CN107937437B - Light-operated expression vector of insect cell and application - Google Patents

Abstract

The invention discloses a light-operated expression vector of insect cells and application thereof.A positive plasmid pMIB-C120-mCherry-GFP-EL222 and a control plasmid pMIB-C120-mCherry-GFP are successfully constructed on an insect cell expression vector pMIB/V5-HisA by an applicant based on a VP-EL222 light-operated transcription activation system; after transfection of Sf9 cells, green fluorescent protein was expressed and red fluorescent protein was not expressed. After being stimulated by blue light irradiation, the red fluorescent protein is expressed; after Sf9 cells were transfected with the control plasmid pMIB-C120-mCherry-GFP, green fluorescent protein was expressed and red fluorescent protein was not expressed. Upon stimulation by blue light irradiation, the red fluorescent protein was still not expressed due to the absence of the EL222 transcription factor. The light-operated induced expression system is successfully applied to insect cells for the first time, and the application range of the technology is expanded. The verification method plasmid constructed by the invention also provides a flexible and easy-to-use method for utilizing the light-operated induced expression technology.

Description

A light-controlled expression vector for insect cells and its application

technical field

The invention belongs to the field of biological engineering, and particularly relates to a light-controlled expression vector of insect cells and its application.

Background technique

With the development of transgenic technology, controllable gene expression regulation system has become an indispensable tool in biomedical research and biotechnology. In the past few decades, chemically regulated gene expression systems have been widely used to temporally regulate gene expression. However, precise spatial and temporal control of gene expression is difficult due to the free diffusion of these small-molecule inducers, their intractable elimination, and their potential off-target effects on cellular function. However, light is an ideal inducer of gene expression, which is highly controllable, non-toxic, and can achieve precise temporal and spatial control of target gene expression. Thus, light-controlled gene expression systems represent a promising tool for precise control of gene expression. Nevertheless, the current major light-controlled expression systems still have many weaknesses that limit their application, such as: the toxicity of the protein itself, the narrow regulatory range, and the slow-response activation and deactivation. Here we present a new light-controlled expression system that does not have the aforementioned drawbacks and has demonstrated broad applicability in mammalian cells and the model animal zebrafish. The system will not only provide innovative tools for basic research on life sciences such as cell biology, developmental biology, and neurobiology, but also be widely used in bioengineering, diagnosis and treatment of human diseases and other applications.

The light control system is a light-regulated gene expression system based on the C120 DNA binding sequence and the EL222 transcription factor (Motta-Mena et al., 2014; Rivera-Cancel, G et al., 2012). The EL222 transcription factor, originally derived from a bacterial photo-oxygen-pressure protein, dimerizes and binds to C120 DNA when exposed to blue light. The light-dependent transcriptional activation of this system requires only a few components, a light-sensitive domain LOV, and a helix-turn-helix (H-T-H) DNA binding domain. The LOV domain binds the HTH domain in the dark, covering the HTH4α site necessary for binding to DNA for dimerization. Blue light irradiation triggers a photochemical reaction: the flavoprotein complex in the LOV domain disrupts the interaction between LOV and HTH, dimerizes EL222, and exerts DNA-binding protein activity. These reactions are reversed in the dark, and the reverse reaction will occur soon after the blue light irradiation is stopped, which is the basis of the rapid deactivation. This system has a large tunable expression range, agile activation and deactivation, and a sufficiently high linear correlation with light intensity for controlled expression of target proteins. Taken together, the studies demonstrate the broad versatility of the C120-EL222 system and its advantages as an optogenetic tool.

At present, light control systems based on C120-EL222 have been widely used in mammalian cells, showing obvious advantages of light control systems in terms of time and space manipulation (Nash et al., 2011; Motta-Mena et al. ., 2014). Further, scientists tried to verify the feasibility of the light-controlled system in zebrafish. Although light-controlled expression can be achieved, the application of this system in zebrafish is limited due to its toxicity. Next, through genetic engineering, scientists optimized a more suitable light control system for zebrafish, namely the TAEL (TA4-EL222) system (Reade, A. et al., 2017). The span of the C120-EL222 system from mammalian cells to zebrafish expands the application range of this light-controlled system, and also provides a powerful tool for fish research.

