CN101805750B - Construction and application of farnesyl pyrophosphoric acid synthetase RNA (Ribonucleic Acid) interference recombinant lentivirus vector - Google Patents

Abstract

The present invention provides the construction of a recombinant lentiviral vector targeting farnesyl pyrophosphate synthase RNA interference. The most effective target sequence of FDS gene RNAi is screened out in the tool cell 293T cells, and the double-stranded DNA of the most effective target sequence is synthesized and connected to pGCSIL - GFP vector, the recombinant vector was successfully constructed through restriction sequencing. Studies have shown that the constructed RNA interference vector LV-sh-FDS can down-regulate the expression of FDS mRNA level in neonatal rat cardiomyocytes, and can also down-regulate the expression of cardiac hypertrophy markers such as cell area and marker genes beta-MHC and BNP. While down-regulating FDS, it can also effectively inhibit the activity of RhoA, and can be used in the preparation of drugs for treating cardiac hypertrophy diseases. It can also be used in the preparation of drugs for regulating cholesterol metabolism.

Description

Construction and application of farnesyl pyrophosphate synthase RNA interference recombinant lentiviral vector

technical field

The invention belongs to the technical fields of molecular biology, biomedicine and genetic engineering, and mainly relates to the construction of an RNA interference recombinant lentiviral vector (LV-sh-FDS) for the gene of Farnesyl pyrophosphate synthase (FDS) and its Applications in cardiac hypertrophy and regulation of cholesterol metabolism.

Background technique

Current research shows that small G protein is involved in the occurrence of cardiac hypertrophy. RhoA belongs to the superfamily of small G proteins, and has two forms: a GDP-bound inactive state and a GTP-bound active state. At present, many studies have shown that RhoA is involved in the occurrence of cardiac hypertrophy, such as angiotensin II (Ang II)-induced cardiac hypertrophy. Studies have found that overexpression of Rho-GDI (Rho-GDP separation inhibitor) and Rho inhibitor C3exeoezyme can significantly inhibit Ang II-induced cardiac hypertrophy.

Farnesylpyrophosphate synthase (FDS) is a key enzyme in the mevalonate pathway, which is the only way for cholesterol synthesis in mammals. At the same time, FDS is also a key enzyme in the isoprene pathway, and the isoprenoid intermediate produced by the decomposition provides an important substrate for the activation of RhoA and its function. Our previous experiments showed that in the cultured neonatal rat cardiomyocytes induced by 1 μM Ang II in the model of cardiac hypertrophy and in the hypertrophic myocardium of spontaneously hypertensive rats (SHRs) (this model contains high concentrations of Ang II in the myocardium), FDS inhibitor A Sodium lenronate can inhibit cardiac hypertrophy by significantly inhibiting the activity of RhoA. These fully demonstrated the possible important role of FDS in regulating RhoA activity in the myocardium and in cardiac hypertrophy. In addition, our study also showed that in the cultured neonatal rat cardiomyocytes incubated with 1μM Ang II for 12-48h, the FDS gene was up-regulated, especially at 24h. At the same time, the up-regulation of FDS gene was also found in the hypertrophic myocardium of spontaneously hypertensive rats (SHRs) at 18 weeks. These previous studies suggested a possible important role of FDS in cardiac hypertrophy.

FDS also shows considerable importance in the research of other fields. Biological studies have shown that by inhibiting the expression of FDS, the growth rate of the parasite decreases until finally it dies. In addition, some studies have shown that FDS is highly expressed in rat prostate cancer cells. Another study showed that down-regulating the expression of FDS can target and treat tumor cells through the immune surveillance mediated by Vγ9Vδ2 T cells.

All these evidences suggest that inhibition of FDS gene expression may prevent Ang II-associated cardiomyocyte/tissue hypertrophic responses.

RNA interference (RNA interference, RNAi) technology is a common means of inhibiting gene expression, also known as gene silencing. The principle of RNA interference is that when double-stranded RNA homologous to the endogenous mRNA coding region is introduced into the cell, the mRNA is degraded, resulting in the silencing of gene expression.

The process of RNA interference mainly has two steps: 1. The double-stranded RNA is cut into short double-stranded RNA of 21-23 base pairs by a cell-derived double-stranded RNA-specific nuclease, namely small interfering RNA (small interference RNA, siRNA) 2. The antisense strand of siRNA and nuclease form a silencing complex (RNA-induced silencing complex, RISC). mRNA cleavage. RNA interference technology is currently widely used in gene therapy and research, and those siRNA/shRNAs that have proved effective in target experiments can be further developed into RNAi drugs. shRNA (short hairpin RNA) consists of two short inverted repeat sequences (one of which is complementary to the target gene), and the middle loop sequence is separated to form a hairpin structure. In vivo shRNA is processed into siRNA to effectively degrade the target gene and inhibit its expression. However, in order to apply this technology to clinical treatment, it is necessary to solve important problems such as the persistence and expression efficiency of RNA interference fragments.

