CN105331635B

CN105331635B - Inducible lentivirus expression system and construction method and application thereof

Info

Publication number: CN105331635B
Application number: CN201510751609.7A
Authority: CN
Inventors: 苟德明; 康康; 黄炼; 李洁璇; 牛燕琴; 张利敏; 吴伊可; 姚丽君
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2015-11-05
Filing date: 2015-11-05
Publication date: 2021-02-02
Anticipated expiration: 2035-11-05
Also published as: CN105331635A

Abstract

The invention discloses an inducible lentivirus expression system, which comprises a target gene expression frame and a rtTA expression frame, wherein the target gene expression frame comprises an inducible promoter containing a tetracycline cis-response element and a target gene, and the rtTA expression frame comprises the promoter, rtTA, a connecting peptide and a screening gene; the connecting peptide is P2A connecting peptide; the screening gene is Puro; the inducible promoter is TetO6 or TRE 3G. The invention also discloses a construction method of the inducible lentivirus expression system, which comprises the following steps: constructing a pLVX-Puro vector; synthesizing and cloning a TetON3G gene; construction of pLVX-rtTA 3; the subcloned EF1a promoter; subcloning red fluorescent protein RFP; subclones contain tetracycline cis-responsive element inducible promoters TRE3G or TETO 6. After the inducible expression system provided by the invention is introduced into cells, the target gene can be induced and expressed at the maximum level while keeping low background expression, and the inducible expression system has sensitive response to an inducer, high efficiency and small molecular weight, and can be widely applied to gene function research.

Description

Inducible lentivirus expression system and construction method and application thereof

Technical Field

The invention belongs to the field of functional genomics research, and particularly relates to an inducible lentivirus expression system and a construction method and application thereof.

Background

The overexpression technology is a common means for researching gene functions, and the inducible overexpression accurately controls the inducible expression of a target gene by adding an inducer, is a powerful tool for researching the gene functions, and is particularly well applied to transgenic animals.

There are four major classes of inducible expression systems that have been developed: a Cre-loxP system (Cre-loxP recombinase system), a tetracycline (Tet) induction system, an ecdysone induction system, and a lactose induction system. Among them, the tetracycline inducible regulation system is the most widely used inducible expression system, and is divided into: the tTA system (Tet-off) and the rtTA system (Tet-on). Among them, Tet-on has been widely used in transgenic mouse models. However, rtTA (trans-tetracycline controlled transcription factor) system has the disadvantages of insufficient sensitivity to inducer, high background level (leaky expression) and low inducible expression level.

For this reason, researchers have focused on the improvement of rtTA, the second generation rtTA-S2 and rtTA-M2 systems developed, to some extent, to reduce the leaky expression of the inducible system and to improve the efficiency of induction (Urlinger S, Baron U et al, Proc Natl Acad Sci, 2000).

The background expression level of the target gene is further reduced by fusing a VP16 minimal transcription activation region with rtTA-M2 (

M R, Gohla G et al, FEBS, 2002). Qu et al further improved stability, reduced leakage, and 10-fold increased sensitivity to inducer Dox over original rtTA by a second generation rtTA2S-M2 system constructed by optimizing codons and other means (Qu Z, Thottassery J V et al, Gene, 2004). The third generation rtTA3 induction system constructed by Das et al can improve the sensitivity to Dox 25 times higher than that of original rtTA and improve the target gene expression level 5 times higher in the high concentration Dox induction state while keeping the background expression unchanged in the non-induced state (DasAT, Zhou X et al, Journal of biological Chemistry, 2004). Similarly, the third generation TetON3G type Tet-ON system is further site-modified ON the basis of rtTA2S-M2 system and combined with an improved TRE3G inducible promoter regulated and controlled by TetON3G, so that the leakage phenomenon can be further reduced, and the reduction of the leakage phenomenon can be realizedInducer Dox was dosed to 1/10 on rtTA2S-M2 only and was able to attenuate potential cytotoxicity (Fan X, Petitt M et al, Endocrinology, 2012).

In conclusion, researchers are dedicated to the improvement of rtTA, and the developed new system reduces the leakage expression of an induction system to a certain extent and improves the induction efficiency, but the effect is limited. The Tet-ON inducible expression system, especially the inducible system established by using a lentiviral vector, is still to be improved and developed.