Based on this, the applicant established a light-controlled gene expression system based on VP-EL222 in Sf9 insect cells, and skillfully integrated the elements of the C120-EL222 light-controlled system and the fluorescent protein reporter gene into an insect cell expression vector , so that the test of the efficacy of the light control system is more rapid and stable, and the regulation of exogenous protein expression in insect cells is more flexible. For the first time, we successfully realized the light-controlled expression of the C120-EL222 system in insect cells, which expanded the scope of application of this light-controlled system.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a light-controlled expression vector pMIB-C120-MCS-GFP-EL222 applied to insect cells, the nucleotide sequence of the vector is shown in SEQ ID NO. 1, and the vector is skillfully integrated The regulatory elements and fluorescent protein reporter genes of the C120-EL222 light control system were used, and the opIE1 constitutive promoter was used to regulate the expression of the GFP reporter gene tandem EL222 transcription factor; the light control promoter C120 DNA sequence was used to regulate the expression of exogenous genes, which improved the The flexibility and ease of operation of this light control system. At the same time, the vector also retains the blasticidin resistance gene, which can be used to screen stable cell lines with light-controlled expression. A GFP reporter gene is newly introduced to facilitate real-time monitoring of the transfection efficiency of the vector. Fast and efficient sorting of light-controlled expressing stable cell lines by flow cytometry.

Another object of the present invention is to provide an application of a light-controlled expression vector for insect cells. After inserting exogenous protein into pMIB-C120-MCS-GFP-EL222, it is transfected into insect cell Sf9, which can realize the expression of functional protein. Light-controlled expression.

For achieving the above object, the technical scheme adopted in the present invention is:

A method for preparing a light-controlled expression vector of insect cells with exogenous proteins, comprising:

1) The exogenous gene replaces the Luc gene in the pC120-Luc plasmid through the EcoR1 and Xba1 restriction sites to obtain the pC120-exogenous gene plasmid;

2) using the pC120-foreign gene plasmid as a template, amplify the C120-foreign gene fragment, and insert it into the BspH1 and Age1 restriction sites of the pMIB/V5-his vector to obtain the pMIB-C120-foreign gene plasmid;

3) The nucleotide fragment shown in SEQ ID NO.2 was artificially synthesized, cloned and placed in the psimple-T vector to obtain the psimple-T/PFT2AN vector, and the fluorescent protein reporter was introduced through the Nhe1 restriction site in psimple-T/PFT2AN gene, and then introduced the EL222 gene through the BsiwI restriction site in psimple-T/PFT2AN to obtain the psimple-T/fluorescent protein-EL222 plasmid;

4) Insert the fluorescent protein-EL222 fragment in the psimple-T/fluorescent protein-EL222 plasmid into the pMIB-C120-exogenous gene plasmid through the PvuII and BglII restriction sites to obtain pMIB-C120-exogenous gene-fluorescence Protein-EL222 plasmid vector;

The pMIB/V5-his vector is pMIB/V5-his A or pMIB/V5-his B or pMIB/V5-his C.

Those skilled in the art can prepare insect cell light-controlled expression vectors expressing different exogenous proteins according to the actual situation and the above-mentioned methods; or directly insert the exogenous proteins into the light-controlled expression vector pMIB-C120-MCS-GFP-EL222 (MCS after C120 carries a variety of enzyme cleavage sites for the insertion of exogenous genes) to prepare insect cell light-controlled expression vectors carrying exogenous proteins.

The application of a light-controlled expression vector for insect cells includes using the light-controlled expression vector to construct a light-controlled expression platform for insect cells, or using the vector to control the expression of proteins.

In the above-mentioned scheme, specifically, the pMIB-C120-exogenous gene-GFP-EL222 plasmid vector is transfected into insect cells, and then subjected to blue light irradiation or dark treatment as required to control the expression of exogenous proteins .

Compared with the prior art, the present invention has the following advantages:

1. The components of the C120-EL222 light control system are cleverly integrated into an insect cell expression vector, providing a paradigm for plasmid construction.

2 The light-controlled expression application of the C120-EL222 light-control system was realized in insect cells for the first time, which expanded the new application scope of the light-control system.

3. The components of the original C120-EL222 light control system are simplified from multi-plasmid carrying to single-plasmid carrying, which greatly improves the positive ratio of effector cells after transfection, and also makes the test of the light control system more rapid and stable. The test index of this light control system is: after blue light induction, in Sf9 cells expressing green fluorescent protein GFP, the expression of red fluorescent protein mCherry can be detected to reflect the working efficiency of light control elements.

