Construction method and application of blue light-operated repressor protein LOV-cI expression vector

Abstract

The invention relates to an expression vector construction method and application of a blue light-operated repressor protein LOV-cI fusion expression element for regulating a pR photopromoter. The invention provides a construction method of an LOV-cI expression vector, which comprises the following steps: fusing a target gene with a cI repressor protein gene and an LOV structural domain to construct a gene expression cassette; and inoculating the gene expression cassette into a bacterial expression vector. Compared with the prior art, the invention provides the LOV-cI-containing expression vector, which has the characteristics of closing target gene expression through illumination induction and inhibiting illumination to promote gene expression, the expression rate is increased by 4-6 times, and the LOV-cI-containing expression vector can be applied to timed, quantitative and accurate regulation and control of key protease expression in a metabolic pathway, and can be combined with a nano drug delivery system to improve the drug delivery efficiency. And the gene has huge potential in gene therapy of malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and can play a very important role in gene and cell-based therapies. More specifically, it relates to a method for constructing an expression vector for regulating a pR light promoter by a blue light light-controlled repressor protein LOV-cI fusion expression element and its application. Background Art

[0002] Commonly used gene expression regulation systems in the field of optogenetics include the BphP1-QPAS1 near-infrared light system, the VVD blue light sensitive photoswitch gene expression system and the VVD variant MAGNET system. The BphP1-QPAS1 system can dimerize or depolymerize components under the control of near-infrared light, thereby converting light signals into biological signals inside cells and relocating certain components inside cells. The light-oxygen-voltage sensing domain (LOV) is a widely used light-sensitive receptor protein that can be found in various organisms, including plants, algae and bacteria. It binds to endogenous chromophores and can detect blue light. Due to its small relative molecular mass, it is soluble, and the riboflavin auxiliary factors it requires are present in most types of cells. It can be used in combination with a variety of signal molecules to regulate signal pathways. These characteristics make LOV an ideal tool molecule for constructing different signal pathways of light-controlled cells. In previous studies, Glotzer et al. developed tunable light-induced dimerization tags (TULIPs), and Niopek et al. used the fusion expression of the LOV domain and the NLS nuclear localization signal peptide to regulate the activity of the nuclear localization signal, thereby regulating the process of transcription factors entering the cell nucleus and achieving the purpose of specific light-controlled gene expression. At present, optogenetic tools still have great limitations. For example, the control system based on blue light activation is difficult to penetrate dense human tissue; the molecular weight of optogenetic (light-controlled) protein switches including red / far-red light optogenetic systems is large, and the gene coding sequence required for overexpression is long, making it difficult to use some gene therapy vectors (such as AAV) to effectively load and transduce into cells. In addition, the poor sensitivity of light-sensitive reactions and slow light reaction kinetics (i.e., slow light activation and inactivation) are also one of the common shortcomings of current photosensitive proteins.

[0003] To solve the above problems, this patent designs and develops an optogenetic vector system based on LOV-cI. LOV is a photosensitive protein that senses blue light found in plants, algae and bacteria. PR Promoter is a strong promoter that controls early transcription in Escherichia coli λ phage. Whether it is transcribed or not determines whether λ phage enters the lytic cycle or lysogenic cycle. The cI repressor protein encoded by the CI gene can cause Escherichia coli infected with λ phage to enter the lysogenic state. The Ja helix at the C-terminus of the Lov protein is covalently bound to the λ phage PR promoter. Under dark conditions, the Jα helix and the Lov body fit tightly, and blue light excitation destroys the contact between the Jα helix and the Lov domain, restoring the repressor activity of the cI protein. Summary of the invention

[0004] The technical problem to be solved by the present invention is to construct an expression vector in which a blue light light-controlled repressor protein LOV-cI fusion expression element regulates a pR light promoter.

[0005] The purpose of the present invention is to provide a method for constructing a blue light light-controlled repressor protein LOV-cI.

[0006] Another object of the present invention is to provide a blue light-controlled repressor protein LOV-cI.

[0007] Another object of the present invention is to provide a blue light-controlled repressor protein LOV-cI that has potential universality for light regulation of transcription factors with DNA binding domains.

