CN105936917A - GRP78 gene targeted RNA interference recombinant lentivirus vectors and construction method - Google Patents

Abstract

The invention relates to construction, screening and application of GRP78 (glucose regulated protein 78) gene targeted RNA interference recombinant lentivirus vectors. With use of an RNA interference technique, aiming at different target sequences of a GRP78 gene, four lentivirus eukaryotic expression vectors for expressing siRNA in 293 cells are constructed, an interference lentivirus is obtained through co-transfecting the 293 T cells with pLv-GRP78shRNA, pMD2.G and pSPAX2 and culturing. The GRP78 gene targeted RNA interference recombinant lentivirus vectors can efficiently and specifically inhibit the expression of the GRP78 gene, and can be used for preparing related gene medicines for treating tumor.

Description

Targeting GRP78 gene RNA interference recombinant lentivirus vector and construction method thereof

Technical field

The present invention relates to targeting GRP78 (glucose regulated protein 78, GRP78) The RNA interference recombinant lentivirus vector of gene and structure thereof, belong to biomedicine technical field.

Background technology

GRP78 (glucose regulated protein 78, GRP78), also known as immunoglobulin Heavy-chain binding protein, the quality control of protein synthesis, Ca in endoplasmic reticulum^{2 +}The regulation of stable state play extremely important Role.Research shows, deprives when anoxia, aminoacid occurs in body, protein Misfolding, Ca^{2 +}Steady output rate, cholesterol close When becoming the multiple abnormal conditions such as limited, cell will start er stress (endoplasmic reticulum stress, ERS) reaction, with the living environment that reply is abnormal, and GRP78 plays an important role in ERS path.Meanwhile, GRP78 height table Reach in kinds of tumor cells, er stress and the living environment bearing high load capacity are fled from for tumor cell and protein closes The pressure become has important effect.Previously research ［ 2-3 ］ shows, adenosine (adenosine) is a kind of important cellular metabolism Product, plays apoptosis-induced effect to kinds of tumor cells.And GRP78 participate in this signal process but, above-mentioned apoptosis lead to In road, the concrete function of GRP78, the most still knows little about it.

RNA perturbation technique energy selective degradation mRNA, silencing of target genes, in the expression of post-transcriptional level suppressor gene, thus can It is used for carrying out the research of gene function and the research of drug targets.RNA interference mechanism research shows, double stranded rna molecule first by Intracellular rna enzyme Dicer be degraded into 21-23 base size small molecules interference RNA (small interfering RNA, SiRNA).SiRNA is considered as the main effects thing of RNA interference.Slow virus carrier is as one of conventional viral vector, and it is special Point is that immunogenicity is low, can infect split coil method and nondividing phase cell, self-contained fragment can be integrated into host cell gene group, And stably express siRNA, long-term inhibition of gene expression in mammal various types of cells.

Summary of the invention

The present invention provides the RNA interference recombinant viral vector structure of a kind of targeting GRP78 gene, screening and application thereof.

The present invention is based on RNA perturbation technique, and for the different target sequences of GRP78 gene, constructing four can be at 293T Expressing the slow virus carrier for expression of eukaryon pLv-GRP78shRNA of siRNA in cell, this carrier is that the slow virus of self inactivation carries Body, its signal collection of illustrative plates is shown in Fig. 1, the specific suppression GRP78 gene expression of energy.

Technical scheme is as follows:

The siRNA recombinant slow virus expression vector of a kind of targeting GRP78 gene, is to treat for tumor GRP78 gene associations Property material, slow virus be by pLv-GRP78shRNA, pMD2.G and pSPAX2 carrier cotransfection 293T cell cultivate obtain；Its In, described pLv-GRP78shRNA recombinant vector is connection double chain DNA fragment in the multiple clone site of plv-shRNA carrier Building and obtain, restriction enzyme site is BamHI and EcoRI, and described double chain DNA fragment is the one in following sequence:

(1) GRP78-homo-U1 oligonucleotide sequence 1:

Positive-sense strand: 5 ' GATCCCTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACAAGTTT TTG3 '

Antisense strand: 5 ' AATTCAAAAACTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACA AGG3 '

(2) GRP78-homo-U2 oligonucleotide sequence 2:

Positive-sense strand: GATCCAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAATCTTTT TTG

Antisense strand: AATTCAAAAAAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAAT CTG

(3) GRP78-homo-U3 oligonucleotide sequence 3:

Positive-sense strand: GATCCGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATTTCTTT TTG

Antisense strand: AATTCAAAAAGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATT TCG

(4) GRP78-homo-S4 oligonucleotide sequence 4:

Positive-sense strand: GATCCGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGCTCTTT TTG

Antisense strand: AATTCAAAAAGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGC TCG.

According to the present invention, the construction method of the siRNA recombinant slow virus expression vector of described targeting GRP78 gene, bag Include step as follows:

A. according to GRP78 mRNA sequence, designing synthetic dsdna fragment, described double chain DNA fragment is described GRP78- One in homo-U1, GRP78-homo-U2, GRP78-homo-U3 and GRP78-homo-U4 nucleotide sequence；

The most then it is connected to described double chain DNA fragment the multiple clone site of plv-shRNA carrier is built into pLv- GRP78shRNA recombinant vector；

The most described pLv-GRP78shRNA recombinant vector is cultivated with pMD2.G and pSPAX2 carrier cotransfection 293T cell, Obtain the recombined lentivirus vector system of targeting GRP78 gene RNA interference.

The inhibition of GRP78 gene is reflected by the recombined lentivirus vector in order to disturb targeting GRP78 gene RNA Fixed, pLv-GRP78shRNA is carried out fluorescence quantitative PCR detection destination gene expression after packaging plasmid cotransfection 293T cell Level, filters out effective interference plasmid.

Accompanying drawing explanation

Fig. 1 is Lentiviral plv-shRNA result schematic diagram in embodiment.

Fig. 2 GRP78 RNA interfering slow virus carrier wink turns 293T cell 48h picture (fluorescence, visible ray).Wherein (a) is Common light microscopic (100 ×)；B () is fluorescence light microscopic (100 ×).

GRP78 expression under Fig. 3 disturbance sequence.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited Scope.

Main material: 293T cell is purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences；E. coli competent DH5 α is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Slow virus carrier plv-shRNA is purchased from Clontech company, containing people U6 Promoter, encoding green fluorescent protein reporter gene；Various restriction endonuclease, T4DNA ligase and Taq DNA polymerization Enzyme is U.S.'s NEB Products；SYBR Green I q RT-PCR Mixs purchases work biotechnology service raw with Shanghai Company；Hyclone and DMEM culture fluid are purchased from Gibico company of the U.S..

The slow virus carrier of embodiment 1 targeting GRP78 gene builds

1. the design of nucleic acid oligomer and synthesis

4 interference sequence (see Table 1) of design targeting GRP78 gene (NM_005347), and synthesize corresponding single stranded DNA

Table 1 targeting GRP78 gene RNA interference sequence

Sequence names	Sequence information	Original position
			U1	CTTGTTGGTGGCTCGACTCGA	1081
U2	AGATTCAGCAACTGGTTAAAG	1109
			U3	GAAATCGAAAGGATGGTTAAT	1609
U4	GAGCGCATTGATACTAGAAAT	1669

3 ' ends of shRNA oligonucleotide are TTTTT, and as the termination signal of RNA polymerase III type promoter U6, they are 5 ' and 3 ' years old End is the restriction enzyme site of BamHI and EcoRI respectively, and the loop structure in plv-shRNA template has selected CTCGAG. Each single stranded DNA synthesis fragment is as follows:

(1) GRP78-homo-U1 oligonucleotide sequence 1:

Positive-sense strand: 5 ' GATCCCTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACAAGTTT TTG3 '

Antisense strand: 5 ' AATTCAAAAACTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACA AGG3 '

(2) GRP78-homo-U2 oligonucleotide sequence 2: positive-sense strand: GATCCAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAATCTTTTTTG

Antisense strand: AATTCAAAAAAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAAT CTG

(3) GRP78-homo-U3 oligonucleotide sequence 3:

Positive-sense strand: GATCCGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATTTCTTT TTG

Antisense strand: AATTCAAAAAGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATT TCG

(4) GRP78-homo-S4 oligonucleotide sequence 4:

Positive-sense strand: GATCCGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGCTCTTT TTG

Antisense strand: AATTCAAAAAGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGC TCG.

