Expression element regulated by protocatechuic acid and application thereof

Abstract

The invention provides an expression element regulated by protocatechuic acid and application of the expression element. Specifically, the invention relates to an expression element responding to PCA regulation, a transcription expression cassette containing the expression element, a recombinant expression vector suitable for prokaryotes, a method for enhancing target gene expression regulation and a method for preparing protein. The expression element responding to PCA regulation in the invention comprises a nucleotide sequence as shown in SEQ ID NO.3-5 and a nucleotide sequence as shown in SEQ ID NO.8, and is a PCA-induced chimeric promoter and repressor protein PcaV which is started to express by PlacI. Through optimization, the expression element responding to PCA regulation realizes reversible PCA responsive protein expression with low background expression and high induction efficiency, so that regulation of recombinant protein production is improved, and the expression element has high application value.

Description

Technical Field

[0001] The present invention belongs to the fields of synthetic biology and biotechnology, etc., and relates to a protocatechuic acid (PCA)-regulated expression element and its application, specifically to a polynucleotide sequence responsive to PCA and a recombinant expression vector containing the polynucleotide, and provides a method for enhancing the regulation of target gene expression. Background Art

[0002] With the development of synthetic biology technology, a large number of "sensing-response" systems have been designed to regulate the expression of exogenous genes. For example, the quorum sensing system can adjust the gene expression intensity by regulating the population density of microorganisms. Only when the microorganism density exceeds a certain threshold will the exogenous gene be expressed. However, it is difficult to achieve precise control of biological behavior and recombinant protein release by this pure biological method. In recent years, some studies have used optogenetics as a non-invasive control system to precisely initiate the expression of target genes in microorganisms in space and time. Nevertheless, the poor tissue penetration ability of light limits its application scenarios. Ultrasonic waves have also received extensive attention in recent years due to their good penetration ability. In addition, both light regulation and ultrasonic regulation require additional equipment, increasing the cost required for regulating recombinant protein production.

[0003] Chemical inducers are considered a promising control method because of their good penetrability and the lack of limitation by additional equipment. The chemical inducers initially used to regulate gene expression in cells are mainly divided into four categories: isopropyl-β-D-1-thiogalactoside, salicylate, tetracycline, and L-arabinose. However, due to its toxicity, isopropyl-β-D-1-thiogalactoside is not suitable for in vivo applications; excessive use of salicylate or tetracycline may cause various potential side effects, such as metabolic disorders and drug resistance; while L-arabinose will be metabolized by multiple enzymes in some microorganisms, so a larger dose is required to play a role.

[0004] In summary, developing an expression element regulated by an ideal small molecule is crucial for solving the current problem of insufficient regulation of recombinant protein production. PCA is a metabolite of tea polyphenols, mainly absorbed into the systemic circulation through oral administration, and plays various beneficial pharmacological effects in the human body, such as antioxidant and neuroprotective effects, etc. Since regulating recombinant protein expression by PCA has the characteristics of being green and safe, it has become an ideal small molecule for regulating protein expression. Summary of the Invention

[0005] The present invention uses prokaryotic synthetic biology technology to achieve precise control of the expression of target proteins in prokaryotic host cells through a polynucleotide sequence responsive to PCA. The summary of the present invention is as follows:

[0006] In a first aspect, there is provided an expression element responsive to PCA regulation, comprising a repressor protein PcaV and a promoter polynucleotide sequence that responds to a small molecule derived from tea polyphenols - PCA to express a recombinant protein. Among them, the expression element is selected from any one of the following (i)-(iv): (i) Comprising the nucleotide sequences shown in SEQ ID NOs. 3 to 5 plus the nucleotide sequence shown in SEQ ID NO. 8; (ii) Comprising the reverse complementary sequence of the nucleotide sequences shown in SEQ ID NOs. 3 to 5 plus the nucleotide sequence shown in SEQ ID NO. 8; (iii) The reverse complementary sequence of a sequence that can hybridize with the nucleotide sequence shown in (i) or (ii) under high-stringency hybridization conditions or very high-stringency hybridization conditions; (iv) Having at least 90%, optionally at least 95%, preferably at least 97%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (i) or (ii).

[0007] In a second aspect, there is provided a transcription expression cassette, which, among others, contains the expression element responsive to PCA regulation described in the first aspect; optionally, the transcription expression cassette further contains a recombinant protein coding sequence and a PCA transporter protein PcaK sequence, and the above sequences are operably linked to the polynucleotide having expression regulatory activity.

