CN101684478B - A method for constructing recombinant lentiviral vector expressing small interfering RNA in tandem - Google Patents

Abstract

The invention discloses a method for constructing a tandem expression siRNA recombinant lentiviral vector, the main steps are as follows: (1) construction of a single-target siRNA recombinant lentiviral vector: a, chemically synthesized oligonucleotide chain; b, sense strand and antisense strand are annealed; c, the annealed fragment is connected to the carrier (2) construction of the siRNA recombinant lentiviral vector with double targets: a, amplify U6/H1- siRNA expression cassette; b, connecting the expression cassette to the siRNA recombination lentiviral vector of a single target site digested by Xhol; c, enzyme digestion identification and screening of clones inserted forward of the expression cassette; (3) multi-target tandem siRNA recombination Construction of lentiviral vector: insert the U6/H1-siRNA expression cassette into the dual-target siRNA recombinant lentiviral vector to construct a three-target siRNA recombinant lentiviral vector, and obtain multi-target tandem siRNA recombinant lentiviral vector by the same method Viral vector. The invention is suitable for simultaneously expressing siRNA for multiple target genes, and suitable for the research of long-term silencing of multiple target genes.

Description

A method for constructing recombinant lentiviral vector expressing small interfering RNA in tandem

technical field

The present invention relates to the field of molecular biology. More specifically, it relates to a method for constructing a recombinant lentiviral vector expressing small interfering RNA in tandem. the

Background technique

RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS) triggered by double-stranded RNA. At present, the mechanism of RNA interference has been basically clarified: endogenous or exogenous double-stranded RNA is cut by Dicer in the cytoplasm into small interfering RNA (siRNA) of 21-23 nt with 2 bases protruding from the 3' end, which is related to RNA-induced silencing complex (RNA-induced silencing complex, RISC) binding. The siRNA is melted in RISC, where the antisense strand guides RISC to target its complementary mRNA, and RISC cleaves the mRNA in the middle of the complementary sequence, thereby inhibiting the expression of the target gene. the

Although it has only been 11 years since the discovery of the RNAi phenomenon, RNAi as a gene silencing tool has been widely used in basic biological research and application fields. At present, the five commonly used methods for preparing siRNA include: 1) chemical synthesis; 2) in vitro transcription; 3) degradation of long dsRNAs by RNase III enzyme; 4) expression of siRNA expression cassette prepared by PCR in cells; 5) expression of siRNA The vector expresses siRNA in cells. The above methods have their own advantages and disadvantages: the siRNAs or siRNA expression cassettes prepared by the first four methods can be transfected or electroporated into cells, which can achieve transient gene silencing, but are not suitable for proteins with a long half-life. The siRNA expression vector can use the antibiotic marker on the vector to establish a relatively stable long-term gene silencing cell line, which can continuously inhibit the expression of the target gene. Most siRNA expression vectors use type III RNA polymerase promoters to manipulate siRNA expression in mammalian cells, and commonly used type III promoters include human and mouse U6 promoters and human H1 promoters. At present, many biological companies have developed siRNA expression vectors based on U6 or H1 promoters. Representative products include: Ambion's pSilencer series; Promega's siLentGene series; Invitrogen's BLOCK-iT series, etc. the

siRNA expression vectors can be divided into two types: plasmid vectors and viral vectors. For some difficult-to-transfect cells, plasmid vectors have the disadvantage of low transfection efficiency. In recent years, many biological companies have begun to develop viral vectors to express siRNA. Inconvenience caused by low dyeing efficiency. Currently commonly used viral vectors include: retroviral vectors, adenoviral vectors, and lentiviral vectors. Among them, lentiviral vectors have advantages such as being able to infect dividing and non-dividing cells, and being able to integrate into the host genome, so they are more and more widely used. A representative siRNA lentiviral vector is pLentiLox3.7, which contains a mouse U6 promoter to manipulate siRNA expression. the

However, the currently developed siRNA lentiviral expression vectors based on type III promoters are only suitable for the expression of single-target siRNA, and more and more basic research and applied research need to simultaneously silence multiple target genes. For this problem, the present invention designs a method for constructing a lentiviral vector expressing siRNA in tandem, which is suitable for experimental research and application research of long-term silencing of multiple target genes. the

