Construction body for co-expression of multiple proteins, application of construction body and multi-protein co-expression method

Abstract

The invention belongs to the technical field of biology, and discloses a construct for co-expression of multiple proteins, application of the construct and a multi-protein co-expression method. The construct provided by the invention comprises a target genome, the target genome comprises two or more coding genes, and two adjacent coding genes are connected through a connecting fragment; the connecting fragment comprises a termination codon and an initiation codon along a direction from 5'to 3 ', and the termination codon and the initiation codon share a partial sequence. When the construction body is applied to co-expression of a plurality of proteins, the simultaneous, independent and efficient expression of the plurality of proteins can be realized under the condition of maintaining the protein structure and function, and the construction body has a good application prospect in the genetic engineering technology.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a construct for the co-expression of multiple proteins, its application, and a method for the co-expression of multiple proteins. Background Technology

[0002] Polycistronic genes are genes that can drive the expression of multiple proteins on a single RNA transcript, enabling the simultaneous expression of multiple proteins. In genetic engineering, polycistronic genes can be tandemly linked to construct polycistronic expression vectors, thereby achieving the co-expression of multiple proteins, simplifying experimental procedures and improving expression efficiency.

[0003] Currently, the linker fragments used in polycistronic vectors to tandemly express multiple protein-coding genes mainly include 2A peptide-coding genes and IRES fragments. 2A peptides are short viral peptides, ranging from 18 to 22 amino acids in length. During translation, they undergo self-cleavage to maintain translation, mediating protein-protein cleavage and enabling the simultaneous and independent expression of multiple proteins. However, different types of 2A peptides exhibit significant differences in cleavage efficiency, and the N-terminal amino acid residues resulting from 2A peptide self-cleavage are retained in the upstream protein, while the C-terminal amino acid residues are retained in the downstream protein, potentially affecting protein structure and function, and even leading to loss of protein function. IRES fragments are internal ribosome entry sites that fold into a structure similar to initiation tRNA, directly binding to ribosomes to independently translate mRNA sequences other than the translation initiation site, thus enabling the independent translation of multiple genes onto the same mRNA. However, IRES sequences are generally 500–600 bp in length, limiting the number of protein-coding genes that can be carried on polycistronic vectors, and the presence of IRES sequences may affect the structure of the transcribed mRNA, thereby impacting protein translation efficiency.

[0004] Therefore, it is of great significance to obtain a construct that can simultaneously, independently and efficiently express multiple proteins while maintaining their structure and function. Summary of the Invention

[0005] The primary objective of this invention is to provide a construct for the co-expression of multiple proteins. This construct connects two adjacent coding genes through a linker fragment with a specific structure. When applied to the co-expression of multiple proteins, it can achieve the simultaneous, independent, and efficient expression of multiple proteins while maintaining their structure and function, and has promising applications in genetic engineering technology.

[0006] A second objective of this invention is to provide the application of the above-described constructs for the co-expression of multiple proteins in genetic engineering technology.

[0007] A third objective of this invention is to provide a method for co-expressing multiple proteins.

[0008] Specifically, the construct for co-expression of multiple proteins provided by the present invention includes a target genome, which includes two or more coding genes, and adjacent coding genes are connected by a linker fragment; the linker fragment includes a stop codon and a start codon along the 5' to 3' direction, and the stop codon and start codon share a portion of the sequence.

[0009] Furthermore, the connection segment includes multiple independently configured stop codons, start codons, or multiple sets of stop codons and start codons sharing a common partial sequence.

[0010] Furthermore, the linker fragment includes one or more nucleotide fragments with sequences as shown in SEQ ID NO:1-4.

[0011] Furthermore, the nucleotide sequence of the linker fragment is shown in SEQ ID NO:1.

[0012] Furthermore, the construct is DNA and / or RNA.

[0013] Furthermore, the construct also includes a promoter located upstream of the target genome.

[0014] Furthermore, the promoter includes one or more of the following: CMV promoter, CAG promoter, T7 promoter, Lac promoter, Trp promoter, Tac promoter, SV40 promoter, CBA promoter, and EF1α promoter.

[0015] Furthermore, the promoter comprises a nucleotide fragment with a sequence as shown in SEQ ID NO:7 and / or a nucleotide fragment with a sequence as shown in SEQ ID NO:10.

[0016] This invention provides the application of the above-mentioned construct for the co-expression of multiple proteins in genetic engineering technology.

[0017] The method for co-expression of multiple proteins provided by the present invention includes: expressing the above-mentioned construct in a protein expression system to achieve co-expression of multiple proteins.