[0008] In a third aspect, there is provided a recombinant expression vector applicable to prokaryotes. The recombinant expression vector is an artificially designed and synthesized dual plasmid system, and its nucleotide sequence is as shown in SEQ ID NOs. 1 and 2; among them, it contains the expression element responsive to PCA regulation described in the first aspect, or the transcription expression cassette described in the second aspect.

[0009] In a fourth aspect, there is provided the use of the expression element responsive to PCA regulation described in the first aspect, the transcription expression cassette described in the second aspect, or the recombinant expression vector of prokaryotes described in the third aspect in the preparation of proteins, or in the preparation of reagents or kits for the preparation of proteins; preferably, the protein is a secretable cytokine; more preferably, the protein is tumor necrosis factor-α.

[0010] In a fifth aspect, there is provided a method for enhancing the expression regulation of a target gene. Among them, the method includes operably linking the expression element responsive to PCA regulation described in the first aspect to the target gene; optionally, simultaneously introducing the PCA transporter protein PcaK sequence described in the second aspect.

[0011] The principle of gene expression regulation by the recombinant expression vector of the present invention is that in the absence of PCA, PcaV will bind to the operator O of the PCA-inducible chimeric promoterpcaV Upstream, it represses the expression of downstream genes. When PCA is present in the environment, PCA competitively binds to the operator binding site on PcaV, preventing PcaV from binding to the promoter and activating the expression of downstream genes; while the PcaK transporter can actively transport PCA into prokaryotic cells, enhancing the sensitivity of the recombinant expression vector to PCA.

[0012] The gene circuit responsive to PCA of the present invention is composed of a PCA-inducible chimeric promoter, a recombinant protein sequence, a PcaV sequence, and a PcaK sequence.

[0013] Among them, the gene circuit responsive to PCA can initiate the expression of recombinant protein with a minimum response to 0.5 μM PCA.

[0014] Among them, the recombinant protein sequence is initiated for expression by a PCA-inducible chimeric promoter.

[0015] Among them, the chimeric promoter for expressing the recombinant protein can be the fusion sequence of the P T7A1 promoter and two operators O pcaV , the fusion sequence of the P Tac promoter and two operators O pcaV , or the fusion sequence of the P lacI promoter and two operators O pcaV as shown in the nucleotide sequences of SEQ ID NO. 3 - 5.

[0016] Among them, the promoter for expressing the PcaK sequence is the weak promoter P lacI or the strong promoter P Tac , as shown in the nucleotide sequences of SEQ ID NO. 6 or 7.

[0017] Among them, the PcaV sequence is initiated by the promoter P lacI , as shown in the nucleotide sequence of SEQ ID NO. 8.

[0018] The gene elements of the protein expression system in the present invention are simple and have tunability in various prokaryotic cells.

[0019] Among them, the microbial host cells include but are not limited to E. coli MG1655, E. coli Bl21(DE3), and E. coli Nissle 1917.

[0020] The present invention provides a novel strategy for the specific, efficient, and precise regulation of microbial gene expression. Specifically, the present invention realizes the controllable expression of recombinant proteins in engineered bacteria by utilizing the polynucleotide sequence responsive to PCA.

[0021] The gene expression element regulated by protocatechuic acid in the prokaryote provided by the present invention can be constructed in a prokaryotic expression vector through genetic engineering technology, thereby regulating the expression of the target gene; the gene expression system in the prokaryote provided by the present invention has controllability, safety, and precision, and the chassis cells can be various types of prokaryotic cells, such as E.coli MG1655, E.coli Bl21(DE3), E.coli Nissle 1917, etc. Brief Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the protein expression system regulated by PCA.

[0023] Figure 2 It is the PCA-inducible chimeric promoter in the optimized protein expression system regulated by PCA.

[0024] Figure 3 It is to optimize the expression intensity of the PcaK sequence in the protein expression system regulated by PCA.

[0025] Figure 4 It is the PCA concentration-dependent working condition of the optimized protein expression system regulated by PCA in different bacteria.

[0026] Figure 5 It is to induce the secretion of TNF-α cytokine in the protein expression system regulated by PCA in E. coli MG1655.

[0027] Figure 6 It is to reversibly control the secretion of TNF-α in the protein expression system regulated by PCA in E. coli MG1655. Detailed Embodiments

[0028] The present invention will be further described in conjunction with the drawings and embodiments. The protection scope of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the concept of the present invention, the changes and advantages that those skilled in the art can think of are all included in the present invention. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and public common sense in the art, and the present invention has no special limitations.

