cells not transfected with pLKO.1-VPS11-shRNA were set as blank controls. Then it was reverse transcribed into a cDNA template, and the designed specific primers were used to detect whether the mRNA of VPS11 was transcribed:

Upstream primer (SEQ ID NO:7): 5'-CTGCTTCCAAGTTCCTTTGC-3'

Downstream primer (SEQ ID NO: 8): 5'-ACCCGTAGTTTGTAGGCTTG-3';

The main steps of total RNA extraction are:

1) Aspirate the medium in the cell culture dish, wash the cells gently with PBS, and then aspirate the PBS;

2) Add 1mL of Trizol to every ^10cm2 Petri dish;

3) Directly blow the cells with a gun in the culture dish several times to lyse the cells, and transfer the mixture into a clean RNase-free centrifuge tube;

4) Stand at room temperature for 5 minutes to completely lyse the nucleoprotein complex;

5) Add 0.2mL of chloroform for every 1mL of Trizol, and cover the centrifuge tube carefully;

6) Vigorously vortex for 15s;

7) Stand at room temperature for 2-3 minutes;

8) Centrifuge at 12000×g for 15 minutes at 4°C;

9) Tilt the centrifuge tube carefully at 45°, and carefully transfer the upper aqueous phase solution to another centrifuge tube;

10) Add isopropanol equal to the volume of the upper layer of water (helps to eliminate the interference of small fragments) or 2 to 3 times the volume of absolute ethanol, and mix well;

11) Stand at room temperature for 10 minutes;

12) Centrifuge at 12000×g for 10 minutes at 4°C; (RNA will form a gelatinous precipitate after centrifugation and adhere to the bottom or wall of the tube.)

13) Discard the supernatant, and add 1 mL of 80% ethanol freshly prepared with DEPC-treated double-distilled water for every 1 mL of Trizol. Vortex the sample briefly, then centrifuge at 7500×g at 4°C for 5 minutes; carefully pour off the eluate, and use a 20 μL pipette to suck off the excess liquid at the bottom of the tube, keeping the tip of the pipette away from the RNA; and place it in a clean environment for several minutes Allow the RNA pellet to dry (do not allow the RNA to dry completely as this will make the RNA less soluble; partial dissolution of the RNA will result in A260/280 < 1.6);

14) Use an appropriate amount (20-50 μL) of DEPC water warmed to 55°C to dissolve the RNA precipitate, then place it at 55-60°C for several minutes (to help dissolve), and quantify;

15) Proceed to follow-up experiments, or store the RNA solution at -80°C.

Reverse transcription step: measure the concentration of the extracted total RNA, add 2 μg total RNA, 1 μl oligo(dT), add DEPC water to 12 μl, and incubate at 68° C. for 5 minutes. Immediately place the product on ice for 2-5 minutes after taking it out, add 4μl Reaction Buffer, 2μl 10mM dNTP Mix, 1μl Reverse Transcriptase, MMLV-Reverse and 1μl RNase Inhibitor respectively. After incubating at 42° C. for 60 minutes, the obtained product was diluted to obtain a cDNA template. PCR amplification was carried out with the specific primers we designed. The reaction conditions of the amplification were: pre-denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 59°C for 30 seconds, extension at 68°C for 1 minute and 30 seconds, and amplification for 35 seconds. cycle, followed by a full extension at 72°C for 10 minutes. The system is 10ul, and the preparation is as follows:

The real-time PCR detection results of this embodiment are shown in FIG. 2 . In Figure 2, it can be seen from Figure 2 that, compared with the control group (Hela cell group transfected with pLKO.1 empty load), the mRNA of VPS11 in the Hela cell group (experimental group) transfected by pLKO.1-VPS11-shRNA obviously decased.

2. Western blot detection of target gene expression

Collect normal Hela cells and the stable VPS11-knockdown Hela cell line provided in this example for protein detection at the same time: First, remove the medium and add 4°C PBS to scrape the cells with a cell scraper and put them into a 15ml centrifuge tube. Centrifuge at 2000 for 5 minutes, remove the supernatant, then add 1ml 4°C PBS to each tube to blow the cell pellet evenly, then transfer to a 1.5ml EP tube and centrifuge again and remove the supernatant; then start to lyse the cells, mix RAPA strong lysate with protease inhibitors, Mix at a ratio of 100:1 and add to the cell pellet. Shake and lyse at 4°C for 1 hour, then centrifuge at 14,000 rpm at 4°C, take the supernatant as the sample; finally measure the protein concentration of the sample, add Loading buffer and boil for 8 minutes, then balance and load the sample Use SDS-polypropylene gel electrophoresis to run the gel; electrophoresis for 3 hours, transfer to the membrane for 2 hours, after that, antibody incubation, primary antibody for 2 hours, secondary antibody for 1 hour, and color exposure, wherein the primary antibody is Abgent’s VPS11 antibody ( AP16504b).

The results of western blot are shown in Figure 3, actin is the internal reference, vps11 represents the VPS11 protein, and the pLKO-empty lane and the pLKO-vps11 lane represent the results of the control group and the experimental group respectively; it can be seen from Figure 3 that the protein expression of VPS11 is higher than that of normal cells Significantly decreased, indicating that the target gene (that is, the DNA encoding VPS11-shRNA) can be continuously transcribed in the stably knocked-down VPS11 cell line provided by the present invention, and produce siRNA to degrade the mRNA of VPS11, thereby reducing the expression level of VPS11 .

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (7)

1. a small molecules interference RNA, it is characterised in that: described small molecules interference RNA has and translation syntaxin The nucleotide sequence that the messenger RNA of VPS11 is complementary, a length of 19～27bp；The nucleotide sequence of described small molecules interference RNA is such as Shown in SEQ ID NO:1 or SEQ ID NO:2.

2. a shRNA, for having the single stranded RNA of loop-stem structure, it is characterised in that arbitrary the one of the coding DNA of described shRNA The nucleotides sequence of bar chain is classified as the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

3. the DNA encoding shRNA, it is characterised in that the nucleotides sequence of arbitrary chain of described DNA is classified as SEQ ID Nucleotide sequence shown in NO:3 or SEQ ID NO:4.

4. a recombinant vector, it is characterised in that described recombinant vector is at the multiple clone site of pLKO.1 plasmid, pEN-hH1c The multiple clone site of plasmid or the recombination site of pDSL-hpUGIP plasmid insert coding shRNA's as claimed in claim 3 The recombinant vector that DNA obtains.

5. a recombinant slow virus, it is characterised in that be recombinant vector as claimed in claim 4 with envelope vector psPAX2 and The recombinant slow virus that package carrier pMD2.G cotransfection mammalian cell obtains.

6. a host cell, it is characterised in that include small molecules interference RNA as claimed in claim 1, such as claim 2 Described shRNA, the coding DNA of shRNA as claimed in claim 3, recombinant vector as claimed in claim 4 and such as right Require at least one in the described recombinant slow virus of 5.

7. small molecules interference RNA as claimed in claim 1, shRNA as claimed in claim 2, as claimed in claim 3 In the coding DNA of shRNA, recombinant vector as claimed in claim 4 and recombinant slow virus as claimed in claim 5 at least A kind of application in the medicine of preparation suppression syntaxin VPS11 gene expression.