Disclosure of Invention

The invention provides an inducible lentivirus expression system, aiming at further reducing the leakage phenomenon and improving the inducible expression level.

An inducible lentivirus expression system comprises a target gene expression frame and an antisense tetracycline transcription activator (rtTA) expression frame, wherein the target gene expression frame comprises an inducible promoter containing a tetracycline cis-response element and a target gene, and the rtTA expression frame comprises the promoter, rtTA, connecting peptide and a screening gene. The screening gene is Puro.

Furthermore, the inducible promoter is TetO6 or TRE3G, and the promoter can be selected from common CMV, PGK, EF1a, Ubc and SV 40; the rtTA is rtTA3 or TetON 3G.

Further, the inducible promoter is TetO6, and the rtTA is TetON 3G.

Furthermore, when TetON3G was expressed using the PGK promoter, the induction efficiency of the target gene was the highest.

Preferably, the linker peptide is a P2A linker peptide. In order to reduce the size of the lentiviral expression vector, P2A connecting peptide is adopted to simultaneously express the antisense tetracycline transcriptional activator and the resistance selection gene Puro.

The invention also provides a construction method of the novel inducible lentivirus expression system, which comprises the following steps:

(1) constructing a pLVX-Puro vector:

the system selects pLVX-Puro vector as a slow virus framework vector, firstly, Tth111I, SacII and BsmBI sites on Puro sequence are mutated through site-directed mutagenesis technology, and BamHI and SpeI sites are respectively introduced before and after the Puro cDNA sequence;

(2) synthesis and cloning of the TetON3G gene:

gene synthesis is carried out according to the published TetONG sequence, XbaI site ON the sequence is subjected to site-specific mutagenesis, and a multiple cloning site is introduced into the upstream of the Tet ON3G sequence: ClaI-MluI-XbaI-AgeI-EcoRI-XhoI, a connecting peptide P2A and SmaI and BamHI sites are introduced at the downstream of the TetON3G sequence, the SmaI site is positioned between TetON3G and P2A, and BamHI positioned behind P2A mediates fusion connection with the Puro sequence. The complete sequence of the synthetic gene is shown in SEQ ID NO. 1. The synthesized gene fragment is subjected to double enzyme digestion by ClaI-BamHI, and then is cloned to a reconstructed pLVX-Puro vector through the same enzyme digestion site, and the new cloning vector is named as pLVX-TetON 3G;

(3) construction of pLVX-rtTA 3:

taking pTRIPz (thermo fisher) vector as a template, obtaining a rtTA3 fragment (the nucleotide sequence of which is shown as SEQ ID NO.2) through PCR amplification, carrying out enzyme digestion by BstBI-SmaI to replace the TetON3G on the pLVX-TetON3G vector, and obtaining a new cloning vector pLVX-rtTA 3;

(4) subcloned EF1a promoter:

using pCDH-CMV-MCS-EF1-copGFP (SBI) vector as template, amplifying to obtain EF1a promoter fragment (the nucleotide sequence is shown as SEQ ID NO.3), carrying out XhoI-BstBI double enzyme digestion, and then respectively cloning to pLVX-TetON3G or pLV X-rtTA3, wherein the obtained new vectors are respectively named as pLVX-EF1a-TetON3G or pLVX-EF1a-rtTA 3;

(5) subcloning red fluorescent protein RFP:

amplifying to obtain an RFP cDNA sequence (the nucleotide sequence of the RFP cDNA sequence is shown as SEQ ID NO.4) by taking a pTRIPz (thermo fisher) vector as a template, cloning to pLVX-EF1a-TetON3G or pLVX-EF1a-rtTA3 after AgeI-EcoRI double enzyme digestion, and respectively naming the obtained new vector as pLVX-RFP-EF1a-TetON3G or pLVX-RFP-EF1a-rtTA 3;