[0008] Another object of the present invention is to provide a blue light-controlled repressor protein LOV-cI, which can be used for the precise regulation of the timing and quantitative expression of key proteases in metabolic pathways.

[0009] Another object of the present invention is to provide a blue light-controlled repressor protein LOV-cI, which, combined with a nano drug delivery system, has great potential in gene therapy of malignant tumors.

[0010] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0011] Through the fusion expression of the light oxygen voltage sensitive domain (LOV) and its Jα helix with the λ phage repressor protein cI, a blue light light-controlled LOV-cI fusion expression element was constructed to regulate the pR light promoter to achieve light-controlled gene expression. By evaluating the performance of the light-controlled gene expression system, the results showed that the lov-cⅠ, loosen, and fasten systems had light-off expression characteristics, while the light-inhibited gene expression was 4 to 6 times. The Coomassie brilliant blue staining results further showed that the maximum induction rates of the pDawn transformation system, Fasten2 system and chemical induction were similar, which revealed the possibility of LOV domain light regulation of cI repressor protein. In particular, the addition of the cⅠ-pR cis-trans element not only reversed the induction effect of blue light, but also increased the expression of the entire system due to the characteristics of the pR strong λ promoter.

[0012] Therefore, in the first aspect, the present invention provides a method for constructing a lov-cⅠ expression vector, which comprises fusing the target gene with the cI repressor protein gene and the Lov domain to construct a gene expression cassette; and then connecting the gene expression cassette to a bacterial expression vector for construction.

[0013] Furthermore, when constructing the gene expression cassette, the target gene is also fused with a regulatory sequence; the regulatory sequence includes but is not limited to a terminator, an enhancer, a leader sequence, an intron, and other regulatory sequences that are operably linked to the target gene.

[0014] Furthermore, the expression vector is a pDawn plasmid.

[0015] Furthermore, the target gene is the meGFP gene.

[0016] It is well understood by those skilled in the art that in order to increase and strengthen the response rate and functional substances by genetic engineering methods, the target gene can also be other genes.

[0017] Based on this research, in a second aspect, the present invention provides an expression vector containing LOV-cI.

[0018] In some specific embodiments, the present invention provides a LOV-cI expression vector whose nucleotide sequence has 80% identity with the sequence shown in loosen1 / 2 or fasten1 / 2; preferably, it has 85% identity, more preferably, it has 90% identity, more preferably, it has 95% identity, and most preferably, it has 99% identity.

[0019] In a third aspect, the present invention provides a LOV-cI expression vector prepared by the above-mentioned method for constructing the LOV-cI expression vector.

[0020] Furthermore, the nucleotide sequence of the vector is (a), (b) or (c); (a) the nucleotide sequence as shown in loosen1 / 2 or fasten1 / 2; (b) a nucleotide sequence that hybridizes with the nucleotide sequence shown in loosen1 / 2 or fasten1 / 2; (c) a nucleotide sequence encoding a nucleotide sequence having 80% or more homology to the nucleotide sequence shown in loosen1 / 2 or fasten1 / 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the mechanism of Lov-cI light-inhibitory gene expression system, the light-oxygen-voltage domain (LOV) is shown in light blue, the bacteriophage cI repressor protein is shown in light yellow, the bacteriophage pR promoter DNA is shown in gray, and the Jα helix at the C-terminus of the LOV domain is shown in orange. (A) Under dark conditions, the Jα helix and the LOV body fit tightly together, (B) light excitation destroys the contact between the Jα helix and the LOV domain, restoring the repressor activity of the cI protein. Figure 2 The results of mEGFP protein expression induced by 20h white light in the pDawn system and lov-cⅠ system. Figure 3 This is the result of mEGFP protein expression induced by 20h white light in the lov-cⅠ second generation loosen system. Figure 4 This is the result of mEGFP protein expression induced by 20h white light in the lov-cⅠ third-generation fasten system. Figure 5 These are the results of Coomassie Brilliant Blue staining of bacterial total protein in the chemical-induced and light-induced expression systems. DETAILED DESCRIPTION

[0022] Example 1 Promoter, terminator and gene cloning Based on the T4 primer design principle, the homology arm amplification primers were designed as shown, and the homology arm primers were used for PCR amplification. The amplification system and procedure are as follows: the amplification system is: amplification template 1.5μL; upstream primer 2μL; downstream primer 2μL; Max DNA Polymerase25μL; ddH2O 19.5μL; The amplification reaction program was as follows: 98°C, 5 min; 98°C, 10 s; 58°C, 15 s; 72°C, 1 min; 34 cycles; 72°C, extension for 5 min.