2. nucleic acid oligomer annealing

By the nucleic acid oligomer of synthesis respectively with without enzyme water dissolution, concentration is 20 μMs.Take corresponding positive-sense strand and antisense strand solution, according to Following proportioning preparation annealing reaction system

Component	Volume (μ L)
		Annealing buffer	5
Positive-sense strand (20 μMs)	5
		Antisense strand (20 μMs)	5
Water is to final volume	50

PCR instrument makes annealing treatment according to following program: 95 DEG C of 5min; 85℃ 5min; 75℃ 5min; 70 ℃5 min;4 DEG C of preservations.

3.pl-shRNA vector linearization

Take 2 μ g plv-shRNA carriers, carry out enzyme action process according to following system:

Component	Volume (μ L)
		10 × buffer	5
BamHI	1
		EcoRI	1
Water is to final volume	50

37 DEG C of enzyme action 1 hour, 1.5% sepharose electrophoresis, use DNA purification kit to reclaim, nucleic acid-protein concentration analyzer measures Reclaim quality.

The structure of 4.plv-GRP78-shRNA carrier

Utilize T4DNA ligase, carry out carrier coupled reaction according to following system:

Component	Volume (μ L)
		10 × T4 connects buffer	2
Carrier recovery	2
		shDNA	2
T4DNA ligase	1
		Water is to final volume	20

20 DEG C connect overnight, convert matter big pumping rod bacterium competence DH5 α, select positive colony and send to order-checking.The product that this step obtains Thing is plv-GRP78-shRNA.

Embodiment 2 RNA interference turns 293T cell in slow virus wink

By 4 kinds of interference slow virus plasmids, use the method transfection 293T cell of transient transfection.293T cell uses containing 10%FBS's DMEM in high glucose culture medium, is placed in 37 DEG C, cultivates in 5%CO2 incubator.Transfect first 1 day digestion 293T cell, counting, cell is connect Plant to 35mm culture dish, each culture dish inoculation 6 × 10⁵Individual cell；The secondary daily DMEM culture medium without FBS is prepared respectively Calcium turns reagent and RNA interference plasmid, adds 16 μ l calcium and turn reagent in every 84 μ l culture medium, adds 4 μ g matter in every 100 μ l culture medium Grain, turns solution by calcium after mixing respectively and plasmid solution mixes gently, and room temperature stands 10 minutes, is added dropwise in culture dish, 37 DEG C, in 5%CO2 incubator, cultivate 24h, change the fresh DMEM in high glucose culture medium containing 10% FBS and continue to cultivate 48h, collect Cell, extracts cell RNA.

The RT-PCR detection of the extraction of embodiment 3 total serum IgE, the synthesis of cDNA and RNA interference effect

Take 1 μ g RNA reverse transcription cDNA respectively, use the expression of fluorescence quantitative PCR method detection GRP78, with beta-actin As internal reference, the primer sequence that quantitative fluorescent PCR is used is shown in Table 2, uses 2-△ △ CT relative quantitation method to calculate different RNA The GRP78 expression of interference sequence sample.Filter out the RNA interference sequence that expression is minimum.Testing result (see figure 3) table The GRP78 gene expression dose of bright U4 sequence is minimum, illustrates that the RNA interference effect of U4 sequence is best.