At present, the vectors for gene therapy mainly include non-viral vectors and viral vectors. However, non-viral vectors cannot satisfy long-term expression, and this defect is undoubtedly filled by viral vectors. In recent years, lentivirus-mediated RNA interference technology has made a good start in research. Lentiviral vector is a kind of retrovirus, which has the basic structure of retrovirus, but has the component characteristics of different retroviruses. The virus can transfect both dividing cells and non-dividing cells. The viral genome can be integrated into the host, enabling long-term stable gene expression, and the lentivirus has low immunogenicity, which makes the lentivirus the best carrier for RNA interference, and is currently widely used in gene expression regulation, gene therapy and other fields . The third-generation replication-deficient lentiviral vector we choose is a suicide virus (SIN), which can be expressed in vivo for a long time and has high safety. Therefore, the lentiviral vector-mediated RNA interference effect exists in the target cells for a long time, which can create conditions for better interference.

Contents of the invention

One object of the present invention is to provide an interference vector, that is, a recombinant lentiviral vector (LV-sh-FDS) for RNA interference of the Farnesyl pyrophosphate synthase (FDS) gene. The vector contains the lentiviral backbone plasmid recombinant vector pGCSIL-sh-FDS, and the recombinant vector is connected to the double-stranded DNA fragment for shRNA in the MCS of the p-GCSIL-GFP backbone plasmid vector, and the target sequence for it is: 1#-GACAGCTTTCTACTCTTTC, the information of the synthesized DNA fragment is:

1#-1: 5'-CcggaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAAGCTGTCttTTTTTg-3',

1#-2: 5'-aattcaaaaaaaGACAGCTTTCTACTCTTTTCTCTTGAAGAAAGAGTAGAAAGCTGTCtt-3'.

The vector can specifically reduce the expression of the FDS gene after effectively transfecting cells or tissues, so that it can be applied to gene therapy or gene function research. The most important feature of the LV-sh-FDS provided by the present invention is to provide a targeted FDS inhibitory effect, and use a recombinant lentiviral vector as a carrier, so that the interference effect can be sustained. The entire preparation process uses plasmids to avoid adenovirus contamination by traditional methods. The target gene FDS targeted in the present invention is a key enzyme in the mevalonate pathway and is also a key enzyme in the isoprene pathway. The prenylated intermediates produced are small G protein family including RhoA activation and functional provide an important substrate. The mevalonate pathway is the only pathway for cholesterol synthesis in mammals. By inhibiting the expression of FDS and reducing the activity of RhoA, it can effectively inhibit Ang II-related cardiac hypertrophy. Additionally, cholesterol production in the mevalonate pathway was also reduced by reducing FDS expression. In the present invention, by screening the most effective FDS interference sequence, synthesizing its double-stranded DNA, connecting it to the lentiviral backbone plasmid vector, and co-transfecting tool cells (293T cells) with the helper plasmid, the FDS interference recombinant lentiviral vector: LV-sh- FDS.

Another object of the present invention is to provide a preparation method of the carrier, which is achieved through the following steps:

(1) Synthesis, screening and identification of effective sequences for FDS gene interference;

(2) Construction and identification of the FDS gene interference lentiviral backbone plasmid recombinant vector pGCSIL-sh-FDS;

(3) Packaging, collection, purification, identification and titer determination of FDS gene interference recombinant lentiviral vector LV-sh-FDS.

The screening of the effective interference sequence of the FDS gene in step (1) is mainly divided into the following steps: (a), purchase FDS cDNA (imaGenes, Germany), and successfully clone the GFP-FDS-FLAG fusion protein particle by restriction enzyme sequencing; (b) According to the principle of RNA interference sequence design, four RNA interference (RNAi) targets for FDS were designed, and the target sequences (Target Seq) were 1#: 5'-GACAGCTTTCTACTCTTTC-3; 2#: 5'-CACGCTAATGCCCTGAAGA- 3'; 3#: 5'-CTGTAGGAGGCAAGTACAA-3; 4#: 5'-CTGGTGGAAC CAAG GAAAC-3' (FDS mRNA NM_031840 GenBank GI: 13929205), and synthesize their double-stranded DNA respectively, the sequences are:

1#-1: 5'-GATCCCaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAAGCTGTCttTTTTTGGAT-3'

1#-2: 5'-AGCTATCCAAAAAaaGACAGCTTTCTACTCTTTCTCTCTTGAAGAAAGAGTAGAAAGCTGTCttGG-3'

2#-1: 5'-GATCCC-aaCACGCTAATGCCCTGAAGATTCAAGAGATCTTCAGGGCATTAGCGTGttTTTTTGGAT-3'

2#-2: 5'-AGCTATCCAAAAAaaCACGCTAATGCCCTGAAGATCTCTTGAATCTTCAGGGCATTAGCGTGttGG-3'

3#-1: 5'-GATCCCaCTGTAGGAGGCAAGTACAATTCAAGAGATTGTACTTGCCTCCTACAGtTTTTTGGAT-3'

3#-2: 5'-AGCTATCCAAAAAaCTGTAGGAGGCAAGTACAATCTCTTGAATTGTACTTGCCTCCTACAGtGG-3'

4#-1: 5'-GATCCCaaCTGGTGGAACCAAGGAAACTTCAAGAGAGTTTCCTTGGTTCCACCAGttTTTTTGGAT-3'