Contents of the invention

The purpose of the present invention is to provide a method for constructing a recombinant lentiviral vector expressing siRNA in tandem, which overcomes the limitation that the existing siRNA expression vector is only suitable for expressing siRNA of a single target, and can simultaneously express multiple target genes siRNA, and is suitable for the establishment of stable cell lines, so it is suitable for the experimental research and application research of long-term silencing of multiple target genes. the

The construction method of the tandem expression siRNA recombinant lentiviral vector comprises the following steps:

1. Promoter modification of lentiviral vector:

The lentiviral vector transformed by the applicant is pLentiLox3.7 (Rubinson and Dillon, Nature Genetics, 2003). The vector contains a mouse U6 promoter to control the expression of siRNA. In order to avoid homologous recombination caused by a single promoter of tandem siRNA, resulting in the loss of the siRNA expression frame, the applicant replaced the mouse U6 promoter of the vector with a human U6 promoter and a human H1 promoter respectively, and the transformed lentivirus The vectors are respectively named pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6 CCTCCM 209135; Escherichia coli Stbl3/pLLH1 CCTCC M 209136, date of deposit: June 25, 2009, depository unit: China Type Culture Collection Center, address: Wuhan, China .Wuhan University)

2. Construction method of tandem expression siRNA recombinant lentiviral vector

2.1 Construction of single-target siRNA recombinant lentiviral vector

2.1.1 Chemically synthesized oligonucleotide chains, the format is as follows:

Sense strand: 5'-(19N)-(TTCAAGAGA)-(N91)-TTTTTTTC-3'

Antisense strand: Complementary to the sense strand with TCGA added at the 5' end to create Xho1 cohesive overhangs

Note: 19N represents the 19-base sequence of the siRNA target, and N19 represents the reverse complementary sequence of 19N;

2.1.2 Anneal the sense strand and antisense strand to form a fragment with Xho1 sticky end and blunt end; 

2.1.3 Connect the above fragments to the vector pLLU6 or pLLH1 (the vector is double-digested by KspA1 and Xho1);

2.1.4 Transform Escherichia coli Stbl3 competent cells;

2.1.5 Screen positive clones and identify them by sequencing;

2.2 Construction of siRNA recombinant lentiviral vector with dual targets

2.2.1 Use PCR to amplify the "U6/H1-siRNA" expression cassette from the single-target siRNA recombinant lentiviral vector, and use Sal1 and Xho1 double enzyme digestion to make it have Xho1 sticky ends

For the "U6-siRNA" expression cassette, the PCR primer sequences are as follows:

Upstream primer: U6-siR-1 (5'-TAACGC GTCGAC CCCCAGTGGAAAGACGCGCA-3')

Sal1

Downstream primer: U6-siR-2 (5'-ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3')

Xho1

For the "H1-siRNA" expression cassette, the PCR primer sequences are as follows:

Upstream primer: H 1-siR-1 (5'-TAACGC GTCGAC AATTCATATTTGCATGTCGC-3')

Sal1

Downstream primer: H1-siR-2 (5'-ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3')

Xho1

2.2.2 Connect the "U6/H1-siRNA" expression cassette to the single-target siRNA recombinant lentiviral vector digested by Xhol, and transform Escherichia coli Stbl3 competent cells, and screen positive clones by colony PCR;

2.2.3 Enzyme digestion identification and screening of "forward fragment insertion" clones

Since the insertion of the "U6/H1-siRNA" expression frame fragment may be inserted in both forward and reverse directions, in order to be suitable for the construction, screening and identification of tandem siRNA recombinant lentiviral vectors, this step only selects clones inserted in the forward direction. The specific identification method is: use Xba1 and Xho1 to double digest the clone to be tested, set the vector without inserting the fragment as the control, if the size of the digested fragment is larger than the control, it is "forward insertion"; if the size of the digested fragment is the same as the control, Then it is "reverse insertion";