[0023] The specific sequences of the primers are shown in the following table:

[0024] Example 2 Construction of expression vector The pDawn plasmid was double-digested with BamHI and HindⅢ, and HindⅢ and BamHI restriction sites were added to both ends of the mEGFP fragment by PCR. After digestion, the vector was purified to obtain a linearized vector; the concentrations of the linearized plasmid vector and the fragments amplified in Example 1 were measured, and the linearized vector and the connection fragment were added to the T4 system for connection; the T4 system was: T4 Enzyme Premix 2.5μL, T4 Enzyme 1μL, connection fragment 1μL, linearized vector 2μL, dH2O was added to 25μL; the connection conditions were: 16°C, 12h.

[0025] The connected vector was transferred into competent E. coli DH5α for transformation. Transformation steps: adjust the temperature of the constant temperature water bath to 42°C in advance. Take out the competent bacteria from the -80°C ultra-low temperature freezer, immediately melt it in a 37°C water bath and insert it on ice, ice bath for 10 minutes, add 1μl of the connected plasmid mixture, gently shake it and place it on ice for 25 minutes, gently shake it and insert it into a 42°C water bath for 45 seconds for heat shock, then quickly put it back into the ice and let it stand for 3 to 5 minutes. In the clean bench, add 700μl of sterile LB medium (without antibiotics) to each tube and mix gently, culture it at 37°C on a shaker for 1 hour, then collect the bacteria, spread it, culture it at 37°C for 24 hours, select it with kanamycin, and pick the positive clones.

[0026] Example 3 Expression verification of the vector of the lov-cⅠ primary system The DHpα helix was used as a connection node to connect the lov domain and the cⅠ repressor protein. As the primary light-inducible gene expression system of lov-cⅠ, the mEGFP protein was used as the downstream reporter gene of the pR promoter to measure the changes in the expression level of the mEGFP protein, in order to measure the light-on and light-off performance of the lov-cⅠ light-inducible expression system and the maximum induced expression amount.

[0027] The vectors were divided into four groups: pDawn negative control vector without mEGFP reporter gene, pDawn positive control vector with mEGFP, lov-cⅠ negative control vector without mEGFP reporter gene, and lov-cⅠ experimental group vector with mEGFP. The four groups of DH5α glycerol bacteria that had completed sequencing and carried the target vector were shaken and revived, and all the culture bottles were darkened by wrapping tin foil. After shaking and reviving overnight, 200ul of bacterial solution was taken from each of them and added to 20ml LB medium with kanamycin concentration of 50ug / ml at a ratio of 1:100, and cultured overnight for 20h. Each of the above four groups of vectors was treated with darkness and light respectively.

[0028] The expression of mEGFP fluorescent protein was measured using a multifunctional microplate reader. The bacterial solution to be tested was mixed before measurement. Each tube was repeated 3 times. 100ul of bacterial solution was added to a 96-well microplate. The excitation wavelength range used to measure the fluorescence value was 485±20nm, and the emission wavelength range was 528±20nm. The OD600 absorbance value corresponding to each well was recorded to measure the number of bacteria. Finally, the results of mEGFP protein expression induced by white light for 20h in the pDawn system and lov-cⅠ system were measured. Figure 2 .