[0029] Materials and Methods

[0030] All primers used for PCR were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Hangzhou, China). Escherichia coli DH5α strain, Escherichia coli BL21(DE3) strain, and Escherichia coli MG1655 strain were all purchased from Beijing Tsingke Biotechnology Co., Ltd. (Beijing, China). All enzyme-linked immunosorbent assay (ELISA) kits were provided by Hangzhou Linko Biotechnology Co., Ltd. (Hangzhou, China). Protocatechuic acid (PCA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 2×Hieff Canace Gold PCR Master Mix was purchased from Nanjing Novoprotein Biotechnology Co., Ltd. (Nanjing, China).

[0031] Example 1: Plasmid construction

[0032] In the present invention, the main molecular cloning techniques for constructing the plasmid vectors required for the synthetic gene circuit and the construction methods are shown in Table 1 in detail; the principle of the expression element responsive to PCA regulating the expression of recombinant proteins in cells is shown in detail Figure 1 .

[0033] The specific method is as follows:

[0034] (1) Use software Snapgene to design the target plasmid and the primers required for polymerase chain reaction (PCR), and construct the target plasmid by methods such as enzymatic digestion ligation and homologous arm recombination. The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Hangzhou, China).

[0035] (2) Prepare the target gene fragment by PCR or linearize the vector: Add 25 μL of 2×Hieff Canace Gold PCR Master Mix high-fidelity enzyme premix, 2.5 μL of forward primer (FP), 2.5 μL of reverse primer (RP), 0.2 μL of the parental plasmid to a 0.2 mL centrifuge tube, and then add ddH 2 O to make up the volume to 25 μL. Mix it well, briefly centrifuge it, and then put it into the PCR instrument. Set the PCR program.

[0036] (3) Prepare the target gene fragment by enzymatic digestion or linearize the vector: In this example, restriction endonucleases from Thermo Fisher were mainly used for the enzymatic digestion reaction. After preparing a 50 μL enzymatic digestion system, react at 37 °C for 1 - 2 h

[0037] (4) Anneal to prepare the DNA fragment: Add 3 μL of FP, 3 μL of RP, 2.5 μL of ddH₂O, 1 μL of T4 PNK buffer, and 0.5 μL of T4 PNK kinase to a 0.2 mL centrifuge tube. Mix them well, briefly centrifuge, and then place them in a PCR instrument. Set the program as needed.

[0038] (5) Purify the DNA product: After identifying and isolating the target band by agarose gel electrophoresis, the DNA fragment is recovered using a DNA gel extraction kit to extract PCR, restriction digestion, or annealing products.

[0039] (6) Ligate the target gene and vector: In this example, a seamless cloning kit is mainly used for ligation. The method is as follows: Add 5 μL of Seamless Coloning Mix, 50 ng of vector fragment, and an insert fragment with a molar ratio of 3:1 to a 0.2 mL centrifuge tube, and then add ddH 2 ₂O to make up to 10 μL; mix them well, briefly centrifuge, and then incubate at 50 °C for 15 min to obtain a plasmid library containing the target plasmid.

[0040] (7) Transformation and monoclonal screening: Transform the above plasmid library into DH5α competent cells, gently flick the centrifuge tube to mix, add the system, and place it on ice for 30 min. After standing, place the above centrifuge tube in a 42 °C water bath for heat shock for 45 s, then immediately place it in an ice bath and stand for 2 - 5 min. Add 500 - 900 μL of LB liquid medium without antibiotics to the centrifuge tube on a sterile workbench, and then incubate the centrifuge tube at 220 rpm and 37 °C for 30 min - 1 h. After centrifuging at 4000 rpm for 1 min, remove the excess medium until there is still 50 - 100 μL of liquid left. Gently pipette and mix, drop it on a solid LB culture plate with the corresponding resistance, spread it evenly with a triangular spreader, dry it, invert the culture plate, and incubate it overnight at 37 °C.

[0041] Sequencing and plasmid purification were both completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0042] Example 2: Preparation of genetically engineered Escherichia coli

[0043] Escherichia coli MG1655, BL21(DE3), and Nissle1917 were all cultured in LB medium. The above microbial cell lines all used the CaCl 2 -mediated chemical transformation method.