4#-2: 5'-AGCTATCCAAAAAaaCTGGTGGAACCAAGGAAACTCTCTTGAAGTTTCCTTGGTTCCACCAGttGG-3'; and synthesized negative control (NC), the DNA fragment information is as follows:

NC-1: 5'-GATCCCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTT-3';

NC-2: 5'-AGCTAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAGGG-3'. Then connect to the digested plasmid pGC-U6/Neo/DsRed (the plasmid vector contains RFP red fluorescent marker, which is convenient for observation). Construction framework of pGC-U6/Neo/DsRed recombinant vector

NO. 5’ STEMP Loop STEMP

1#-1 GATCCC aaGACAGCTTTCTACTCTTTTC TTCAAGAGA GAAAGAGTAGAAAGCTGTCtt TTTTTGGAT

1#-2 AGCTATCCAAAAA aaGACAGCTTTTCTACTTCTTTC TCTCTTGAA GAAAGAGTAGAAAGCTGTCtt GG

2#-1 GATCCC aaCACGCTAATGCCCTGAAGA TTCAAGAGA TCTTCAGGGCATTAGCGTGtt TTTTTGGAT

2#-2 AGCTATCCAAAAA aaCACGCTAATGCCCTGAAGA TCTCTTGAA TCTTCAGGGCATTAGCGTGtt GG

3#-1 GATCCC aCTGTAGGAGGCAAGTACAA TTCAAGAGA TTGTACTTGCCTCCTACAGt TTTTTGGAT

3#-2 AGCTATCCAAAAA aCTGTAGGAGGCAAGTACAA TCTCTTGAA TTGTACTTGCCTCCTACAGt GG

4#-1 GATCCC aaCTGGTGGAACCAAGGAAAC TTCAAGAGA GTTTCCTTGGTTCCACCAGtt TTTTTGGAT

3#-2 AGCTATCCAAAAA aaCTGGTGGAACCAAGGAAAC TCTCTTGAA GTTTCCTTGGTTCCACCAGtt GG

NC-1 GATCCCC TTCTCCGAACGTGTCACGT TTCAAGAGA ACGTGACACGTTCGGAGAA TTTTTT

NC-2 AGCTAAAAAA TTCTCCGAACGTGTCACGT TCTCTTGAA ACGTGACACGTTCGGAGAA GGG

(c) The constructed FDS fusion protein and four RNAi vector plasmids with different targets or negative control plasmids (NS, coding disordered sequence, no interference effect) were co-transfected into tool cell 293T cells in good growth state, 36 After 48 hours, immunofluorescence and immunoblotting were used to observe the expression of GFP/RFP and detect the expression of FLAG protein to judge the interference effect of different targets.

In step (2), the lentiviral vector construction system (Jikai, Shanghai) adopted in the present invention consists of the backbone plasmid pGCSIL-GFP, the helper plasmid pHelper 1.0 carrying the virus gag, pol, and rev genes, and the helper plasmid containing VSV-G pHelper 2.0 composition. The construction and identification steps of the FDS interference plasmid pGCSIL-sh-FDS In the previous (1) results of foreign source screening, the most effective sequence was the double-stranded DNA whose shRNA was designed and synthesized for 1#. 1: 5'-CcggaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAAGCTGTCttTTTTTg-3'; 2: 5'-aattcaaaaaaaGACAGCTT TCTAC TCTTTCTCTCTTGAAGAAAGAGTAGAAAGCTGTCtt-3', ligated with the pGCSIL-GFP lentiviral backbone plasmid vector. After ligation, the product was transformed into DH5 Escherichia coli and identified by PCR and sequencing (Shanghai Meiji Company). The primers for positive clones identified by PCR were Primer: Up: 5'-CCTATTTCCCATGATTCCTTCATA-3'; Down: 5'-GTAATACGGTTATCCACGCG-3. Lentiviral Backbone Plasmid Recombination Vector Construction Framework:

NO. 5' STEMP Loop STEMP 3' 1 Ccgg aaGACAGCTTTTCTACTTCTTTC TTCAAGAGA GAAAGAGTAGAAGCTGTCtt TTTTTg 2 aattcaaaaa aaGACAGCTTTTCTACTTCTTTC TCTCTTGAA GAAAGAGTAGAAGCTGTCtt

Ligation product PCR identification cycle conditions:

Step (3) FDS interferes with the packaging, collection, purification, identification and titer determination of the recombinant lentiviral vector LV-sh-FDS. High-purity and endotoxin-free extraction of three plasmids pGCSIL-GFP, pHelper 1.0 and pHelper 2.0, preparation of three DNA solutions to absorb pGCSIL-sh-FDS (20μg), pHelper 1.0 (15μg) and pHelper 2.0 (10μg) by Invitrogen Lipofectamine 2000 instructions for co-transfection of 293T cells, while establishing a positive control for recombinant virus. After 8 hours of transfection, the complete medium was replaced, and the culture medium was continued for 48 hours at 37° C. in a 5% CO ₂ incubator, and the cell supernatant rich in lentiviral particles was collected.