4. The constitutive expression of GFP reporter gene is introduced into the light-controlled plasmid, which facilitates the screening of positive cells by a flow cytometer with a green fluorescent label, and greatly improves the efficiency of screening stable light-controlled cell lines.

Description of drawings

Fig. 1 Map of insect cell expression vector pMIB/V5-His.

Figure 2 is a schematic diagram of a light-controlled expression system based on C120-EL222.

Figure 3 is the basic flow chart of the construction of the light-controlled expression vector pMIB-C120-mCherry-GFP-EL222 in insect cells.

Figure 4. The electrophoresis chromatogram of the restriction enzyme digestion identification of the constructed vector.

Figure 5. Schematic diagram of functional verification of insect cell light-controlled expression vector in sf9 insect cells.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Example 1:

Construction of Insect Cell Expression Vector pMIB-C120-mCherry-GFP-EL222

1. Experimental materials

pMIB/v5-hisA vector Invitrogen pC120-Luc plasmid Gift from Prof. Kevin HGardner (Motta-Mena et al., 2014) pVP-EL222 plasmid Gift from Prof. Kevin HGardner (Motta-Mena et al., 2014) Gel Extraction kit OMEGA bio-tek Plasmid mini kit Plasmid Recovery Kit OMEGA bio-tek E.coli DH-5α competent ATCC restriction endonuclease NEB Corporation CIP NEB Corporation T4DNA ligase NEB Corporation Gel Imager BioRad Corporation

List of primers used for plasmid cloning:

2. Experimental method

1) Using the pLV-mCherry (purchased from Addgene) vector as a template, use primers E-mch-F and X-mch-R to amplify the mcherry gene fragment, and replace the Luc in the pC120-Luc plasmid through the EcoR1 and Xba1 restriction sites gene to obtain the pC120-mcherry plasmid.

2) Using the pC120-mcherry plasmid as a template, the C120-mcherry fragment was amplified using primers BspH-C120-F and Age-Mch-R, and inserted into the BspH1 and Age1 restriction sites of the pMIB/V5-hisA vector to obtain pMIB- C120-mcherry plasmid.

3) In order to facilitate the construction of the multi-element integration vector plasmid, a DNA fragment PFT2AN was artificially synthesized, and the fragment was composed of 457 base pairs (shown in SEQ ID NO. 2), cloned and placed in the psimple-T vector to obtain the plasmid psimple -T/PFT2AN. Using primers Nhe-GFP-F and GFP-Nhe-R, the pEGFP-N1 (purchased from Clontech) vector was used as a template to amplify the GFP gene, and the GFP reporter gene was introduced through the Nhe1 restriction site in psimple-T/PFT2AN, The EL222 gene was amplified with primers Bsiw-EL-F and EL-Bsiw-R, and the EL222 gene was introduced through the BsiwI restriction site in psimple-T/PFT2AN to obtain the psimple-T/GFP-EL222 plasmid.

4) Insert the GFP-EL222 fragment in the psimple-T/GFP-EL222 plasmid into the pMIB-C120-mcherry plasmid through the PvuII and BglII restriction sites to obtain the pMIB-C120-mCherry-GFP-EL222 plasmid vector

5) The vector pMIB-C120-mCherry-GFP-EL222 was directly digested with BsiwI enzyme. After purification, the pMIB-C120-mCherry-GFP plasmid was obtained by ligation and transformation as a negative control plasmid for EL222 function loss;

According to the vector construction strategy in Figure 3, the positive plasmid pMIB-C120-mCherry-GFP-EL222 and the negative control plasmid pMIB-C120-mCherry-GFP were constructed, which were verified to be correct by enzyme digestion (Figure 4). The EcoRI/XbaI digestion of the pMIB-C120-mCherry-GFP-EL222 functional vector releases a band of about 750bp, which is the mCherry band, and the digestion of BsiwI releases the EL222 band of about 900bp; EcoRI of the pMIB-C120-mCherry-GFP control vector The band of about 750bp released by /XbaI is the mCherry band, and there is no band in the enzyme digestion of BsiwI. The identification results were in line with expectations, and all cloned plasmids were sequenced and verified, and the sequences were correct.