[0044] (1) First, prepare the chemical competent cells of Escherichia coli MG1655, BL21(DE3), and Nissle1917; (2) Use the heat shock method to co-transform the plasmid system for PCA regulation into chemically competent cells; (3) Screen positive strains using solid LB medium containing working concentrations of chloramphenicol and ampicillin; (4) Amplify engineered Escherichia coli using liquid LB medium containing working concentrations of chloramphenicol and ampicillin.

[0045] Example 3: Induction efficiency of different chimeric promoters on the reporter gene EGFP in the PCA-regulated protein expression system in Escherichia coli.

[0046] In this example, with EGFP as the reporter gene, the effect of using different PCA-inducible chimeric promoters on the induction efficiency of EGFP in the PCA-regulated protein expression system was explored.

[0047] The specific method is as follows:

[0048] First step, construct plasmids containing different chimeric promoters and transform these plasmids into Escherichia coli.

[0049] Second step, the screened engineered Escherichia coli strains were cultured in LB liquid medium containing ampicillin and chloramphenicol at 37 °C and 220 rpm for 12 hours. Adjust the concentration of the engineered Escherichia coli to OD 600 ≈0.5 (in the logarithmic growth phase).

[0050] Third step, add an equal volume but different concentrations of PCA mother liquor to stimulate it, and then incubate it at 37 °C and 1000 rpm for 3 h.

[0051] Fourth step, the bacterial samples after PCA stimulation were centrifuged at 6000 rpm for 3 minutes, washed twice with 1×PBS, and then resuspended in 100 μL of PBS.

[0052] Fifth step, take three parallel samples from each group and transfer them to a black 96-well plate, immediately place them in an enzyme-linked immunosorbent assay (ELISA) reader to detect the reporter gene; then transfer them to a transparent 96-well plate and immediately place them in an ELISA reader to detect the OD 600 value.

[0053] The results showed that the fusion promoter of P Tac and the operon O pcaV (P Tac -O pcaV ) exhibited the lowest basal expression level (about 1.19% of the basal expression compared with the P T7A1 -O pcaV system), as well as the highest expression level after stimulation (compared with the P T7A1 -O pcaVThe system increased by approximately 45.4 times, so we selected P Tac -O pcaV as the optimal ribosomal site sequence. See the experimental data in Figure 2 , and all data are expressed as mean ± standard deviation; n = 3 independent replicate experiments.

[0054] Example 4: In Escherichia coli, exploration of the induction efficiency of the reporter gene EGFP by different PcaK expression intensities in the PCA-regulated protein expression system.

[0055] In this example, with EGFP as the reporter gene, we explored the effect of the lack of the PcaK transporter protein or the use of different promoters to drive PcaK expression on the induction efficiency of EGFP in the PCA-regulated protein expression system.

[0056] The specific method is as follows:

[0057] First step, construct plasmids lacking the pcaK sequence or with different promoters driving PcaK expression, and transform these plasmids into Escherichia coli.

[0058] Second step, the engineered Escherichia coli strains after screening were cultured in LB liquid medium containing ampicillin and chloramphenicol at 37 °C and 220 rpm for 12 hours.

[0059] Third step, adjust the concentration of the engineered Escherichia coli to OD 600 ≈0.5 (in the logarithmic growth phase), add an equal volume but different concentrations of PCA mother liquor for stimulation, and then incubate at 37 °C and 1000 rpm for 3 h.

[0060] Fourth step, the bacterial samples after PCA stimulation were centrifuged at 6000 rpm for 3 minutes, the precipitate was washed twice with 1×PBS, and then resuspended in 100 μL of PBS.

[0061] Fifth step, take three parallel samples from each group and transfer them to a black 96-well plate, immediately place them in a microplate reader to detect the reporter gene. Then transfer them to a transparent 96-well plate and immediately place them in a microplate reader to detect the OD 600 value.

[0062] The results showed that although both promoters could improve the efficiency, when exposed to low concentration PCA (1 μM), the PCA induction loop encoded by P lacI -PcaK showed more sensitive transgene expression (about 6.5 times), so we selected P lacI to initiate the expression of pcaK. See the experimental data in Figure 3 , and all data are expressed as mean ± standard deviation; n = 3 independent replicate experiments.

[0063] Example 5: PCA-regulated protein expression system's PCA concentration-dependent performance in different bacteria.

[0064] In this example, EGFP was used as the reporter gene to explore whether the PCA-regulated protein expression system could function in different bacteria.