Another object of the present invention is to provide the application of the gene drug LV-sh-FDS in cardiac hypertrophy. In the Ang II (1 μM) induced cardiomyocyte hypertrophy model, the application of LV-sh-FDS effectively inhibited the expression of FDS, and effectively reversed the hypertrophic response of cardiomyocytes to angiotensin II (specifically, including the cell area decrease, reduction of hypertrophy marker genes such as β-MHC and BNP, etc.). In spontaneously hypertensive rats, intramyocardial injection of LV-sh-FDS not only reduced the expression level of FDS in myocardial tissue, but also reduced the expression of hypertrophic marker genes such as β-MHC and BNP, and partially reversed the cardiac index including whole heart/ Body weight (HW/BW), left ventricle weight/body weight (LVW/BW), and echocardiographic indexes IVSD, EF, and FS% were improved to varying degrees.

In addition to being used in gene therapy, LV-sh-FDS can also be used in research related to small G proteins. For example, as detected by western and Rho activity kits, LV-sh-FDS pre-incubated cardiomyocytes can effectively inhibit Ang II ( 1μM) induced RhoA activity increased, but the expression of total RhoA protein did not change, thus revealing the relationship between FDS and RhoA in Ang II (1μM)-induced cardiac hypertrophy.

LV-sh-FDS can also be used to study the function of the key enzyme FDS in the mevalonate pathway, for example, to find the total cholesterol level (TC), low-density lipoprotein (LDL-C) was decreased, and FDS in the liver tissue was also inhibited.

The carrier of the invention can specifically reduce the expression of FDS gene after effectively transfecting cells or tissues, so that it can be applied to gene therapy or gene function research. The most important feature of the LV-sh-FDS provided by the present invention is to provide a targeted FDS inhibitory effect, and use a recombinant lentiviral vector as a carrier, so that the interference effect can be sustained. The entire preparation process uses plasmids to avoid adenovirus contamination by traditional methods. The target gene FDS targeted in the present invention is a key enzyme in the mevalonate pathway and is also a key enzyme in the isoprene pathway. The prenylated intermediates produced are small G protein family including RhoA activation and functional provide an important substrate. The mevalonate pathway is the only pathway for cholesterol synthesis in mammals. By inhibiting the expression of FDS and reducing the activity of RhoA, it can effectively inhibit Ang II-related cardiac hypertrophy. Additionally, cholesterol production in the mevalonate pathway was also reduced by reducing FDS expression.

At the same time, the LV-sh-FDS of the present invention has a fluorescent marker EGFP to facilitate detection of transfection efficiency. Experiments have shown that LV-sh-FDS can effectively transfect cardiomyocytes and myocardial tissue. Cell level transfection experiments show that when the MOI is 20, the transfection efficiency of cardiomyocytes can reach more than 80%. At the same time, the route of administration can be various ways such as direct myocardial injection or intravenous administration and coronary artery administration.

The drug efficacy experiment of the present invention shows that LV-sh-FDS can effectively inhibit the expression of FDS gene in cardiomyocytes and myocardial tissue.

The benefits of the present invention are:

1 The present invention designs four effective interference sequences for the FDS target gene according to the online principle. In order to overcome the low transfection efficiency of primary cells and the lack of commercial antibodies for the target gene, four FDS interference plasmids and FDS fusion gene plasmids are constructed by co-transfection After transfecting 293T cells, the most effective interference fragments were screened out by detecting marker proteins, and verified in target cells, laying a good experimental foundation for further research on FDS genes.

2. In the present invention, on the basis of constructing the most effective FDS interference plasmid, the recombinant lentiviral interference vector construction system is further constructed to obtain LV-sh-FDS by packaging, which not only overcomes the low transfection efficiency of non-viral vectors, but also avoids the The recombinant adenovirus has disadvantages such as immunogenicity and short expression time. This recombinant lentiviral vector is a "suicide" virus that is relatively safe and can be integrated into the host genome, making the interference effect more sustainable, and can be widely used in vivo Gene therapy and gene function research.

3. The LV-sh-FDS in the present invention is marked with EGFP to express "enhanced fluorescent protein" after transfection, which is convenient for detection after transfection.

4. The LV-sh-FDS constructed by the present invention can effectively transfect the cultured cardiomyocytes, the mRNA level of FDS is significantly suppressed (78.56%), and the hypertrophic response of cardiomyocytes induced by Ang II (1 μM) has been significantly inhibited. After myocardial injection of LV-sh-FDS in spontaneously hypertensive rats for 11 weeks, the mRNA level of FDS was suppressed (37.25%), and the hypertrophy genes β-MHC and BNP were significantly suppressed, and the cardiac index (HW/BW, LVW /BW) was partially reversed, while the echocardiographic indexes (IVSD, EF, FS%) were partially improved. It is predicted that the carrier provides an important experimental resource for the study of cardiac hypertrophy.

5. LV-sh-FDS myocardial injection of the present invention effectively transfects myocardial tissue, while the hypertrophy response is improved, serum TC LDL-C is also reduced to varying degrees, and the level of FDS in liver tissue can also be effectively suppressed, indicating that LV-sh-FDS may pass Blood circulation affects the expression of FDS in the liver, and then regulates cholesterol metabolism, which indicates that the carrier can be implemented in various ways such as myocardial injection, intravenous injection, and coronary artery injection.