Each step of the above vector construction adopts the conventional molecular cloning method:

1) Design specific primers to obtain fragments with specific restriction sites at the ends by PCR amplification; at the same time, digest the vector and fragments separately, and purify the digested products; use T4 ligase, and connect the vector fragments at 16°C overnight;

2) Transform, shake, and plate (all ampicillin-resistant plates used in the experiment), place the plate in a 37°C oven overnight; pick and shake the bacteria. Cultivate the bacteria identified as positive; save the bacterial species (the bacterial species are stored in a 10%-15% glycerol solution, -20°C).

3) Extract the plasmid with a plasmid mini-extraction kit, and determine the concentration; use a reasonable restriction enzyme to digest the extracted plasmid for identification; pick the positive plasmid for sequencing, and use the plasmid for the next step after the sequencing is correct. Plasmids should be stored at -20°C.

In this example, the reporter gene mCherry is used as an exogenous gene to illustrate the light-controlled expression vector provided by the present invention. Those skilled in the art can prepare insect cell light expressing different exogenous proteins according to the actual situation and the above-mentioned method. control expression vector; or directly insert the exogenous protein into the light-controlled expression vector pMIB-C120-MCS-GFP-EL222 (MCS after C120 carries a variety of enzyme cleavage sites for the insertion of exogenous genes), to prepare carrying Insect cell light-controlled expression vector for exogenous protein.

Example 2:

Functional verification of insect cell light-controlled expression vector pMIB-C120-mCherry-GFP-EL222 in Sf9 insect cells: 1. Experimental materials

2. Experimental methods and results

2.1 Subculture of Sf9 cells: Use Grace's Insect Cell Medium plus serum and double antibody medium to culture Sf9 cells adherently in a cell incubator at 28°C. When the cells grow to about 90%, directly blow the cells up gently. pass on.

2.2 Sf9 insect cells were transfected with light-controlled expression vector in insect cells. There are two plasmids to be transfected in this experiment: pMIB-C120-mCherry-GFP-EL222 (functional plasmid) and pMIB-C120-mCherry-GFP (negative control plasmid).

1) Sf9 cells were seeded in a 6-well plate at 8×10 ⁵ cells/well 24 hours in advance, and transfection was carried out when the cell confluence reached 70-80%. Prepare 10ml plate medium before transfection: 1.5ml Grace cell culture medium (containing 10% FBS)+8.5ml Grace cell culture medium (without FBS). Antibiotics should not be present in the plate medium.

2) Place the Sf9 cells in the 6-well plate to be transfected on the ultra-clean table, remove the medium in the cells in the 6-well plate, and add 2 ml of the above fresh plate medium.

II Reagent)于100μl Grace’s培养基。稀释1～3μg目标质粒于100μl Grace’s培养基。3) Dilute 2-6 μl of transfection reagent (

II Reagent) in 100 μl Grace's medium. Dilute 1-3 μg of the target plasmid in 100 μl Grace's medium.

4) Mix the diluted DNA and transfection reagent evenly and incubate at room temperature for 15-30min.

5) Add 210 μl of the mixture of plasmid and transfection reagent to the cells to be transfected, and incubate at 28°C for 4-6 hours.

6) Aspirate off the transfection reagent mixture, add 2ml of Sf9 cell complete culture medium (containing double antibody and 10% FBS), continue to culture in a 28°C incubator, and track the cell state after 24h. According to different needs, the cultured cells were given different light conditions.

A total of four groups of transfection experiments were designed. The positive plasmid pMIB-C120-mCherry-GFP-EL222 was transfected in two groups of light and dark, and the negative control plasmid pMIB-C120-mCherry-GFP was transfected in two groups of light and dark. The blue light irradiation conditions were: irradiation for 20s and dark for 60s as a cycle.

2.3 After transfection of Sf9 cells with plasmids pMIB-C120-mCherry-GFP-EL222 and pMIB-C120-mCherry-GFP, they were cultured in a 28°C incubator, and 24 hours after transfection, blue light irradiation and dark conditions were applied, respectively. The expression of red fluorescence was observed under a fluorescence microscope after 24h and 48h of irradiation stimulation (Fig. 5).