(6) subclones contain tetracycline cis-responsive element-inducible promoters TRE3G or TETO 6:

respectively digesting pLVX-RFP-EF1a-TetON3G or pLVX-RFP-EF1a-rtTA3 by XbaI-AgeI, then respectively inserting the synthesized inducible promoter TRE3G or TETO6 (the nucleotide sequences are respectively SEQ ID NO5 and SEQ ID NO 6) promoter, and respectively naming the obtained new vectors as

pLVX-TRE3G-RFP-EF1a-TetON3G；pLVX-TRE3G-RFP-EF1a-rtTA3

pLVX-TETO6-RFP-EF1a-TetON3G；pLVX-TETO6-RFP-EF1a-rtTA3；

CMV, PGK, Ubc and SV40 promoters are obtained by PCR amplification, after XhoI-BstBI enzyme digestion, EF1a promoters on pLVX-TETO6-RFP-EF1a-TetON3G vectors are respectively replaced, and the obtained new vector is named as

pLVX-TETO6-RFP-CMV-TetON3G，

pLVX-TETO6-RFP-PGK-TetON3G，

pLVX-TETO6-RFP-Ubc-TetON3G，

pLVX-TETO6-RFP-SV40-TetON3G。

In the scheme, restriction sites AgeI and EcoRI are arranged at two ends of the target gene and are used for constructing inducible expression systems of different target genes.

The invention also provides application of the novel inducible lentivirus expression system, and the inducible lentivirus expression system is used for inducible expression of endogenous genes or exogenous genes.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a novel high-efficiency lentivirus induced expression system, which can maintain low background expression and maximally induce and express a target gene after being introduced into cells, has sensitive response to an inducer, high efficiency and small molecular weight, and can be widely applied to gene function research.

Drawings

FIG. 1 is a map of a novel inducible gene expression vector constructed according to the present invention.

FIG. 2 comparison of four combinations of promoters and trans-acting factors (TetO6-rtTA3, TetO6-TetON3G, TRE3G-rtTA3, TRE3G-TetON3G) in the expression of target genes (the second and fourth columns in FIG. 2 show the red fluorescence expression, the red fluorescence protein expression gradually decreases from top to bottom in the second column, and the system induction level gradually decreases).

FIG. 3 shows the results of the effect of positive insertion on inducible expression efficiency and background omission.

FIG. 4 compares the effect of four different promoters on induction efficiency.

FIG. 5 shows the immunoblotting and quantitative analysis results of HA-Ago2 induced by the novel inducible gene expression vector.

FIG. 6 is a quantitative analysis of the immunoblotting result of the novel inducible gene expression vector for inducible expression of the endogenous protein Ago 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. The methods, reagents, etc. used in the present invention are well known to those skilled in the art, and will not be described herein, except as specifically described. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The sources of experimental materials used in the present invention are shown in Table 1.

TABLE 1 Experimental materials and sources

Example 1 construction of vectors and functional analysis of different inducible promoters and antisense tetracycline transcriptional activator

In this example, after combining two inducible promoters TetO6 and TRE3G with two antisense tetracycline transcriptional activators rtTA3 and TetON3G, systematic comparative analysis is performed to analyze the induced expression quantity and the background expression level of the target gene of the corresponding inducible vector formed by four combinations (TetO6-rtTA3, TetO6-TetON3G, TRE3G-rtTA3 and TRE3G-TetON3G) involved in the formation. The preparation steps are as follows:

(1) reconstruction of pLVX-Puro vector

Selecting pLVX-Puro (Clontech) vector as a lentiviral backbone vector, firstly mutating Tth111I, SanII and BsmBI sites on a Puro sequence by a site-directed mutagenesis technology, then using the pLVX-Puro vector as a template, amplifying Puro fragments by using an upstream primer (SEQ ID NO: 7) and a downstream primer (SEQ ID NO: 8), and then cloning into the pLVX-Puro vector which is subjected to double enzyme digestion and then filling in by BspMI-Tth 111I to obtain a reconstructed pLVX-Puro vector backbone;

(2) synthesis and cloning of TetON3G gene

The gene was synthesized according to the published TetON3G sequence and the XbaI site on the sequence was mutated and a multiple cloning site was introduced upstream of the TetON3G sequence: ClaI-MluI-XbaI-AgeI-EcoRI-XhoI, a connecting peptide P2A and SmaI and BamHI sites are introduced at the downstream of the TetON3G sequence, the SmaI site is positioned between TetON3G and P2A, and BamHI positioned behind P2A mediates fusion connection with the Puro sequence. The complete sequence of the synthetic gene is shown in SEQ ID NO. 1. The synthesized gene fragment is subjected to double enzyme digestion by ClaI-BamHI, and then is cloned to a reconstructed pLVX-Puro vector through the same enzyme digestion site, and the new cloning vector is named as pLVX-TetON 3G;