6. The present invention can effectively transfect cardiomyocytes. When the MOI is 20, the transfection efficiency can reach more than 80%. In vivo studies, myocardial injection of LV-sh-FDS can be highly expressed, which is suitable for gene function research and gene expression in vivo. applications in the field of therapy.

Description of drawings

Figure 1 is a schematic diagram of the pGC-U6/Neo/DsRed vector structure.

Fig. 2 is a schematic diagram of the structure of the pEGFP-C1 vector.

Fig. 3 is a schematic diagram of the structure of the lentiviral backbone plasmid pGCSIL-GFP vector.

Figure 4 is a picture of the effective target interference effect of the exogenous sieve target.

Fig. 5 is an agarose gel electrophoresis identification map of pGCSIL-sh-FDS.

Fig. 6 is a sequence identification map of pGCSIL-sh-FDS.

Figure 7 is a picture of the transfection efficiency of LV-sh-FDS in cardiomyocytes and myocardial tissue.

Figure 8 is a picture of LV-sh-FDS effectively down-regulating FDS mRNA in cardiomyocytes/tissues.

Fig. 9 is a picture showing that LV-sh-FDS effectively improves the hypertrophic response of cardiomyocytes related to angiotensin II.

Figure 10 is a picture of LV-sh-FDS improving myocardial hypertrophy in spontaneously hypertensive rats.

Fig. 11 is the echocardiogram of spontaneously hypertensive rats affected by LV-sh-FDS.

Fig. 12 is a picture showing the effect of LV-sh-FDS on the activity and expression of RhoA.

Figure 13 is a picture of LV-sh-FDS affecting liver FDS mRNA in spontaneously hypertensive rats.

Detailed ways

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

Example 1: FDS fusion gene plasmid and interference plasmid co-transfection tool cell screening FDS interference most effective target sequence siRNA:

1. According to the design principle, 4 interference targets were designed according to the online RNAi series design software, and the double-stranded DNA was synthesized and connected to the linearized pGC-U6/Neo/DsRed vector (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd., see Figure 1) After the correct clone was identified, the plasmid was extracted for future use.

The target sequence (Target Seq) is as follows:

#1: GACAGCTTTCTACTTCTTTC

2#: CACGCTAATGCCCTGAAGA

#3: CTGTAGGAGGCAAGTACAA

#4: CTGGTGGAACCAAGGAAAC

At the same time, the respective DNA synthesis fragment information is as follows:

1#-1: 5'-GATCCCaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAAGCTGTCttTTTTTGGAT-3'

1#-2: 5'-AGCTATCCAAAAAaaGACAGCTTTCTACTCTTTCTCTCTTGAAGAAAGAGTAGAAAGCTGTCttGG-3'

2#-1: 5'-GATCCC-aaCACGCTAATGCCCTGAAGATTCAAGAGATCTTCAGGGCATTAGCGTGttTTTTTGGAT-3'

2#-2: 5'-AGCTATCCAAAAAaaCACGCTAATGCCCTGAAGATCTCTTGAATCTTCAGGGCATTAGCGTGttGG-3'

3#-1: 5'-GATCCCaCTGTAGGAGGCAAGTACAATTCAAGAGATTGTACTTGCCTCCTACAGtTTTTTGGAT-3'

3#-2: 5'-AGCTATCCAAAAAaCTGTAGGAGGCAAGTACAATCTCTTGAATTGTACTTGCCTCCTACAGtGG-3'

4#-1: 5'-GATCCCaaCTGGTGGAACCAAGGAAACTTCAAGAGAGTTTCCTTGGTTCCACCAGttTTTTTGGAT-3'

4#-2: 5'-AGCTATCCAAAAAaaCTGGTGGAACCAAGGAAACTCTCTTGAAGTTTCCTTGGTTCCACCAGttGG-3', and synthesized negative control (NC), the DNA fragment information is as follows:

NC-1: 5'-GATCCCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTT-3';

NC-2: 5'-AGCTAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAGGG-3'.

2. Preparation of FDS Fusion Protein Particle Vector

Purchase the FDS cDNA library (imaGenes, Germany), use the PCR method to capture the target gene and the expression vector pEGFP-C1 Vector (clonetech, #632465, see Figure 2) respectively, perform double enzyme digestion and directional ligation at the C-terminal of the expression vector Add the FLAG tagged protein, and transform the product into competent cells for PCR identification and sequencing analysis to compare and correctly construct the successful fusion protein expression plasmid vector pEGFP-FDS-FLAG, and perform ultra-pure endotoxin-free extraction for future use.