2.2.4 Sequencing identification

2.3 Construction of multi-target tandem siRNA recombinant lentiviral vector

Insert the "U6/H1-siRNA" expression cassette into the dual-target siRNA recombinant lentiviral vector to construct a three-target siRNA recombinant lentiviral vector. With this construction strategy, a multi-target tandem siRNA recombinant lentiviral vector can be obtained. Viral vector. the

In a set of application examples for interfering with HIV, the applicant constructed a tandem siRNA recombinant lentiviral vector targeting three targets, and the experimental results confirmed that the lentiviral vector can work normally, and the tandem siRNA expression cassette works independently ( See Example 3). the

The beneficial effects of the present invention are: (1) The lentiviral vector expressing siRNA in tandem provided by the present invention is suitable for silencing multiple target genes at the same time, and is suitable for basic research and applied research with such needs, such as: in the field of AIDS treatment, Using siRNA to simultaneously target multiple viral genes or target different targets of the same viral gene can effectively prevent or delay virus escape; (2) the present invention is suitable for the establishment of stable cell lines, so that the target gene can be stably silenced for a long time; (3) It is easy to realize the directional insertion of the siRNA expression cassette; (4) The lentiviral vector for tandem expression of siRNA provided by the present invention provides two kinds of promoters U6 and H1 for selection, which can avoid multiple times of the same promoter to a certain extent Homologous recombination caused by duplication results in loss of the siRNA expression cassette. the

Description of drawings

Figure 1 is a schematic diagram of the promoter modification of the lentiviral vector. Replace the mouse U6 promoter on pLentiLox 3.7 with the human U6 and human H1 promoters from the pSilencer plasmid to obtain pLLU6 and pLLH1 vectors. the

Fig. 2 is a flowchart of the construction of a recombinant lentiviral vector expressing small interfering RNA in tandem. First, the chemically synthesized oligonucleotide fragments encoding siRNA were connected to the pLLU6 and pLLH1 vectors through KspA1 and Xho1 restriction sites to obtain single-target siRNA recombinant lentiviral vectors; then, PCR amplified "U6/H1- siRNA” expression cassette, which was connected to the single-target siRNA recombinant lentiviral vector cut by Xho1, so as to obtain the double-target siRNA recombinant lentiviral vector; finally, a recombinant lentiviral vector expressing siRNA in tandem was constructed according to this strategy. the

Figure 3 is an example of the application of lentiviral vectors expressing small interfering RNA in tandem. In this example, the HIV genes rev, pol, tat and the host cell membrane protein CCR5 required for virus infecting cells were selected as targets, and single-target, double-target and triple-target siRNA recombinant lentiviral vectors were constructed. The result of this example shows that the lentiviral vector for tandem expression of small interfering RNA provided by the present invention can work normally, and the tandem siRNA expression cassette works independently. the

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments. the

Example 1: Promoter transformation of lentiviral vectors

The lentiviral vector transformed by the applicant is pLentiLox3.7 (Rubinson and Dillon, Nature Genetics, 2003). The vector contains a mouse U6 promoter to control the expression of siRNA, and also contains a reporter gene EGFP for flow cytometry sorting. In order to avoid homologous recombination caused by a single promoter of the tandem siRNA, resulting in the loss of the siRNA expression box, the applicant replaced the mouse U6 promoter of the vector with a human U6 promoter and a human H1 promoter (human U6 promoter and the human H1 promoter were obtained from Ambion’s products pSilencer2.0_U6 and pSilencer3.0_H1 by PCR), and the transformed lentiviral vectors were named pLLU6 and pLLH1 respectively (Escherichia coli Stbl3/pLLU6CCTCC M209135; Escherichia coli Stbl3/pLLH1 CCTCC M 209136 ). The schematic diagram of promoter transformation is shown in Figure 1. The specific transformation process is as follows:

1.1 PCR amplification of U6 and H1 promoter fragments;

For the U6 promoter, the PCR primers are as follows:

Upstream primer: U6-1 (5'-ACA TCTAGA CCCCAGTGGAAAGACGCGCAG-3')

Xba1

Downstream primer: U6-2 (5'-ACG GTTAAC GATCCCGCGTCCTTTCCCAA-3')

KspA1

For the H1 promoter, the PCR primers are as follows:

Upstream primer: H1-1 (5'-CGG TCTAGA AATTCATATTTGCATGTCGCTA-3')