[0065] The specific steps are as follows:

[0066] First, an optimized plasmid system was constructed and these plasmids were transformed into E. coli MG1655, E. coli BL21(DE3), or E. coli Nissle.

[0067] Second, the screened engineered E. coli strains were cultured in LB liquid medium containing ampicillin and chloramphenicol at 37 °C and 220 rpm for 12 hours.

[0068] Third, the concentration of the engineered E. coli was adjusted to OD 600 ≈0.5 (in the logarithmic growth phase), and an equal volume of PCA mother liquor with different concentrations was added for stimulation, followed by incubation at 37 °C and 1000 rpm for 3 h.

[0069] Fourth, the bacterial samples after PCA stimulation were centrifuged at 6000 rpm for 3 minutes, the precipitate was washed twice with 1×PBS, and then resuspended in 100 μL of PBS.

[0070] Fifth, three parallel samples from each group were transferred to a black 96-well plate and immediately placed in a microplate reader to detect the reporter gene. Then they were transferred to a transparent 96-well plate and immediately placed in a microplate reader to detect the OD 600 value.

[0071] The results showed that PCA exhibited good induction effects on genetically engineered E. coli MG1655, E. coli BL21(DE3), or E. coli Nissle, indicating the universality of this PCA-regulated protein expression system in different strains. The experimental data are shown in Figure 4 , and all data are presented as mean ± standard deviation; n = 5 independent replicate experiments.

[0072] Example 6: Detection of PCA-regulated anti-tumor cytokine secretion in engineered E. coli MG1655. In this example, non-pathogenic E. coli MG1655 was used as the chassis cell to explore whether PCA regulation could effectively regulate the secretion of the anti-tumor cytokine TNF-α from genetically engineered MG1655.

[0073] The specific steps are as follows:

[0074] First, construct a plasmid system containing the TNFA sequence as shown in SEQ ID NO.9, and transform these plasmids into E. coli MG1655.

[0075] Second, the screened engineered MG1655 strain was cultured in LB liquid medium containing ampicillin and chloramphenicol at 37 °C and 220 rpm for 12 hours.

[0076] Third, adjust the concentration of engineered MG1655 to OD 600 ≈0.5 (in the logarithmic growth phase), add an equal volume but different concentrations of PCA stock solution for stimulation, and then incubate at 37 °C and 1000 rpm for 3 h.

[0077] Fourth, the bacterial samples after PCA stimulation were centrifuged at 6000 rpm for 3 minutes, and the supernatant samples were taken.

[0078] Fifth, take three parallel samples from each group and use an ELISA kit (Linkage Biotechnology) to measure the concentration of TNF-α. Specifically, (1) Add 300 μL of 1× wash buffer to each well of the ELISA plate, soak for 30 s, and then gently pat on the absorbent paper until the wash buffer is removed. (2) Add 100 μL of the above culture supernatant and incubate at 300 rpm and room temperature for 2 h. (3) Wash each well with 300 μL of 1× wash buffer, and then completely remove the wash buffer, repeat 6 times. (4) Add 100 μl of diluted detection antibody to each well and incubate at 300 rpm and room temperature for 45 min. (5) After washing 6 times, add 100 μL of the chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 5 - 30 min. (6) After determining that it can be terminated, add 100 μL of stop solution to each well. (7) Detect the absorption wavelengths at 450 nm, 570 nm, and 630 nm with an enzyme-linked immunosorbent assay reader, and then process the data.

[0079] The results showed that genetically engineered MG1655 significantly induced the production of TNF-α in a PCA dose-dependent manner: when induced with 0.5 μM PCA, the expression increased by 32-fold; when induced with 100 μM PCA, the expression increased by 70-fold. The experimental data are shown in Figure 5 , and all data are expressed as mean ± standard deviation; n = 3 independent replicate experiments.

[0080] Example 7: Detection of the reversibility of PCA-regulated anti-tumor cytokine secretion in genetically engineered non-pathogenic bacteria MG1655.

[0081] In this example, non-pathogenic Escherichia coli MG1655 was used as the chassis cell to explore whether the PCA regulation could reversibly regulate the secretion of the anti-tumor cytokine TNF-α from genetically engineered MG1655, so as to control the expression concentration of TNF-α within the safety window.

[0082] The specific steps are as follows:

[0083] First, construct a plasmid system containing the TNFA sequence and transform these plasmids into E. coli MG1655.