3. Exogenous co-transfection of 293T cells to screen for the most effective target sequence of FDS RNAi

Cultivate tool cells (i.e. 293T cells) in a good growth state, construct FDS overexpression fusion gene plasmids (1 μg), RNAi vector plasmids (1 μg) for different targets and negative reference plasmids (1 μg), according to Invitrogen’s Instructions for use of lipofectamine 2000 Co-transfect 293T cells, observe the expression of GFP/RFP under a fluorescent microscope after 36 hours, and preliminarily judge the interference effect of different targets. At the same time, the experimental group with a transfection efficiency greater than 70% can enter the follow-up detection, 48 hours Afterwards, Western blot was used to detect the expression of FLAG protein at the same time, and further confirmed that the most effective target was No. 1 (see Figure 4). Figure 4 A: Immunofluorescence observation of the changes of GFP/RFP co-transfected with overexpression plasmid and interference plasmid in 293 cells, where a: green fluorescence (GFP), b: red fluorescence (RFP), c: plain light microscope; B: Western -blot detection of FLAG protein changes after co-transfection of overexpression plasmid and interference plasmid protein in 293 cells, where OV: overexpression plasmid transfection group, NC: negative virus transfection group, α-Tubilin: internal reference.

Example 2: Construction and identification of lentiviral recombinant plasmid pGCSIL-sh-FDS

The most effective target sequence obtained by exogenous screening target is No. 1: GACAGCTTTCTACTCTTTC, the double-stranded DNA of its shRNA is synthesized, and the information of the synthesized fragment is as follows:

1: CcggaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAAAGCTGTCttTTTTTTg;

2: aattcaaaaaaaGACAGCTTTCTACTCTTTTCTCTTGAAGAAAGAGTAGAAAGCTGTCtt.

Ligated to the linear pGCSIL-GFP vector (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd., see Figure 3) after being digested by AgeI and EcoRI, ligation reaction system: 1 μl of vector DNA (100ng/μl) recovered by enzyme digestion, Annealed double-stranded DNA (100ng/μl) 1μl, 10×T4 phage DNA ligase buffer 1μl, T4 phage DNA ligase 1μl, dd H2O 7μl, ligate at 4°C for 12h, then culture at 37°C for 16h to transform into DH5 large intestine Bacillus, positive clones were extracted and identified by PCR and sequencing. The primers of the positive clones identified by PCR were Primer: Up: 5'-CCTATTTCCCATGATTCCTTCATA-3'; Down: 5'-GTAATACGGTTATCCACGCG-3, the PCR product identification results (Figure 5) and sequencing results (Figure 6) of bacterial clones confirmed that pGCSIL- The insertion direction and sequence of sh-FDS are correct. After sequencing, positive clone plasmids were extracted, and the obtained recombinant plasmid pGCSIL-sh-FDS was used to prepare lentiviral vectors.

In Figure 5, lane 1 is negative control (ddH ₂ O), lane 2 is negative control (empty vector group) 306bp, lane 3 is Marker: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb , 1.5kb, 1kb, 750bp, 500bp, 250bp, lanes 4-8 are 343bp positive clones ligated into pGCSIL-shFDS vector.

PCR cycling conditions (Table 2):

Example 3: Preparation of FDS gene RNA interference recombinant lentiviral vector (LV-sh-FDS)

The lentiviral vector construction system (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.) consists of the backbone plasmid vector pGCSIL-GFP, the helper plasmid pHelper1.0 carrying the virus gag, pol, and rev genes, and the helper plasmid pHelper containing VSV-G 2.0 composition.

Prepare recombinant virus plasmids encoding lentiviral particles and helper plasmids, namely pGCSIL-sh-FDS, pHelper1.0 and pHelper2.0 plasmids, and perform high-purity endotoxin-free extraction respectively. Draw the plasmid pGCSIL-sh-FDS (20 μg), pHelper 1.0 (15 μg) and pHelper 2.0 (10 μg), carry out co-transfection according to Invitrogen Company Lipofectamine 2000 instructions, successfully package FDS) gene RNA interference recombinant lentivirus (LV-sh -FDS), and set up a positive control at the same time, that is, pGCSIL-NS (negative reference) (20 μg), pHelper 1.0 (15 μg) and pHelper 2.0 (10 μg) co-transfect 293T cells to construct a negative control recombinant lentiviral vector,

After 8 hours of transfection, the complete medium was replaced, and the culture medium was continued for 48 hours at 37° C. in a 5% CO ₂ incubator, and the cell supernatant rich in lentiviral particles was collected. Centrifuge at 4000g for 10min at 4°C to remove cell debris and filter the supernatant with a 0.45μM filter to obtain lentivirus for use in general cell experiments. If you want to obtain a higher concentration of lentivirus, you can further concentrate and purify it to obtain a high-titer lentivirus concentrate, subpackage the virus concentrate for long-term storage at -80 degrees, and take one of them to measure the biological titer of the virus.

Example 4: FDS Gene RNA Interference Recombinant Lentiviral Vector (LV-sh-FDS) was used in gene therapy research of angiotensin II-related cardiac hypertrophy model.