(3) construction of pLVX-rtTA3

Amplifying rtTA3 fragment (SEQ ID NO: 2) by using an upstream primer (SEQ ID NO: 9) and a downstream primer (SEQ ID NO: 10) by using a pTRIPz vector as a template, carrying out double enzyme digestion on a PCR product by using BstBI-SmaI, then cloning the PCR product into a pLVX-TetON3G vector through the same enzyme digestion site, and naming the new cloning vector as pLVX-rtTA 3;

(4) subcloned EF1a promoter

Using pCDH-CMV-MCS-EF1-copGFP (SBI) vector as template, using upstream primer (SEQ ID NO: 11) and downstream primer (SEQ ID NO: 12) to amplify EF1a fragment (SEQ ID NO: 3), using XhoI-BstBI to double-enzyme-cut PCR product, then respectively cloning into pLVX-TetON3G or pLVX-rtTA3 vector by using identical enzyme-cutting sites, and finally naming new cloning vector as pLVX-EF1a-TetON3G or pLVX-EF1a-rtTA 3;

(5) subcloning of Red fluorescent protein RFP

Using pTRIPz (thermo fisher) vector as a template, amplifying an RFP fragment (SEQ ID NO: 4) by using an upstream primer (SEQ ID NO: 13) and a downstream primer (SEQ ID NO: 14), double-digesting a PCR product by AgeI-EcoRI, then cloning the PCR product into a pLVX-EF1a-TetON3G vector or a pLVX-EF1a-rtTA3 vector respectively through the same digestion site, and naming the new cloning vector as pLVX-RFP-EF1a-TetON3G or pLVX-RFP-EF1a-rtTA 3;

(6) subclones containing tetracycline cis-responsive element inducible promoters TRE3G or TETO6

Respectively using XbaI-AgeI to enzyme-cut pLVX-RFP-EF1a-TetON3G or pLVX-RFP-EF1a-rtTA3 vectors, then respectively inserting synthesized inducible promoter TRE3G (SEQ ID NO: 5) or TETO6 promoter (SEQ ID NO: 6) into a skeleton vector through the same enzyme-cutting sites, and obtaining new vectors which are respectively named as

pLVX-TRE3G-RFP-EF1a-TetON3G，pLVX-TRE3G-RFP-EF1a-rtTA3，

pLVX-TetO6-RFP-EF1a-TetON3G，pLVX-TetO6-RFP-EF1a-rtTA3。

TABLE 2 primers required for amplification of different vector sequences

(7) Analysis of red fluorescence expression level of different inducible promoter and trans-acting factor vector

Four vector induction efficiency analyses were performed on the recombinant vectors pLVX-TRE3G-RFP-EF1a-TetON3G, pLVX-TRE3G-RFP-EF1a-rtTA3, pLVX-TETO6-RFP-EF1a-TetON3G and pLVX-TETO6-RFP-EF1a-rtTA3 prepared in (1) - (6), and the specific steps were as follows:

293T cells were cultured at 37 ℃ in 5% CO in DMEM supplemented with 10% FBS₂The day before transfection, cells were seeded in a 24-well plate at a cell density of 1.0X 10⁵Culturing each cell/hole for 12-24 h.

When the cell density reaches 60-80%, four vectors of pLVX-TRE3G-RFP-EF1a-TetON3G, pLVX-TRE3G-RFP-EF1a-rtTA3, pLVX-TETO6-RFP-EF1a-TetON3G and pLVX-TETO6-RFP-EF1a-rtTA3 and the green fluorescent protein plasmid are co-transfected into cells by PEI transfection reagent. The recombinant vector and the green fluorescent vector required by the transfection system are respectively 500ng and 100 ng; after the plasmids are uniformly mixed, NaCl (150mM) is supplemented to ensure that the final volume of the Solutiona is 25 mu l; the required transfection reagent PEI for each set was 4.8. mu.l and NaCl (150mM) for SolutionB was 20.2. mu.l. In the experiment, red fluorescent protein is used as a target gene, and green fluorescent protein is used as a normalization internal reference.