1) After the functional plasmid pMIB-C120-mCherry-GFP-EL222 was transfected into sf9, two groups were cultured under blue light irradiation and dark conditions. 24 hours after transfection, light was applied (blue light irradiation for 20s and then 60s for a cycle) and dark. conditions, observed under a fluorescence microscope after 24 hours of illumination, and obtained the results (A in Figure 5). The results showed that the green fluorescent protein GFP was continuously expressed after transfection, and the red fluorescent protein mCherry was strongly expressed in GFP-positive cells after blue light irradiation. The above results demonstrate that the C120-EL222 light control system can have specific and efficient functional activity in Sf9 cells.

2) After the negative control plasmid pMIB-C120-mCherry-GFP was transfected into Sf9, the two groups were also cultured under blue light irradiation and dark conditions. 24 hours after transfection, they were given light (blue light irradiation for 20s and then 60s for a cycle) and dark conditions. , and observed under a fluorescence microscope after 24 hours of illumination to obtain the results (B in Figure 5). The results showed that the green fluorescent protein GFP was continuously expressed after transfection, but in the absence of EL222 light-controlled transcription factor, there was no red fluorescent protein mCherry expression under blue light irradiation and dark conditions. The above results prove that the C120-EL222 light control system has a high degree of specificity and rigor of transcriptional activation in Sf9 cells, and there is basically no leakage expression.

3) After the plasmid pMIB-C120-mCherry-GFP-EL222 was transfected into Sf9, two groups were cultured under blue light irradiation and dark conditions. 24 hours after transfection, light (blue light irradiation for 20s and 60s for a cycle) and dark conditions were applied. , and observed under a fluorescence microscope after 48 hours of illumination to obtain the results (C in Figure 5). An additional 24 h of blue light exposure slightly increased the number of red fluorescent cells, but the increase was not obvious. The above results proved that in Sf9 cells, the expression of red fluorescent protein mCherry reached a high level after 24 hours of blue light irradiation in the C120-EL222 light control system. high level.

In summary, the C120-EL222 light control system can have specific high-efficiency functional activity and high rigor in Sf9 cells, and can continuously activate the expression of exogenous target proteins.

Those skilled in the art can easily understand that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection scope of the present invention.

sequence listing

<110> Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences

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cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc 60

cctgcggacg gtgccgacag gttcttctcg atctgcatcc tgggatcaaa gccatagtga 120

aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg 180

tgtgggaggg ccagactttg aattttgacc ttctcaagtt ggcgggagac gtggagtcca 240

acccagggcc cgctagcgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga 300

atcctggccc acgtacgcga aacttgttta ttgcagctta taatggttac aaataaagca 360

atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 420

ccaaactcat caatgtatct tatcatgtct gacatct 457

Claims (4)

1. A light-operated expression vector applied to insect cells is disclosed, wherein the nucleotide sequence of the vector is shown in SEQ ID NO. 1.

2. A preparation method of a light-operated expression vector of insect cells with exogenous genes comprises the following steps:

1) the exogenous gene replaces the Luc gene in the pC120-Luc plasmid through EcoR1 and Xba1 enzyme cutting sites to obtain a pC 120-exogenous gene plasmid;

2) amplifying a C120-exogenous gene fragment by taking the pC 120-exogenous gene plasmid as a template, and inserting the C120-exogenous gene fragment into Bsp H1 and Age1 enzyme cutting sites of a pMIB/V5-his vector to obtain a pMIB-C120-exogenous gene plasmid;

3) artificially synthesizing a nucleotide fragment shown in SEQ ID NO.2, cloning and placing the nucleotide fragment into a psimple-T vector to obtain a psimple-T/PF T2AN vector, introducing a fluorescent protein reporter gene through a NheI enzyme cutting site in the psimple-T/PFT2AN, and introducing an EL222 gene through a BsiwI enzyme cutting site in the psi mple-T/PFT2AN to obtain a psimple-T/fluorescent protein-EL 222 plasmid;

4) and (3) inserting the fluorescent protein-EL 222 fragment in the psimple-T/fluorescent protein-EL 222 plasmid into the pMIB-C120-exogenous gene plasmid through the enzyme cutting sites PvuII and BglII to obtain the psimple-T/fluorescent protein-EL 222 plasmid.

3. Use of the vector of claim 1 or the vector produced by the method of claim 2 to construct a light-regulated expression platform for insect cells.

4. Use of the vector of claim 1 or the vector produced by the method of claim 2 to control the expression of a protein.

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