Xba1

Downstream primer: H1-2 (5'-AGC GTTAAC CGAGTGGTCTCATACAGAACTT-3')

KspA1

1.2 The above promoter fragments were cloned into the pEGM-T vector (purchased from Promega) with the T-A cloning strategy;

1.3 Digest the above recombinant pEGM-T vector with Xba1 and KspA1, and isolate the U6/H1 promoter fragment with cohesive ends;

1.4 Digest pLentiLox 3.7 with Xba1 and KspA1 to obtain a vector with corresponding cohesive ends;

1.5 Connect the U6/H1 promoter fragment to the above vector to obtain promoter-modified lentiviral vectors, which are named pLLU6 and pLLH 1 (Escherichia coli Stbl3/pLLU6CCTCC M 209135; Escherichia coli Stbl3/pLLH1 CCTCC M 209136). the

Example 2: Construction method of tandem expression siRNA recombinant lentiviral vector

2.1 Construction of single-target siRNA recombinant lentiviral vector (see step I in Figure 2)

2.1.1 Chemically synthesized oligonucleotide chains, the format is as follows:

Sense strand: 5'-(19N)-(TTCAAGAGA)-(N91)-TTTTTTTC-3'

Antisense strand: Complementary to the sense strand with TCGA added at the 5' end to create Xho1 cohesive overhangs

Note: 1) 19N represents the 19-base sequence of the siRNA target, and N19 represents the reverse complementary sequence of 19N;

2) (TCAAGAGA) guides the "ring" region of the siRNA precursor shRNA (small hairpin RNA),

The sequence is selected from (Brummelkamp et al., Science, 2002)

2.1.2 Anneal the sense strand and antisense strand to form a fragment with Xho1 sticky end and blunt end; 

2.1.3 Connect the above fragments to the vector pLLU6 or pLLH1 (the vector is double-digested by KspA1 and Xho1);

2.1.4 Transform Escherichia coli Stbl3 competent cells (Stbl3 strains were purchased from Invitrogen);

2.1.5 Screen positive clones and identify them by sequencing;

2.2 Construction of dual-target siRNA recombinant lentiviral vector (see Figure 2 Step II)

2.2.1 Use PCR to amplify the "U6/H1-siRNA" expression cassette from the single-target siRNA recombinant lentiviral vector, and use Sal1 and Xho1 double enzyme digestion to make it have Xho1 sticky ends

For the "U6-siRNA" expression cassette, the PCR primer sequences are as follows:

Upstream primer: U6-siR-1 (5'-TAACGC GTCGAC CCCCAGTGGAAAGACGCGCA-3')

Sal1

Downstream primer: U6-siR-2 (5'-ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3')

Xho1

For the "H1-siRNA" expression cassette, the PCR primer sequences are as follows:

Upstream primer: H 1-siR-1 (5'-TAACGC GTCGAC AATTCATATTTGCATGTCGC-3')

Sal1

Downstream primer: H1-siR-2 (5'-ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3')

Xho1

2.2.2 Connect the "U6/H1-siRNA" expression cassette to the single-target siRNA recombinant lentiviral vector digested by Xhol, and transform Escherichia coli Stbl3 competent cells, and screen positive clones by colony PCR;

2.2.3 Enzyme digestion identification and screening of "forward fragment insertion" clones

Since the insertion of the "U6/H1-siRNA" expression frame fragment has the possibility of both positive and negative directions (the connection direction shown in step II in Figure 2 is the forward direction), in order to be suitable for the construction and screening of tandem siRNA recombinant lentiviral vectors, This step picks only clones that insert in the forward direction. The specific identification method is: use Xba1 and Xho1 to double digest the clone to be tested, set the vector without inserting the fragment as the control, if the size of the digested fragment is larger than the control, it is "forward insertion"; if the size of the digested fragment is the same as the control, Then it is "reverse insertion";

2.2.4 Sequencing identification

Sequencing primer: 5'-CAGTGCAGGGGAAAGAATAGTAGAC-3'

(Rubinson and Dillon, Nature Genetics, 2003);