Mixing solution A and solution B, and standing at room temperature for 10 min.

Adding 50 mul transfection reagent into 293T cell culture solution, gently mixing, culturing in an incubator for 4-8h, replacing fresh culture solution, wherein the experiment is divided into two groups, replacing and adding culture solution containing Dox (Doxycyline, final concentration is 2 mug/ml) inducer in one group, replacing fresh culture solution without Dox in the other group (control group), observing the expression conditions of green fluorescent protein and red fluorescent protein under a fluorescence microscope after plasmid transfection for 48 h.

This example compares the TetO6-rtTA3 system developed by Thermofoisher with the TRE3G-TetON3G system developed by Clontech. Four combinations of inducible promoter and trans-acting factor (TetO6-rtTA3, TetO6-TetON3G, TRE3G-rtTA3, TRE3G-TetON3G) vectors were analyzed on the pLVX-RFP-EF1a-P2A-Puro vector backbone for red fluorescent protein expression levels. As seen in fig. 2, in comparison of the inducible promoter TetO6 with TRE3G, TetO6 mediated that the red fluorescent protein expression level (first, second row and second column in fig. 2) was significantly stronger than the promoter TRE3G (third, fourth row and second column in fig. 2), and the background expression level was not significantly changed. In the comparison of the two antisense tetracycline transcriptional activators, the expression level of red fluorescent protein of the induction system in which TetON3G participates is slightly stronger than that of rtTA 3. The results show that the promoter species have a greater effect on gene expression in this system than the antisense tetracycline transcriptional activator. TetO6-TetON3G was the combination with the highest expression level of red fluorescence selected (FIG. 1).

Example 2 construction and functional analysis of Induction vectors with different insertion orientations

In this embodiment, the forward and reverse insertion directions of the target gene expression cassette are compared and analyzed, and the influence of the forward and reverse insertion directions on the induction system is analyzed through the induction expression efficiency and the background leakage level.

(1) Construction of reverse inducible expression vectors

Taking pLVX-TetO6-RFP-EF1a-TetON3G vector as a template, and amplifying by using an upstream primer (SEQ ID No: 15) and a downstream primer (SEQ ID No: 16) to obtain a TetO6-RFP fragment; a pLVX-TRE3G-RFP-EF1a-TetON3G vector is used as a template, and an upstream primer (SEQ ID No: 17) and a downstream primer (SEQ ID No: 16) are used for amplification to obtain a TRE3G-RFP fragment. The PCR product was double digested with EcoRI-XbaI and then cloned into the vector pLVX-EF1a-TetON3G or pLVX-EF1a-rtTA3, which were also double digested, and the resulting new vectors were named as:

pLVX-RFP-TRE3G-EF1a-TetON3G；pLVX-RFP-TRE3G-EF1a-rtTA3；

pLVX-RFP-TetO6-EF1a-TetON3G；pLVX-RFP-TetO6-EF1a-rtTA3。

TABLE 3 primers required for amplification of different vector sequences

(2) Analysis of Red fluorescence expression level of vectors of different insertion orientations

The comparative analysis of the red fluorescence expression level was performed using the 4 recombinant vectors prepared in (1) of this example and the four recombinant vectors used in (7) of example 1, and the specific steps are as follows:

293T cells were seeded in 24-well plates one day before transfection, and cells were cultured for 12-24h after seeding. The same as in example 1.

When the cell density reached 60% to 80%, the transfection experiment was performed as in example 1.

The expression levels of the green fluorescent protein and the red fluorescent protein of the 4 recombinant plasmids in example 2 and the four recombinant plasmids finally obtained in 1 were analyzed 48h after transfecting the cells, and the specific operation was the same as that of example 1.

In this embodiment, on the basis of the selected TetO6-TetON3G, the influence of positive and negative induction directions on the system induction efficiency and local omission expression is analyzed. As seen from FIG. 3, the induction efficiency of the reverse induction system (the expression level of red fluorescence is shown in the third row and the second column of the fourth row of FIG. 3) is slightly higher than that of the forward induction system (the expression level of red fluorescence is shown in the first row and the second column of the second row of FIG. 3), but the background omission level of the reverse induction system is significantly higher than that of the forward induction system, so that the forward induction system is selected to participate in forming a new induction system.