[0029] Example 4 Expression verification of the vector of lov-cⅠloosen system The hinge connecting the lov domain and cⅠ protein was optimized, the hinge amino acid length was extended, and a second-generation loosen light-inducible expression system with a looser structure was constructed to improve the expression difference of the system under dark and light conditions and reduce leaky expression under light. Therefore, the original 124-amino acid lov domain was extended by 12 and 40 amino acids at its C-terminus to construct the loosen1 and loosen2 light-inducible expression systems, respectively. The specific experimental steps were the same as those for the construction of the lov-cⅠ primary system. The mEGFP protein expression results of the pDawn system, lov-cⅠ primary system, loosen1 and loosen2 systems after 20h white light induction and dark treatment, respectively, are shown in the figure. Figure 3 .

[0030] Example 5 Expression verification of the vector of the lov-cⅠfasten system The lov-cⅠ light-inducible system with higher overall expression level was constructed. The strategy of shortening the hinge between the lov domain and the cⅠ repressor protein was adopted. The original 124-amino acid lov domain was shortened by 11 and 18 amino acids at its C-terminus, respectively, to construct the fasten1 and fasten2 light-inducible expression systems, respectively. In the fasten2 system, the DHpα helix part in the lov-cⅠ system was completely abandoned, and the Jα helix was directly connected to the N-terminus of the cⅠ protein, coupling the lov domain and cⅠ protein functions as much as possible, reducing the activity of the cⅠ protein DNA binding domain, and thus improving the overall protein expression. The specific experimental steps were constructed in the same way as the lov-cⅠ primary system. The mEGFP protein expression results of the pDawn system, the lov-cⅠ primary system, the fasten1 and fasten2 systems after 20h white light induction and dark treatment, respectively, are shown in the figure. Figure 4 The mEGFP band was obvious under IPTG induction conditions, but almost no band under non-induction conditions, with a higher induction rate and lower leakage expression ( Figure 5). After 20 hours of light induction, the mEGFP band of the pDawn light-induced group was obvious, and the leakage expression was low in the dark; while the staining results of the Fasten2 group were basically consistent with the results of the light microplate reader detection. The expression level under dark conditions was significantly higher than that under light conditions, showing the effect of light inhibition of gene expression, and its maximum induction rate was comparable to that of the pDawn system and the chemical induction system, but its background leakage expression was still high, which was the key to limiting its application. The other constructed systems, including loosen1 and loosen2, as well as lov-cⅠ and Fasten1 groups, also showed the effect of light inhibition of gene expression, but the induction rate was not as high as that of the Fasten2 system.

[0031] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. A method for constructing an illumination expression vector based on LOV-cI fusion regulatory element, characterized in that: The target gene is fused with the cI repressor protein gene and the LOV light-sensitive domain to construct a gene expression cassette; the gene expression cassette is then connected to an expression vector under the control of a blue light transcription regulatory factor.

2. The method for constructing a LOV-cI expression vector according to claim 1, characterized in that: When constructing a gene expression cassette, the target gene is also fused to a regulatory sequence; the regulatory sequence includes but is not limited to a terminator, an enhancer, a leader sequence, an intron, and other regulatory sequences that are operably linked to the target gene.

3. The method for constructing a LOV-cI expression vector according to claim 1, characterized in that: The expression vector is a pDawn transformed plasmid under the control of a blue light transcription regulatory factor.

4. The method for constructing a LOV-cI expression vector according to claim 1, characterized in that: The target gene is mEGFP gene.

5. An LOV-cI expression vector obtained by the construction method according to any one of claims 1 to 4.

6. The carrier according to claim 5, characterized in that: The nucleotide sequence of the vector is (a), (b) or (c); (a) the nucleotide sequence as shown in loosen1 / 2; (b) a nucleotide sequence that hybridizes with the nucleotide sequence shown in loosen1 / 2; (c) A nucleotide sequence encoding a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in loosen1 / 2.

7. The carrier according to claim 5, characterized in that: The nucleotide sequence of the vector is (a), (b) or (c); (a) the nucleotide sequence as shown in fasten1 / 2; (b) a nucleotide sequence that hybridizes with the nucleotide sequence shown in fasten1 / 2; (c) A nucleotide sequence encoding a nucleotide sequence having more than 80% homology with the nucleotide sequence shown in fasten1 / 2.

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