DNA fragment and method for expressing multiple genes in biological cell
By introducing expression cassettes into biological cells and using ACX3 IN7 or IGR DNA fragments, the problem of insufficient regulation of multiple gene expression was solved, and independent and efficient expression of multiple genes was achieved.
Patent Information
- Application Number
- CN202410470427.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-18
- Publication Date
- 2025-10-24
AI Technical Summary
There is no systematic study on the efficiency of restarting the window in the current technology, which leads to insufficient regulation of the expression of multiple genes.
By introducing an expression cassette containing multiple genes into a biological cell, the expression of downstream genes is mediated by a specific DNA fragment such as ACX3 IN7 or IGR. This DNA fragment has a starting function and ensures identity through hybridization under strict conditions, thereby achieving the co-expression of multiple genes.
The independent expression of multiple genes was successfully achieved in biological cells, improving the efficiency and regulatory capacity of gene expression.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, DNA fragments and methods for expressing multiple genes in biological cells. BACKGROUND
[0002] Protein production is derived from mature eukaryotic messenger RNA (mRNA), seemingly simple: the protein-coding open reading frame (ORF) of each mRNA is defined by a translation initiation site and a corresponding termination site. This ORF is precisely and accurately recognized by the ribosome translation sequence to produce a functional protein. However, while most eukaryotic mRNAs contain a single "major" ORF encoding a polypeptide product, often multiple peptides are synthesized from a single mRNA. This is often due to "translation reinitiation" (TeRe), when a ribosome begins making a protein from a start site, translation proceeds until it terminates at a stop codon, but then escapes the normal recycling steps and then reinitiates ("re-starts") at a different, downstream site. Reinitiation can enable translation of an ORF from a start codon that is downstream, upstream, or internal to the major ORF, but in all cases, a ribosome can produce multiple proteins from a single mRNA. This form of translational regulation has many important roles, such as rapidly responding to environmental changes (Lu et al., 2004; Liu and Qian, 2014; Young and Wek, 2016; Zhou et al., 2018). Thus, reinitiation is an important part of the rich repertoire of mechanisms that eukaryotes use to regulate translation.
[0003] The distance between the upstream stop codon and the downstream start codon constitutes the "reinitiation window", and the distance varies in different reports. The smallest distance between the stop codon and the start codon is overlapping, i.e., the stop codon and the start codon share the same nucleotide. For example, UGAUG (stop codon is represented in italics, start codon is represented in bold), represents a one-nucleotide gene overlap, while AUGA is a two-nucleotide overlap.
[0004] There is no systematic study on the efficiency of the reinitiation window, but it can be expected that the efficiency of reinitiation will be negatively correlated with the distance between the stop codon and the reinitiation codon, because reinitiation requires the 30S subunit (or 70S ribosome) to scan the mRNA, and the probability of dissociation increases with the distance. Therefore, finding reinitiation windows of different lengths helps to fill the relevant theoretical gap, and thus can be applied to the expression regulation of two interrelated genes in prokaryotes. SUMMARY
[0005] The technical problem to be solved by the present application is how to realize expression of multiple genes.
[0006] To solve the above technical problem, the present application first provides a method for expressing multiple genes in a biological cell, the method comprising: introducing an expression cassette containing multiple genes into a biological cell to realize expression of the multiple genes; the multiple genes are greater than or equal to 2 genes, and each gene in the expression cassette is connected in sequence by a DNA segment;
[0007] The DNA segment is a), b) or c) as follows:
[0008] a) at least contains SEQ ID No. 1 from 45 to 121, and from the 45th and 121st positions of SEQ ID No. 1, the nucleotide sequence of SEQ ID No. 1 is extended to the 5' end and / or 3' end of SEQ ID No. 1, to obtain any one DNA segment with a length of 80 to 141 bp; the DNA segment has the function of starting the expression of downstream genes;
[0009] b) a DNA segment with 75% or more identity with the nucleotide sequence defined in a) and having the function of starting the expression of downstream genes;
[0010] c) a DNA segment that hybridizes to the nucleotide sequence defined in a) or b) under stringent conditions and has the function of starting the expression of downstream genes.