[0018] Furthermore, the protein expression system is an in vivo expression system and / or an in vitro expression system.

[0019] Furthermore, the in vitro expression system includes a cell expression system and / or a cell-free expression system.

[0020] Beneficial effects:

[0021] The construct provided by this invention includes multiple coding genes for guiding protein synthesis, and adjacent coding genes are linked together by a specific linker fragment. The linker fragment specifically includes a stop codon and a start codon along the 5' to 3' direction, and the stop codon and start codon share a partial sequence to partially or completely hide the start codon within the stop codon. The presence of this linker fragment does not significantly affect the structure of the transcribed mRNA, and the coding genes upstream and downstream of it have good translation efficiency. It can achieve the simultaneous, independent, and efficient expression of multiple proteins while maintaining protein structure and function, and has good application prospects.

[0022] In some specific embodiments, the nucleotide sequence of the linker fragment is preferably as shown in SEQ ID NO:1; in this case, compared with other linker fragments, the presence of the linker fragment can effectively improve the translation efficiency of the mRNA transcribed from the construct, and effectively induce the simultaneous expression of multiple proteins, so as to ultimately express a large amount of target protein. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the construct I provided in Embodiment 1 of the present invention;

[0024] Figure 2 This is one of the experimental results (scale bar is 200 μm) showing the protein expression of construct I provided in Example 1 of this invention in HEK293 cells;

[0025] Figure 3 The second flow cytometry result of the protein expression of construct I provided in Example 1 of this invention in HEK293 cells (ratio of GFP protein fluorescence intensity to Tdtomoato protein fluorescence intensity);

[0026] Figure 4 This is a schematic diagram of the structure of the construct II provided in Embodiment 2 of the present invention;

[0027] Figure 5 This is one of the experimental results (luciferase LuxSit) showing the protein expression of construct II provided in Example 2 of this invention in HEK293 cells;

[0028] Figure 6 The second figure shows the experimental results of protein expression of construct II provided in Example 2 of this invention in HEK293 cells (luciferase Gluc);

[0029] Figure 7Figure 3 shows the experimental results of protein expression of construct II provided in Example 2 of this invention in HEK293 cells (luciferase Rluc);

[0030] Figure 8 Figure 4 shows the experimental results of protein expression of construct II provided in Example 2 of this invention in HEK293 cells (β-galactosidase);

[0031] Figure 9 This is a schematic diagram of the structure of builder III provided in Embodiment 3 of the present invention.

[0032] Figure 10 This is one of the experimental results (luciferase Rluc) of construct III provided in Example 3 of the present invention and the RNA obtained from its in vitro transcription in HEK293 cells.

[0033] Figure 11 Figure 2 shows the experimental results of protein expression (β-galactosidase) in HEK293 cells of the construct III provided in Example 3 of this invention and the RNA obtained from its in vitro transcription.

[0034] Figure 12 Figure 3 shows the experimental results of protein expression in HEK293 cells of the construct III provided in Example 3 of this invention and the RNA obtained from its in vitro transcription (luciferase LuxSit).

[0035] Figure 13 Figure 4 shows the experimental results of protein expression in HEK293 cells of the construct III provided in Example 3 of this invention and the RNA obtained from its in vitro transcription (luciferase Gluc). Detailed Implementation

[0036] The specific sequences involved in this invention are shown in Table 1.

[0037] Table 1.

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Based on a profound understanding of the problems in existing genetic engineering techniques caused by the introduction of linker fragments such as the 2A peptide coding gene and IRES sequence in polycistronic vectors, including protein structural errors and loss of function, excessively large polycistronic vectors, low protein translation efficiency, and large differences in translation efficiency among different proteins, the inventors of this invention, through extensive and in-depth research and numerous experiments, creatively discovered that by partially or completely hiding the start codon of a downstream gene within the stop codon of an upstream gene—that is, constructing a linker fragment by sharing a sequence between the stop and start codons—when applied to tandem multiple coding genes, it does not significantly affect the structure of the mRNA transcribed from the polycistronic vector. Furthermore, both the upstream and downstream coding genes exhibit good translation efficiency, enabling the simultaneous, independent, and efficient expression of multiple proteins while maintaining protein structure and function. Based on this, the technical solution of this invention was obtained.

[0046] In this invention, the construct for co-expression of multiple proteins specifically includes: a target genome, which includes two or more coding genes, and adjacent coding genes are connected by a linker fragment; the linker fragment includes a stop codon and a start codon along the 5' to 3' direction, and the stop codon and start codon share a portion of the sequence.