2.3 Construction of multi-target tandem siRNA recombinant lentiviral vector (see step III in Figure 2)

Insert the "U6/H 1-siRNA" expression cassette into the dual-target siRNA recombinant lentiviral vector to construct a three-target siRNA recombinant lentiviral vector. With this construction strategy, a multi-target tandem siRNA recombination can be obtained lentiviral vector. the

Example 3: Application example of lentiviral vector expressing siRNA in tandem

In order to verify whether the lentiviral vector expressing siRNA in tandem can work normally, the applicant explained with an application example of interfering with HIV:

In this example, the applicant selected the HIV genes rev, pol, tat and the host cell membrane protein CCR5 required for virus-infected cells as targets, and used the lentiviral vector for tandem expression of siRNA provided by the present invention to construct a single target Point, double-target, triple-target siRNA recombinant lentiviral vector. The combination of siRNA expression cassettes is shown in the table below:

single target H1 CCR5 U6 rev U6 pol U6 tat double target H1 CCR5_U6 rev H1 CCR5_U6 pol H1 CCR5_U6 tat Three targets H1 CCR5_U6 rev_U6 pol H1 CCR5_U6 rev_U6 tat H1 CCR5_U6 poL_U6 tat

The applicant co-transfected HEK-293T cells with the above siRNA recombinant lentiviral vectors respectively with pNL4-3.Luc.R-E-(Landau NR, Viology, 1995), and detected the relative luciferase activity after 24 hours, as shown in Figure 3 ( A) (Note: pNL4-3.Luc.R-E- is an HIV replicon with a luciferase reporter gene, and its replication level can be represented by luciferase activity). The results showed that each siRNA recombinant lentiviral vector had different inhibitory effects on the replication of HIV, and the combined effects of rev-pol, rev-tat, and pol-tat siRNA were higher than their individual effects, indicating that rev, The pol and tatsiRNA expression cassettes can work independently, and when combined in pairs, the effect has an additive effect. The specific results and analysis data are shown in the table below:

(Note: The CCR5 siRNA expression cassette does not play a role in the above experiments, because the HEK-293T cells used do not express the CCR5 membrane protein, and pNL4-3.Luc.R-E- is liposome transfection into the cells, and the membrane receptor CCR5 is not required mediate)

In order to verify whether the CCR5 siRNA expression cassette can work independently, the applicant established a stable cell line based on GhostCD4/CCR5 using the constructed siRNA recombinant lentiviral vector pLLH1 CCR5 and pLLH1 CCR5_U6 rev_U6 tat (Kewal Ramani V and Littman DR, J Virol, 1999 ) (this cell line can stably express the membrane protein CCR5). Then, the above-established stable cell line was detected by flow cytometry with PE-labeled CCR5 antibody (purchased from eBioscience Company) (the results are shown in Figure 3(B), the gray area is the sample, and the hollow area is the negative control). The results showed that: CCR5 siRNA expressed by pLLH1 CCR5 and pLLH1 CCR5_U6rev_U6 tat could effectively down-regulate the expression level of CCR5, and there was no significant difference between the two (77.5% and 79.9% down-regulation, respectively), thus indicating that: siRNA expressed in tandem in the recombinant lentiviral vector The CCR5 siRNA expression cassette can work independently. the

In summary, the application examples involved in this example illustrate that the lentiviral vectors for tandem expression of siRNA provided by the present invention can work normally, and the tandem siRNA expression cassettes work independently. the

Example 4: Packaging of lentivirus expressing siRNA in tandem. the

4.1 Spread HEK-293T cells (purchased from CCTCC) in a cell culture dish with a diameter of 7.5 cm 24 hours before transfection;

4.2 The confluence of cells should be 40-60% during transfection, and the following plasmids are co-transfected into cells by standard calcium phosphate transfection operation:

4μg siRNA recombinant lentiviral vector

2 μg pLP1

2 μg pLP2

2 μg pVSVG

(Note: pLP1, pLP2, and pVSVG are lentiviral helper packaging plasmids, purchased from Invitrogen); 4.3 Collect virus supernatant (i.e., cell culture medium) at 24 hours, 48 hours, and 72 hours after transfection, and supplement the medium; Mix the virus supernatant collected 3 times afterwards;