Example 3 construction and functional analysis of Induction vectors for different promoters

In the embodiment, the influence of five different antisense tetracycline transcriptional activator promoters on the system induction efficiency is systematically researched, and the influence of the tetracycline transcriptional activator expression level on the gene induction expression level and the background omission level of the vector is further analyzed on the basis of the screened vector skeleton structure.

(1) Construction of different promoter vectors

Amplifying a CMV fragment by using a pIRESneo-FLAG-HA-Ago2(Addgene) vector as a template and an upstream primer (SEQ ID No: 18) and a downstream primer (SEQ ID No: 19); amplifying a PGK fragment by using a pLVX-AmCyan1-C1(Clontech) vector as a template and an upstream primer (SEQ ID No: 20) and a downstream primer (SEQ ID No: 21); using pTRIPz (Thermofeisher) vector as template, and using upstream primer (SEQ ID No: 22) and downstream primer (SEQ ID No: 23) to amplify Ubc fragment; the SV40 fragment was amplified using pmirGLO (Promega) vector as template, and the upstream primer (SEQ ID No: 24) and the downstream primer (SEQ ID No: 25). The PCR product is double-digested by XhoI-BstBI, and then cloned into a pLVX-TetO6-RFP-EF1a-TetON3G vector which is double-digested by the same method, so as to obtain four new recombinant vectors which are respectively named as:

pLVX-TetO6-RFP-CMV-TetON3G；pLVX-TetO6-RFP-PGK-TetON3G；

pLVX-TetO6-RFP-Ubc-TetON3G；pLVX-TetO6-RFP-SV40-TetON3G。

TABLE 4 primers required for amplification of different vector sequences

(2) Analysis of Red fluorescence expression level of vector of different forward inducible promoters

Comparing the four prepared recombinant vectors with pLVX-TetO6-RFP-EF1a-TetON3G, and analyzing the red fluorescence induced expression level and background omission level of the species vector, wherein the specific steps are as follows:

The four recombinant plasmids, pLVX-TetO6-RFP-EF1a-TetON3G plasmid and green fluorescent protein plasmid were revolved around 293T cells, and the expression levels of green fluorescent protein and red fluorescent protein were analyzed 48h after transfection of the cells, the specific operation was the same as example 1.

In this example, five different promoters were analyzed on the basis of the constructed novel inducible lentivirus expression system. From the results in FIG. 4, it can be seen that in the forward inducible system, the induced system composed of Ubc and PGK is involved in maintaining low background expression, and the red fluorescent protein expression level is stronger than that of the system composed of the other three promoters (second column in the second three rows and second row in FIG. 4), wherein the PGK promoter has smaller molecular weight than that of the Ubc promoter, and the red fluorescent induced expression level is slightly stronger than that of the Ubc promoter.

Example 4 application of novel inducible Lentiviral expression System to Induction expression of foreign genes

In the embodiment, the induction system consisting of the screened better PGK and Ubc promoters is used for carrying out the induced expression analysis of the exogenous genes, and the practical value of the system is further verified through the application of the system in the induced expression of the exogenous gene Red Fluorescent Protein (RFP). The induction system can efficiently induce the expression of the red fluorescent protein RFP after adding the Dox stimulation according to the RFP expression conditions before and after the Dox stimulation of the system, the experimental result is the red fluorescent expression level before and after the Dox stimulation of a TetO6-RFP-PGK/Ubc-TetON3G group in figure 4, and the experiment proves that the induction system can be effectively applied to the induction expression of the foreign gene through the induction expression of the novel induction system to the foreign gene.

Example 5 use of novel inducible Lentiviral expression systems for inducible expression of endogenous genes

In this example, the selected induction system of the superior PGK and Ubc promoters was analyzed for the induction expression of endogenous genes. The practical value of the system is further proved by the application of the induction system in the induction expression of the endogenous gene (Ago 2).