[0047] In this invention, the stop codon in the linker fragment refers to a nucleotide fragment that provides a termination signal for the translation of a gene encoding a specific protein. The nucleotide sequence of the stop codon is specifically TAA, TAG, or TGA, and the nucleotide sequence of the start codon is specifically ATG. The key to the linker fragment achieving simultaneous, independent, and efficient expression of multiple proteins lies in at least one set of stop codons and start codons with shared partial sequences. The linker fragment may also include multiple independently set stop codons and start codons or multiple sets of stop codons and start codons with shared partial sequences.

[0048] In this invention, "the target genome includes two or more coding genes" refers to a construct containing multiple protein coding genes tandemly. Specifically, the number of coding genes is adaptively selected based on the types of proteins to be expressed, and can be 2, 3, 4, 5, 6, 7, 8, or any other integer value greater than these. The specific nucleotide sequence of the coding gene is adaptively designed based on the amino acid sequence of the protein to be expressed and the protein expression system used; this invention does not impose any particular limitation on it.

[0049] In some specific embodiments, the linker fragment preferably comprises one or more nucleotide fragments with sequences as shown in SEQ ID NO:1-4. In some preferred embodiments, the nucleotide sequence of the linker fragment is as shown in SEQ ID NO:1; in this case, the translation efficiency of the mRNA transcribed from the construct can be effectively improved, and the simultaneous expression of multiple proteins can be well induced, so as to ultimately express a large amount of the target protein.

[0050] In this invention, the construct refers to a genetic vector carrying a target gene fragment, a commonly used technique in genetic engineering. Those skilled in the art can combine and connect functional fragments such as promoters and terminators with the target genome as needed to obtain the desired construct. That is, the design of the connecting fragments in the construct provided by this invention is key to achieving the technical effect of "simultaneous, independent, and efficient expression of multiple proteins while maintaining protein structure and function." This invention does not specifically limit other parts of the construct structure; constructs designed by those skilled in the art based on the disclosure of this invention are also covered within the scope of protection of this invention.

[0051] In this invention, the construct can specifically be DNA and / or RNA. Those skilled in the art can make adaptive choices according to actual needs, and this invention does not impose any particular limitations. More specifically, specific examples of the construct form can be, but are not limited to, one or more of the following: plasmid vector, phage vector, animal virus vector, viral vector, mRNA, and circRNA.

[0052] In this invention, when the construct is DNA, the construct preferably further includes a promoter located upstream of the target genome. The phrase "located upstream of the target genome" describes the relative positional relationship between the promoter and the target genome on the construct. Upstream and downstream are defined by the order in which they are bound by RNA polymerase; that is, the portion bound by RNA polymerase first is defined as upstream, and the portion bound by RNA polymerase later is defined as downstream. Specifically, "a promoter located upstream of the target genome" means that the promoter's start position is before the start position of the target genome, in which case the promoter is bound by RNA polymerase before the target genome.

[0053] In this invention, the promoter refers to a nucleotide sequence that can be recognized and bound by RNA polymerase to initiate transcription. It is a commonly used technique in genetic engineering, and specific examples include, but are not limited to, one or more of the following: CMV promoter, CAG promoter, T7 promoter, Lac promoter, Trp promoter, Tac promoter, SV40 promoter, CBA promoter, and EF1α promoter. Specifically, the CMV promoter refers to a strong mammalian expression promoter derived from human cytomegalovirus. The CAG promoter refers to a synthetically produced promoter obtained by fusing the CMV promoter, the chicken β-actin gene promoter, and the rabbit β-Globin gene splice acceptor, exhibiting strong initiation capabilities in a wide range of cells. The T7 promoter refers to a promoter derived from T7 bacteriophage, whose transcription requires the participation of T7 RNA polymerase. The Lac promoter refers to a promoter derived from the Lac operon, which can be induced by IPTG or lactose to begin expression. The Trp promoter refers to a promoter derived from the tryptophan operon, whose expression can be regulated by adjusting intracellular tryptophan concentration. The Tac promoter refers to a synthetic promoter obtained by fusing the lac promoter and the Trp promoter, possessing strong initiation capability. The SV40 promoter refers to a strong mammalian expression promoter derived from simian vacuolating virus 40. The CBA promoter refers to a synthetic promoter obtained by fusing the CMV promoter, the chicken β-actin gene promoter, and the SV40 gene intron, possessing strong initiation capability. The EF1α promoter refers to a strong mammalian expression promoter derived from human elongation factor 1α.