[0011] The term "identity" used herein refers to sequence similarity with the natural nucleic acid sequence. "Identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the sequence of the present application including SEQ ID No. 1 from 45 to 121, which is shorter than or the same as SEQ ID No. 1. The identity can be evaluated by naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
[0012] In the above-mentioned application, the stringent condition can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C in 2xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C in lxSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C in 0.5xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing at 65°C in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 65°C in a solution of 6xSSC, 0.5% SDS, and rinsing the membrane once each with 2xSSC, 0.1% SDS and lxSSC, 0.1% SDS; or as follows: hybridization at 68°C in a solution of 2xSSC, 0.1% SDS, and rinsing the membrane twice, each for 5min, and hybridization at 68°C in a solution of 0.5xSSC, 0.1% SDS, and rinsing the membrane twice, each for 15min; or as follows: hybridization at 65°C in a solution of 0.1xSSPE (or 0.1xSSC), 0.1% SDS, and rinsing the membrane.
[0013] The above-mentioned 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
[0014] In the above-mentioned method, the DNA fragment can be the DNA fragment shown in positions 45-121 of SEQ ID No. 1 or the DNA fragment shown in SEQ ID No. 1.
[0015] In the above-mentioned method, the expression cassette can be introduced into the biological cell by an expression vector containing the expression cassette. The vector can be a plasmid, a cosmid, a bacteriophage or a viral vector. Specifically, the plasmid can be a pUC57-Kan vector.
[0016] In the above-mentioned method, the biological cell can be a microbial cell, a plant cell or an animal cell.
[0017] Specifically, the microbial cell can be a bacterium, a yeast, an algae or a fungus.
[0018] Further, the bacterium can be Escherichia coli (such as Escherichia coli K-12 MG1655).
[0019] The DNA fragment also belongs to the protection scope of the present application.
[0020] The present application also provides biological materials related to the DNA fragment, which are any of the following B1) to B4):
[0021] B1) an expression cassette containing the DNA fragment;
[0022] B2) a recombinant vector containing the expression cassette of B1);
[0023] B3) a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2);
[0024] B4) a cell line containing the expression cassette of B1), or a cell line containing the recombinant vector of B2).
[0025] The expression cassette refers to DNA capable of expressing proteins in host cells, which can include not only promoters for initiating gene transcription, but also terminators for terminating gene transcription.
[0026] The recombinant vector containing the expression cassette can be constructed using existing expression vectors.
[0027] In the above biological materials, the vector can be a plasmid, cosmid, bacteriophage or viral vector. The plasmid can be specifically pUC57-Kan vector.
[0028] In the above biological materials, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Escherichia coli (such as Escherichia coli K-12 MG1655).
[0029] The application of the DNA fragment in mediating the expression of multiple genes also belongs to the protection scope of the present application.
[0030] The application of the biological material in the expression of multiple genes also belongs to the protection scope of the present application.
[0031] In the present application, the multiple genes can be greater than or equal to 2 genes, and the maximum number of genes is not limited, as long as each gene in the multiple genes can be expressed, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 genes, etc. In an embodiment of the present application, the multiple genes are GFP gene and RFP gene.
[0032] Experiments prove that the method for expressing multiple genes in biological cells of the present application and the DNA fragment used can successfully realize the co-expression of multiple genes, and has good application prospect.
[0033] The application will be described in further detail below with specific reference to the embodiments. The examples provided are only for the purpose of illustrating the application and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 ACX3 IN7 and IGR can mediate the expression of downstream genes. A) Schematic diagram of three recombinant vector expression cassettes, P represents the promoter, and T represents the terminator; B) Detection of the expression of GFP and RFP in three recombinant bacteria under a fluorescence microscope, GFP-IN-RFP represents GFP-ACX3 IN7-RFP; C) Western blotting detection of the expression of upstream and downstream genes GFP and RFP in different recombinant bacteria. DETAILED DESCRIPTION
[0035] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. In the following examples, the materials, reagents, instruments, etc. used, unless otherwise specified, can be obtained commercially. In the following examples, at least three repeated experiments were set for the quantitative test, and the average value was taken. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.