1. Application of LV-sh-FDS in hypertrophy of cardiomyocytes cultured in vitro

Cut the ventricles of 1-3 day old newborn WKYs mice into small pieces of about 1mm2, and digest them repeatedly with a mixture of 0.125% trypsin and 0.05% collagenase type II, filter them through a 200-mesh stencil and centrifuge at 1000g for 6min to collect cells, 5% After culturing in a CO ₂ incubator at 37°C for 1 h, remove the adherent cells, adjust the cell density to 5×10E5-1×10E6/mL and inoculate them in a six-well plate for culture. According to the virus-to-cell ratio (MOI) of 20, LV-sh-FDS and negative virus particles (control) were added to infect neonatal mouse cardiomyocytes, and the complete medium was changed after 24 hours. Transfection efficiency (efficiency about 80% or more), see Figure 7A, after 72 hours of infection, the serum-free medium was changed for 24 hours, and Ang II (1 μM) was added to incubate for 24 hours, and then the experiment was terminated. The results showed that: LV-sh-FDS effectively inhibited the expression of FDS in neonatal mouse cardiomyocytes, and the inhibitory effect was more obvious when Ang II (1 μM) was co-incubated, negative virus particles had no effect on the expression of FDS, see Figure 8A(i) . At the same time, the experimental results showed that LV-sh-FDS could effectively inhibit the hypertrophic response of cardiomyocytes to Ang II (1 μM), which was manifested by the reversal of cell area and the down-regulation of the expression of hypertrophic genes BNP and β-MHC. See Figure 9; in addition, pre-incubation of LV-sh-FDS can significantly inhibit the increase of RhoA activity induced by Ang II (1 μM) (incubation for 15 min), and the expression of RhoA protein does not change, see Figure 12.

Figure 7A: Immunofluorescence microscope observation of transfected cardiomyocytes (10×) (MOI=20); B: Western-blot detection of GFP protein expression in myocardial tissue, where a: SHR rats without virus solution group, b: SHR rat negative virus transfection group, c: SHR rat interference virus knockout group, d: WKY rat group.

The data in Figure 8 are expressed as mean ± standard error. Figure 8A cell level: 1: non-transfection group, 2: negative virus transfection group, 3: interference virus knockout group, 4: negative virus transfection cardiac hypertrophy model group (AngII incubation), 5: interference virus transfection Myocardial hypertrophy model (Ang II incubation), ^# P＜0.05and ^## P＜0.01vs.2 group. ^** P＜0.01 vs.4group; Figure 8B Myocardial tissue, where b: SHR rat negative virus transfection group, c: SHR rat interference virus knockout group, d: WKY rat group, ^* P<0.05 and ^** P<0.01 vs.b group.

The data in Fig. 9 are presented as mean ± standard error. In Figure 9, A: cell area, B: hypertrophy marker gene β-MHC, C: hypertrophy marker gene BNP; where 1 on the abscissa is the negative virus infection group, and 2 is the interfering virus knockout group; ^## P＜0.01 vs. 1 group (Ang II-) and ^** P<0.01 vs. 1 group (Ang II+).

The data in Figure 12 are presented as mean ± standard error. Figure 1: negative virus transfection group, 2: negative virus transfection myocardial hypertrophy model group (Ang II incubation), 3: interference virus transfection myocardial hypertrophy model group (Ang II incubation). ^# P<0.05 vs. 1 group and ^* P<0.05 vs. 2 group.

2. Study on LV-sh-FDS for myocardial hypertrophy in spontaneously hypertensive rats (SHRs)

A total of 12 7-week-old SHR rats and 6 7-week-old WKYs rats were enrolled in the group. Chloral hydrate (400mg/kg/mg) was injected intraperitoneally for general anesthesia. The supine position was taken, the tracheotomy was performed, and the animal ventilator was connected. Chest cavity, expose the heart, draw 80uL (about 5×10 ⁷ TU) of LV-sh-FDS or negative virus particles, inject the recombinant virus at 3-4 points on the apex of the heart and the wall of the left ventricle, and inject the same dose of normal saline into the myocardium of WKYs rats To normal control. Close the chest and remove the ventilator. After waking up, the rats were fed with common feed in a clean environment for 11 weeks, and then echocardiographic examination was performed. After the rats were weighed, the heart was weighed and the left ventricle was retained for reverse transcription and real-time quantitative PCR detection of FDS, BNP, and β-MHC gene detection. . The results showed that: LV-sh-FDS can significantly down-regulate the transcription of FDS (Fig. 8B), and at the same time the hypertrophy genes BNP and β-MHC are reduced, and the heart index (HW/BW and LVW/BW) is partially relieved, see Fig. 10 . In addition, the results of echocardiography also improved to varying degrees, see Figure 11.

The data in Figure 10 are expressed as mean ± standard error. A of FIG. 10 : HW/BW (heart weight/body weight), B: LVW/BW (left ventricular weight/body weight), C: hypertrophy marker gene β-MHC, D: hypertrophy marker gene BNP. Each figure b: SHR rat negative virus transfection group, c: SHR rat interference virus knockout group, d: WKY rat group; ^# P＜0.05 vs.d group. ^* P<0.05 and ^** P<0.01 vs. b group.

The data in Figure 11 are expressed as mean ± standard error. A of FIG. 11 : IVSD (end-diastolic ventricular septal wall thickness), B: LVPWD (end-diastolic ventricular posterior wall thickness), C: FS (short axis shortening), D: EF (ejection fraction). In each figure b: SHR rat negative virus transfection group, c: SHR rat interference virus knockout group, d: WKY rat negative control group. ^#P <0.05 and ^## P<0.01 vs.d group; ^* P<0.05 and ^** P<0.01 vs.bgroup.