(1) Construction of pLVX-TetO6-FLAG-HA-Ago2-PGK/Ubc-TetON3G vector

Using pIRESneo-FLAG-HA-Ago2 (Addge) vector as a template, using an upstream primer (SEQ ID No: 26) and a downstream primer (SEQ ID No: 27) to amplify a FLAG-HA-Ago2 fragment, using AgeI-EcoRI to double-enzyme-cut a PCR product, cloning the product into a pLVX-TetO6-RFP-PGK/Ubc-TetON3G vector which is subjected to the same double-enzyme cutting, and obtaining two new cloning vectors which are respectively named as follows

pLVX-TetO6-FLAG-HA-Ago2-PGK-TetON3G；

pLVX-TetO6-FLAG-HA-Ago2-Ubc-TetON3G。

(2) Construction of pLVX-TetO6-FLAG-HA-Ago2-UbcrtTA3 vector

The PCR-digested product in (3) of example 1 was cloned into pLVX-TetO6-FLAG-HA-Ago2-Ubc-TetON3G vector, which was also double digested with BstBI-SmaI, and the new cloning vector was named pLVX-TetO6-FLAG-HA-Ago2-Ubc-rtTA 3.

TABLE 5 primers required for amplification of different vector sequences

(3) Protein induction level analysis of inducible expression Ago2 vector

The following steps were performed to compare the induction efficiencies of the 3 recombinant vectors prepared in (1) and (2) of example 5.

293T cells were plated on 12-well plates one day before transfection, and cells were cultured for 12-24h after plating, as in example 1.

The three plasmids in example 4 were transfected into 293T cells. After 48h, cells are lysed, protein samples are collected for protein quantification, 30 mu g of protein samples are taken for sample loading, concentrated gel is run under the condition of 80V constant pressure, and then gel is separated through 120V constant pressure running.

And (3) carrying out constant-current membrane transfer for 2h at 0.25A, placing the membrane in 5% skimmed milk powder sealing solution for sealing for 1h, and washing the membrane twice by using 1 × TBST.

HA, beta-actin primary antibody (1:5000 dilution) in 2% BSA (bovine serum albumin) was added and placed in a shaker at 4 ℃ overnight.

The membrane was washed three times with 1 × TBST the next day, each time for 5min, rabbit/mouse secondary antibody (1:5000 dilution) formulated with 3% nonfat dry milk was added, and hybridization was carried out at room temperature for 1h, followed by color development (or chemiluminescence autoradiography reaction).

Example 5 protein-induced expression analysis was performed on the endogenous gene expression using the two high-efficiency inducible systems selected in example 3. The difference of the two high-efficiency induction systems in the protein expression level is judged by the system induction Ago2 protein expression level, and the high-efficiency induction efficiency of the system is verified in the endogenous gene expression level. From FIGS. 5 and 6, the novel inducible lentiviral expression system with PGK-initiated expression was slightly stronger at the protein expression level than the Ubc-initiated inducible system. The experimental result preliminarily verifies a series of characteristics of low background expression, high induction efficiency and the like of the novel lentivirus induction system on the protein expression level, provides a powerful basis for establishing the novel inducible lentivirus expression system, and preliminarily verifies the high-efficiency induction efficiency of the novel inducible lentivirus of PGK (protein PGK-initiated protein kinase) initiation expression compared with the expression system on the endogenous gene protein level.

By comparing the induced expression efficiency and background omission of the different vectors, the following conclusions can be drawn:

(1) comparing four different combinations, pLVX-TRE3G-RFP-EF1a-TetON3G, pLVX-TRE3G-RFP-EF1a-rtTA3, pLVX-TETO6-RFP-EF1a-TetON3G and pLVX-TETO6-RFP-EF1a-rtTA3, the induced expression effect of pLVX-TETO6-RFP-EF1a-TetON3G is the strongest, and the second is pLVX-TETO6-RFP-EF1a-rtTA3, pLVX-TRE3G-RFP-EF1a-TetON3G, pLVX-TRE3G-RFP-EF1a-rtTA 3. The invention first proposes that the best effect of the combination of TETO6 and TETON3G is achieved.

(2) When the influence of the insertion direction on the induction effect is compared, the background expression level of the reverse induction system is higher, the induction efficiency of the forward induction system is higher, and meanwhile, the background expression level is obviously lower than that of the reverse induction system.