[0084] Second, the engineered MG1655 strain after screening was cultured in LB liquid medium containing ampicillin and chloramphenicol at 37 °C and 220 rpm for 12 hours.

[0085] Third, adjust the concentration of engineered MG1655 to OD 600 ≈0.25 and continuously culture for 26 h. During this period, alternately change the PCA concentration from 0 to 10 μM, and collect the culture supernatant after 1 hour of PCA incubation and at 6 hours and 12 hours after removing PCA for further ELISA detection.

[0086] Fourth, take three parallel samples in each group and use an ELISA kit (Linkage Biotechnology) to measure the concentration of TNF-α. Specifically, (1) Add 300 μL of 1× wash buffer to each well of the ELISA plate, soak for 30 s, and then gently pat on the absorbent paper until the wash buffer is removed. (2) Add 100 μL of the above culture supernatant and incubate at 300 rpm and room temperature for 2 h. (3) Wash each well with 300 μL of 1× wash buffer and then completely remove the wash buffer, repeating 6 times. (4) Add 100 μl of diluted detection antibody to each well and incubate at 300 rpm and room temperature for 45 min. (5) After washing 6 times, add 100 μL of the chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 5 - 30 min. (6) After determining that it can be terminated, add 100 μL of stop solution to each well. (7) Detect the absorption wavelength at 450 nm, 570 nm, and 630 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and then process the data.

[0087] The results showed that genetically engineered MG1655 induced the production of TNF-α in the presence of PCA and gradually stopped producing TNF-α in the absence of PCA, effectively controlling the secretion concentration of TNF-α. The experimental data are shown in Figure 6 , and all data are expressed as mean ± standard deviation; n = 3 independent replicate experiments.

[0088] Table 1. Plasmid vectors and construction methods

[0089] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

Claims

1. An expression element responsive to PCA regulation, characterized in that: The expression element is selected from any one of the following (i)-(iv): (i) comprising the nucleotide sequence shown in SEQ ID NOs. 3 to 5 plus the nucleotide sequence shown in SEQ ID NO. 8; (ii) comprising the nucleotide sequence shown in SEQ ID NOs. 3 to 5 plus the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO. 8; (iii) a reverse complementary sequence of a sequence that can hybridize to the nucleotide sequence shown in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions; (iv) has at least 90%, optionally at least 95%, preferably at least 97%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in (i) or (ii).

2. An expression element responsive to PCA regulation according to claim 1, characterized in that: Includes a PCA inducible chimeric promoter and a repressor protein PcaV sequence.

3. An expression element responsive to PCA regulation as claimed in claim 2, characterized in that: The PCA inducible chimeric promoter is based on the operator O pcaV Different types of fused promoters can form different types of promoters, and their nucleotide sequences are shown in SEQ ID NOs. 3-5.

4. An expression element responsive to PCA regulation according to claim 2, characterized in that: The repressor protein PcaV sequence is composed of P lacI Promote expression, and its nucleotide sequence is shown in SEQ ID NO.

8.

5. A transcription expression cassette, characterized in that: The transcription expression cassette comprises the expression element responsive to PCA regulation as described in claim 1; optionally, the transcription expression cassette also contains a recombinant protein coding sequence and a PCA transporter protein PcaK sequence, and the above sequence is operably linked to the polynucleotide having expression regulation activity.

6. The transcription expression cassette according to claim 5, characterized in that The PCA transporter PcaK sequence is mediated by the promoter P lacI or P tac Promote expression, and its nucleotide sequence is shown in SEQ ID NO.6 or 7.

7. A recombinant expression vector suitable for prokaryotes, characterized in that: The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NOs. 1 and 2; the expression vector comprises the expression element responsive to PCA regulation according to claim 1, or the transcription expression cassette according to claim 2.

8. A method for enhancing the expression regulation of a target gene, characterized in that: The method comprises connecting the expression element responsive to PCA regulation of claim 1 to a target gene; optionally, simultaneously introducing the repressor protein PcaV sequence of claim 3 and the PCA transporter protein PcaK sequence of claim 6.

9. A method for preparing a protein, characterized in that: Select the expression element responsive to PCA regulation as described in claim 1, the transcription expression cassette as described in claim 2, or the recombinant expression vector suitable for prokaryotes as described in claim 5; preferably, the protein is a secreted cytokine; further preferably, the protein is tumor necrosis factor-α.

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