4.4 Detection of virus titer: The virus supernatant was diluted 10 times, and then infected with HEK-293T cells (polybrene (purchased from SIGMA) was added to the culture medium, with a final concentration of 8 μg/ml), and GFP was detected by flow cytometry 48 hours later. Positive cell ratio, and virus titers were calculated as dilutions with ratios between 0.1-10%. the

Example 5: Establishment of stable cell lines using lentivirus expressing siRNA in tandem. the

5.1 Use the lentivirus packaged in Example 4 to infect Ghost CD4/CCR5 cells with a gradient m.o.i. After 48 hours of infection, the ratio of GFP-positive cells is detected by flow cytometry, and those whose ratio is less than 10% are selected for sterile flow sorting (Note: The reason why the ratio is less than 10% is to avoid cells being re-infected by lentivirus and affecting the accuracy of subsequent experiments);

5.2 Subculture the sorted cells until the number of cells is appropriate;

5.3 Check the ratio of GFP positive cells again by flow cytometry to determine the purity of the stable cell line. the

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Claims (3)

1. A transformed lentiviral vector pLLU6, the vector is Escherichia coli StbI3/pLLU6, and the preservation number is CCTCC M 209135. 2. A transformed lentiviral vector pLLH1, the vector is Escherichia coli StbI3/pLLH1, and the preservation number is CCTCC M 209136. 3. A method for constructing a tandem expression siRNA recombinant lentiviral vector, comprising the following steps: (1) Construction of single-target siRNA recombinant lentiviral vector: a. Chemically synthesized oligonucleotide chains, the format is as follows: Sense strand: 5'-(19N)-(TTCAAGAGA)-(N91)-TTTTTTTC-3' Antisense strand: Complementary to the sense strand, TCGA is added at the 5' end to generate Xho1 cohesive overhangs; The 19N represents the 19 base sequences of the siRNA target, and N19 represents the reverse complementary sequence of 19N; b. The sense strand and antisense strand are annealed to form fragments with Xho1 sticky ends and blunt ends; c. connecting the above fragments to the vector pLLU6 according to claim 1 or the vector pLLH1 according to claim 2; d, transform Escherichia coli StbI3 competent cells; e. Screen positive clones and identify them by sequencing; (2) Construction of siRNA recombinant lentiviral vector with dual targets: a. Use PCR to amplify the U6/H1-siRNA expression cassette from the single-target siRNA recombinant lentiviral vector, and use Sal1 and Xho1 double enzymes to make it have Xho1 sticky ends For the U6-siRNA expression cassette, the PCR primer sequences are as follows: Upstream primer: U6-siR-1 (5'-TAACGC GTCGAC CCCCAGTGGAAAGACGCGCA-3') Sal1 Downstream primer: U6-siR-2 (5'-ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3') Xho1 For the H1-siRNA expression cassette, the PCR primer sequences are as follows: Upstream primer: H1-siR-1 (5'-TAACGC GTCGAC AATTCATATTTTGCATGTCGC-3') Sal1 Downstream primer: H1-siR-2 (5'ATACCG CTCGAG CTCGTGAAGCGAGCTTATCGATACC-3') Xho1 b. Connect the U6/H1-siRNA expression cassette to the single-target siRNA recombinant lentiviral vector digested by XhoI, and transform Escherichia coli StbI3 competent cells, and screen positive clones by colony PCR; c. Enzyme digestion to identify the clones with forward insertion of the screening fragment. This step only selects the clones with forward insertion. The specific identification method is: use Xba1 and Xho1 to double enzyme digest the clone to be tested, set the vector without the inserted fragment as a control, and digest If the size of the fragment is larger than that of the control, it is inserted in the forward direction; if the size of the enzyme-digested fragment is the same as that of the control, it is inserted in the reverse direction; d. Sequencing identification; (3) Construction of multi-target tandem siRNA recombinant lentiviral vector: Insert the U6/H1-siRNA expression cassette into the dual-target siRNA recombinant lentiviral vector to construct a three-target siRNA recombinant lentiviral vector. With this construction strategy, a multi-target tandem siRNA recombinant lentiviral vector can be obtained .

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