[0036] Anti-GFP tag mouse monoclonal antibody: Beijing Quanshi Gold Biotechnology Co., Ltd. (Item No.: HT801-01);
[0037] Anti-RFP tag mouse monoclonal antibody: Bioeasyjet (Beijing) Technology Co., Ltd. (Item No.: BE2024-100).
[0038] Example 1, ACX3 IN7 and fragments thereof can mediate the expression of multiple downstream genes
[0039] The inventors found that a DNA fragment (ACX3 IN7, 141 bp, its sequence is SEQ ID No. 1 in the sequence listing) derived from an intron in eukaryotic organism Arabidopsis thaliana and its 45-121 positions (denoted as IGR) can mediate the expression of downstream genes in two relatively independent genes in E. coli.
[0040] In this example, the inventors constructed two independent expression cassettes of fluorescent reporter genes GFP and RFP (GFP-ACX3 IN7-RFP and RFP-ACX3 IN7-GFP) and introduced them into E. coli to test whether the ACX3 IN7 and IGR can mediate the expression of downstream genes. Figure 1GFP gene contains normal stop codon UGA. To check whether ACX3 IN7 and IGR can mediate the expression of downstream genes, ACX3 IN7 and IGR were inserted between the GFP and RFP genes, respectively, and their ability to mediate the expression of downstream genes was checked.
[0041] 1. Construction of recombinant vectors
[0042] pUC-GFP-RFP: The GFP-RFP expression cassette shown in SEQ ID No. 2 in the sequence listing was inserted in the multiple cloning site of pUC57-Kan vector using restriction enzymes Kpnl and Sad.
[0043] In SEQ ID No. 2, positions 1-185 represent the promoter, positions 186-914 represent the GFP gene, positions 915-1592 represent the RFP gene, and positions 1593-1710 represent the terminator.
[0044] pUC-GFP-ACX3IN7-RFP: The GFP-ACX3IN7-RFP expression cassette shown in SEQ ID No. 3 in the sequence listing was inserted in the multiple cloning site of pUC57-Kan vector using restriction enzymes Kpnl and Sad.
[0045] In SEQ ID No. 3, positions 1-185 represent the promoter, positions 186-914 represent the GFP gene, positions 915-1055 represent ACX3 IN7, positions 1056-1733 represent the RFP gene, and positions 1734-1851 represent the terminator.
[0046] pUC-GFP-IGR-RFP: The GFP-IGR-RFP expression cassette shown in SEQ ID No. 4 in the sequence listing was inserted in the multiple cloning site of pUC57-Kan vector using restriction enzymes Kpnl and Sad.
[0047] In SEQ ID No. 4, positions 1-185 represent the promoter, positions 186-914 represent the GFP gene, positions 915-991 represent IGR, positions 992-1669 represent the RFP gene, and positions 1670-1787 represent the terminator.
[0048] 2. Construction of recombinant bacteria
[0049] The pUC-GFP-RFP, pUC-GFP-ACX3IN7-RFP and pUC-GFP-IGR-RFP were introduced into E. coli K-12 MG1655 (Beijing Zhuangmeng International Biotech Co., Ltd.) respectively to obtain recombinant bacteria MG1655 / pUC-GFP-RFP, MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP.
[0050] 3. Detection of protein expression
[0051] The strains to be tested: MG1655 / pUC-GFP-RFP, MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP.
[0052] The strains to be tested were cultured in LB liquid medium (containing kanamycin 50 μg / ml) at 180 rpm and 37°C for 16 h, and then the bacterial bodies were collected.