(3) When the effects of different promoters on the induction efficiency were compared, it was found that the induction efficiency of the target gene was the highest when TetON3G was expressed using the PGK promoter.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A method for constructing an inducible lentivirus expression system is characterized by comprising the following steps:

(1) constructing a pLVX-Puro vector: using pLVX-Puro vector as slow virus frame vector, mutating Tth111I, SacII and BsmBI sites on Puro sequence by site-directed mutagenesis technology, and respectively introducing B amHI and SpeI sites before and after Puro cDNA sequence;

(2) synthesis and cloning of the TetON3G gene: gene synthesis is carried out according to the TetONG sequence, XbaI sites on the sequence are subjected to site-specific mutagenesis, and a multiple cloning site is introduced into the upstream of the TetON3G sequence: ClaI-MluI-XbaI-AgeI-EcoRI-XhoI, introducing connecting peptide and SmaI and BamHI sites at the downstream of the TetON3G sequence, performing double enzyme digestion on the synthesized gene fragment by ClaI-BamHI, and cloning the gene fragment to the reconstructed pLVX-Puro vector through the same enzyme digestion site to obtain pLVX-TetON 3G;

(3) subcloned EF1a promoter: using a pCDH-CMV-MCS-EF1-copGFP vector as a template, amplifying to obtain an EF1a promoter fragment, carrying out XhoI-BstBI double enzyme digestion, and cloning to pLVX-TetON3G to obtain vectors pLVX-EF1a-TetO N3G respectively;

(4) preparation of subcloned red fluorescent protein RFP: amplifying to obtain an RFP cDNA sequence by taking a pTRIPz vector as a template, cloning to pLVX-EF1a-TetON3G after AgeI-EcoRI double enzyme digestion to obtain a vector pLVX-RFP-EF1a-TetON 3G;

(5) subcloning of inducible promoters containing tetracycline cis-responsive elements: the vector pLVX-R FP-EF1a-TetON3G is cut by XbaI-AgeI, and then a synthesized inducible promoter TRE3G or a synthesized inducible promoter TETO6 promoter is respectively inserted into a linear vector to obtain a vector pLVX-TRE3G-RFP-EF1a-TetON3G or pLVX-TETO6-RFP-EF1a-TetON 3G.

2. The method for constructing an inducible lentiviral expression system according to claim 1, wherein the linker peptide in step (2) is P2A; the SmaI site is located between TetON3G and P2A.

3. A method for constructing an inducible lentivirus expression system is characterized by comprising the following steps:

(1) constructing a pLVX-Puro vector: using pLVX-Puro vector as slow virus frame vector, mutating Tth111I, SacII and BsmBI sites on Puro sequence by site-directed mutagenesis technology, and respectively introducing BamHI and SpeI sites before and after Puro cDNA sequence;

(3) construction of pLVX-rtTA 3: using pTRIPz vector as a template, obtaining rtTA3 fragment through PCR amplification, using BstB I-SmaI enzyme to cut enzyme and replace TetON3G on pLVX-TetON3G vector to obtain cloning vector pLVX-rtTA 3;

(4) subcloned EF1a promoter: using a pCDH-CMV-MCS-EF1-copGFP vector as a template, amplifying to obtain an EF1a promoter fragment, carrying out XhoI-BstBI double enzyme digestion, and cloning to pLVX-rtTA3 to obtain a vector pLVX-EF1a-rtTA 3;

(5) preparation of subcloned red fluorescent protein RFP: amplifying to obtain an RFP cDNA sequence by taking a pTRIPz vector as a template, cloning to pLVX-EF1a-rtTA3 after AgeI-EcoRI double enzyme digestion to obtain a vector pLVX-RFP-EF1a-rtTA 3;

(6) subcloning of inducible promoters containing tetracycline cis-responsive elements: the pLVX-R FP-EF1a-rtTA3 is cut by XbaI-AgeI, and then a synthesized inducible promoter TRE3G or a TETO6 promoter is inserted into a linear vector to obtain a vector pLVX-TRE3G-RFP-EF1a-rtTA3 or pLVX-TETO6-RFP-EF1a-rtTA 3.

4. The method for constructing an inducible lentiviral expression system according to claim 3, wherein the linker peptide in step (2) is P2A; the SmaI site is located between TetON3G and P2A.

5. The in vitro use of the inducible lentiviral expression system constructed by the construction method of any one of claims 1 to 4, wherein the inducible lentiviral expression system is used for the inducible expression of an endogenous gene or an exogenous gene.