Table 2.GRP78 and the design of beta-actin primed probe

Primer	Sequence
		F-GRP78	CCCGAGAACACGGTCTTTGA
R-GRP78	ACCACCTTGAACGGCAAGAA
		F-actinb	TATCGCCGCGCTCGTCGTC
R-actinb	TCTTGCTCTGGGCCTCGTC

SEQUENCE LISTING

<110>Tongji University Suzhou academy

<120>targeting GRP78 gene RNA interference recombinant lentivirus vector and construction method thereof

<130> 2016

<160> 12

<170> PatentIn version 3.5

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gatccagatt cagcaactgg ttaaagctcg agctttaacc agttgctgaa tcttttttg 59

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aattcaaaaa agattcagca actggttaaa gctcgagctt taaccagttg ctgaatctg 59

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gatccgaaat cgaaaggatg gttaatctcg agattaacca tcctttcgat ttctttttg 59

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aattcaaaaa gaaatcgaaa ggatggttaa tctcgagatt aaccatcctt tcgatttcg 59

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gatccgagcg cattgatact agaaatctcg agatttctag tatcaatgcg ctctttttg 59

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aattcaaaaa gagcgcattg atactagaaa tctcgagatt tctagtatca atgcgctcg 59

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cccgagaaca cggtctttga 20

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accaccttga acggcaagaa 20

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tatcgccgcg ctcgtcgtc 19

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tcttgctctg ggcctcgtc 19

Claims (3)

1. a siRNA recombinant slow virus expression vector for targeting GRP78 gene, is to treat for tumor GRP78 gene-correlation Property material, interference slow virus be by pLv-GRP78shRNA, pMD2.G and pSPAX2 carrier cotransfection 293T cell cultivate obtain； Wherein,

Described pLv-GRP78shRNA recombinant vector is connection double-stranded DNA sheet in the multiple clone site of pLv-shRNA carrier Section builds and obtains, and restriction enzyme site is BamHI and EcoRI；Described double chain DNA fragment is the one in following sequence:

(1) GRP78-homo-U1 oligonucleotide sequence 1:

Positive-sense strand:

5’GATCCCTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACAAGTTTTTG3’

Antisense strand:

5’ AATTCAAAAACTTGTTGGTGGCTCGACTCGACTCGAGTCGAGTCGAGCCACCAACAAGG3’

(2) GRP78-homo-U2 oligonucleotide sequence 2:

Positive-sense strand: GATCCAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAATCTTTT TTG

Antisense strand: AATTCAAAAAAGATTCAGCAACTGGTTAAAGCTCGAGCTTTAACCAGTTGCTGAAT CTG

(3) GRP78-homo-U3 oligonucleotide sequence 3:

Positive-sense strand: GATCCGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATTTCTTT TTG

Antisense strand: AATTCAAAAAGAAATCGAAAGGATGGTTAATCTCGAGATTAACCATCCTTTCGATT TCG

(4) GRP78-homo-S4 oligonucleotide sequence 4:

Positive-sense strand: chain: GATCCGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGCTCTTT TTG

Antisense strand: AATTCAAAAAGAGCGCATTGATACTAGAAATCTCGAGATTTCTAGTATCAATGCGC TCG.

The construction method of the siRNA recombinant slow virus expression vector of targeting GRP78 gene the most according to claim 1,

Comprise the following steps that

A. according to GRP78 mRNA sequence, designing synthetic dsdna fragment, described double chain DNA fragment is described GRP78- One in homo-U1, GRP78-homo-U2, GRP78-homo-U3 and GRP78-homo-U4 nucleic acid sequence;

The most then it is connected to described double chain DNA fragment the multiple clone site of plv-shRNA carrier is built into pLv- GRP78shRNA recombinant vector；

The most described pLv-GRP78shRNA recombinant vector is cultivated with pMD2.G and pSPAX2 carrier cotransfection 293T cell, Obtain the recombined lentivirus vector system of targeting GRP78 gene RNA interference.

3. the siRNA recombinant slow virus expression vector of the targeting GRP78 gene described in claim 1 is correlated with in preparation treatment tumor Application in property genomic medicine.