Example 5: Research of LV-sh-FDS in Regulation of Cholesterol Metabolism

A total of 12 7-week-old SHR rats and 6 7-week-old WKYs rats were enrolled in the group. Chloral hydrate (400mg/kg/mg) was injected intraperitoneally for general anesthesia. The supine position was taken, the tracheotomy was performed, and the animal ventilator was connected. Chest cavity, expose the heart, draw 80uL (about 5×10 ⁷ TU) of LV-sh-FDS or negative virus particles, inject the recombinant virus at 3-4 points on the apex of the heart and the wall of the left ventricle, and inject the same dose of normal saline into the myocardium of WKYs rats To normal control. Close the chest and remove the ventilator. After waking up, the rats were fed with common feed in a clean environment for 11 weeks, and then the whole blood was collected from the rats. After centrifugation at 3000g for 15 minutes, the serum was separated and stored at -80°C. Take 500uL serum to detect rat serum cholesterol (TC) and low-density lipoprotein (LDL-C). The results showed that LV-sh-FDS myocardial injection effectively transfected myocardial tissue, while the hypertrophic response was improved, serum TC LDL-C was reduced to varying degrees, and FDS mRNA levels in liver tissue were also effectively suppressed, see Table 1 and Figure 13, respectively. It indicates that LV-sh-FDS may affect the expression of FDS in the liver through blood circulation, thereby affecting cholesterol metabolism and decreasing serum cholesterol concentration, and also indicates that the carrier may be implemented in various ways such as myocardial injection, intravenous injection, and coronary injection.

Table 1: LV-sh-FDS affects serum cholesterol data in spontaneously hypertensive rats

Note: The data are expressed as mean ± standard error; b: SHR rat negative virus transfection group; c: SHR rat interference virus knockout group; d: WKY rat negative control group. ; ^#P <0.05 and ^## P<0.01 vs.d group and ^* P<0.05 vs.bgroup.

In Fig. 13, b: SHR rat negative virus transfection group, c: SHR rat interference virus knockout group, d: WKY rat negative control group. ^# P<0.01 vs. d group and ^* P<0.05 vs. b group.

Sequences involved in the present invention

<110> Zhejiang University

<120> Construction and application of farnesyl pyrophosphate synthase RNA interference lentiviral vector

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<221>Target sequence

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<221>Target sequence

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Claims (6)

1. A recombinant lentiviral vector for farnesyl pyrophosphate synthase RNA interference, characterized in that, the carrier contains the lentiviral backbone plasmid recombinant vector pGCSIL-sh-FDS, and the recombinant vector is p-GCSIL-sh-FDS The double-stranded DNA fragment for shRNA is connected to the MCS of the GFP backbone plasmid vector. The target sequence is: 1#-GACAGCTTTCTACTCTTTC, and the information of the synthesized DNA fragment is: 1#-1:5'-CcggaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGTAGAAA GCTGTCt tT TTTTg-3' , 1#-2: 5'-aattcaaaaaaaGACAGCTTTACTCTTTTCTCTCTTGAAGAAAGAG T AGAAAGCTGTCtt-3'. 2. A preparation method for farnesyl pyrophosphate synthase RNA interference recombinant lentiviral vector, characterized in that, according to the FDS mRNA sequence, the double-stranded DNA fragment for shRNA is designed and synthesized as follows: Sense strand: 5'-CcggaaGACAGCTTTCTACTCTTTCTTCAAGAGAGAAAGAGT AGAAA GCTGTCt tT TTTTg-3' Antisense strand: 5’-aattcaaaaaaaGACAGCTTTACTCTTTTCTCTCTTGAAGAAAGAG T A G AAA GCTGTCtt-3’ Then the above DNA fragments were connected to the MCS of the lentiviral backbone plasmid vector pGCSIL-GFP to construct the pGCSIL-sh-FDS recombinant vector, and then the pGCSIL-sh-FDS recombinant vector, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells to obtain the obtained The recombinant lentiviral vector LV-sh-FDS described above. 3. the preparation method for farnesyl pyrophosphate synthase RNA interference recombinant lentiviral vector according to claim 2, is characterized in that, introduces Age I and EcoRI restriction site at the end of synthetic DNA fragment. 4. the preparation method for farnesyl pyrophosphate synthase RNA interference recombinant lentiviral vector according to claim 2, it is characterized in that pGCSIL-GFP is digested with enzymes of Age I and EcoRI, after reclaiming large fragments, it is After ligation with DNA fragments, the competent bacteria were transformed to obtain recombinant positive clones. 5. The application of the recombinant lentiviral vector targeting farnesyl pyrophosphate synthase RNA interference according to claim 1 in the preparation of drugs for the treatment of cardiac hypertrophy. 6. The application of the recombinant lentiviral vector targeting farnesyl pyrophosphate synthase RNA interference according to claim 1 in the preparation of drugs for regulating cholesterol metabolism.

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