[0053] The expression of fluorescent proteins was detected under a fluorescence microscope, and the results are shown in Figure 1 As shown in B of FIG. 6, the three strains all contained GFP fluorescent signals, indicating that the upstream gene GFP gene could be expressed in the three strains. In addition to MG1655 / pUC-GFP-RFP which did not contain RFP fluorescent signals, MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP both contained RFP fluorescent signals, indicating that the downstream gene RFP gene in MG1655 / pUC-GFP-RFP could not be expressed, while the downstream gene RFP gene in MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP could be expressed. This indicated that ACX3IN7 and IGR could mediate the expression of downstream genes.
[0054] In order to further determine whether the upstream and downstream genes GFP and RFP were independently expressed or expressed as a fusion protein, the inventors detected the total protein of the three strains by western blotting, and the antibodies used were anti-GFP tag mouse monoclonal antibody and anti-RFP tag mouse monoclonal antibody.
[0055] The results are shown in Figure 1As shown in Fig. 3, it can be seen that the expression products of the upstream gene GFP can be detected in all three groups of experiments, while the RFP protein of the downstream gene RFP can only be detected in MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP which have the upstream gene linked to the downstream gene by ACX3IN7 and IGR. In addition, the sizes of the GFP proteins detected in the three groups are the same, which are consistent with the size of the wild-type GFP protein, indicating that the translation of the upstream protein GFP stops after encountering its stop codon; while the sizes of the RFP proteins detected in MG1655 / pUC-GFP-ACX3IN7-RFP and MG1655 / pUC-GFP-IGR-RFP are the same, and different from the size of the GFP protein, indicating that the two proteins are independently expressed rather than fusion-expressed in the strains expressing both GFP and RFP proteins.
[0056] In summary, both ACX3IN7 and IGR can mediate the expression of the downstream gene in two independent genes in E. coli.
[0057] The present application has been described in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application is intended to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.
Claims
1. A method of expressing a plurality of genes in a biological cell, comprising: The expression cassette containing multiple genes is introduced into a biological cell to realize expression of the multiple genes; The multiple genes are equal to or greater than 2 genes, and each gene in the expression cassette is connected by a DNA fragment; The DNA fragment is a), b) or c) as follows: a) at least contains the 45th to 121st positions of SEQ ID No. 1, and is extended from the 45th and 121st positions of SEQ ID No. 1 to the 5' and / or 3' end of SEQ ID No. 1 according to the nucleotide sequence of SEQ ID No. 1 to obtain any one DNA fragment with a length of 80 to 141 bp; the DNA fragment has the function of starting the expression of downstream genes; b) the DNA fragment has 75% or more identity with the nucleotide sequence defined in a), and has the function of starting the expression of downstream genes; c) the DNA fragment hybridizes to the nucleotide sequence defined in a) or b) under stringent conditions, and has the function of starting the expression of downstream genes.
2. The method of claim 1, wherein: The DNA fragment is the DNA fragment shown in the 45th to 121st positions of SEQ ID No. 1 or the DNA fragment shown in SEQ ID No.
1.
3. The method according to claim 1 or 2, characterized in that: The expression cassette is introduced into the biological cell by an expression vector containing the expression cassette.
4. The method of any one of claims 1-3, wherein: The biological cell is a microbial cell, a plant cell or an animal cell.
5. The method of claim 4, wherein: The microbial cell is bacteria, yeast, algae or fungi.
6. The method of claim 5, wherein: The bacteria is Escherichia coli.
7. The DNA fragment as claimed in claim 1 or 2.
8. A biological material related to the DNA fragment as claimed in claim 1 or 2, which is any one of the following B1) to B4): B1) an expression cassette containing the DNA fragment as claimed in claim 1 or 2; B2) a recombinant vector containing the expression cassette as claimed in B1); B3) a recombinant microorganism containing the expression cassette as claimed in B1), or a recombinant microorganism containing the recombinant vector as claimed in B2); B4) a cell line containing the expression cassette as claimed in B1), or a cell line containing the recombinant vector as claimed in B2).
9. Use of the DNA fragment as claimed in claim 1 or 2 in mediating expression of multiple genes.
10. Use of the biological material as claimed in claim 8 in expression of multiple genes.