[0054] In some specific embodiments, the promoter preferably includes the nucleotide fragment shown in SEQ ID NO:7 and / or the nucleotide fragment shown in SEQ ID NO:10. In this case, the promoter can work together to connect the fragments, thereby achieving better simultaneous, independent, and efficient expression of multiple proteins.

[0055] Based on the application potential of the above-mentioned constructs in the co-expression of multiple proteins, the present invention further provides the application of the above-mentioned constructs in genetic engineering technology.

[0056] Based on the application potential of the above-mentioned construct in the co-expression of multiple proteins, the present invention further provides a method for the co-expression of multiple proteins. The method specifically includes: expressing the above-mentioned construct in a protein expression system to achieve the co-expression of multiple proteins.

[0057] In this invention, the protein expression system refers to a system capable of protein expression, specifically including the molecular machines, substrates, and energy required for gene replication, translation, and transcription. It is a commonly used technique in genetic engineering, and those skilled in the art can make adaptive selections according to actual needs. This invention does not specifically limit it. Specific examples include, but are not limited to, in vivo expression systems and / or in vitro expression systems. The in vivo expression system refers to a system that achieves protein expression based on an organism. The in vitro expression system specifically includes cell expression systems and / or cell-free expression systems. More specifically, the cell expression system refers to a system that achieves protein expression based on the intact structure and function of cells; depending on the cell type, specific examples of the cell expression system include, but are not limited to, one or more of the following: *E. coli* expression system, *Pichia pastoris* expression system, and CHO cell expression system. The cell-free expression system refers to a system constructed based on cell extracts that can express proteins under extracellular conditions. It can express proteins under in vitro conditions and has advantages such as high efficiency, high flexibility, ease of operation, and easy protein isolation. Specific examples of the cell-free expression system, depending on the source of the cell extract, include, but are not limited to, one or more of the following: Escherichia coli cell-free protein expression system, insect cell lysate protein expression system, malt extract protein expression system, and rabbit reticulocyte protein expression system.

[0058] In this invention, the method of "expressing the construct in a protein expression system" is a commonly used technique in genetic engineering. Those skilled in the art can make adaptive choices according to actual needs, limited to the ability to achieve the co-expression of multiple proteins. This invention does not impose any particular limitations on it.

[0059] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0060] Example 1

[0061] This embodiment illustrates the construction of construct I and its protein expression effect in HEK293 cells (MeisenCTCC, catalog number CTCC-003-0015, hereinafter the same), specifically including:

[0062] 1. Construction of Component I: Component I has the following characteristics: Figure 1The structures shown are illustrated in Table 1 (the nucleotide sequences of each fragment are shown in Table 1). Depending on the different ligation fragments introduced, they can be further divided into constructs I-1 to I-5, as shown in Table 2. Constructs I-1 to I-5 were constructed using the ClonExpress II One Step Cloning Kit (Novizan, catalog number C1112, the same below) and in accordance with the instructions. In other words, constructs I-1 to I-5 are DNA.

[0063] Table 2.

[0064]

[0065]

[0066] 2. Protein expression of construct I: Using liposome Lipofectamine 3000 (Thermo Fisher Scientific, catalog number L3000008), constructs I-1 to I-5 were introduced into HEK293 cells according to the manufacturer's instructions. Cells were then incubated at 37°C with 5% CO2 for 72 hours. Cell morphology was observed using a fluorescence microscope, and the fluorescence intensity of tdTomato and GFP proteins was measured. Specific test results are shown below. Figure 2 and 3 As shown.

[0067] The methods, reagents, and conditions used for introducing each construct into HEK293 cells were kept consistent, as were the methods, reagents, and conditions used for culturing each HEK293 cell and measuring fluorescence intensity.

[0068] Depend on Figure 2 The test results show that, compared with constructs I-4 and I-5, constructs I-1, I-2, and I-3 all have significantly improved GFP protein fluorescence intensity.

[0069] Depend on Figure 3 It can be seen that, compared with constructs I-4 and I-5, the fluorescence signal intensity ratio of GFP protein to tdTomato protein in constructs I-1, I-2, and I-3 is significantly improved. This indicates that the linker designed by hiding the start codon of the downstream GFP protein encoding gene in the stop codon of the upstream tdTomato protein encoding gene, that is, the shared sequence between the upstream stop codon and the downstream heuristic codon, can effectively drive the expression of the downstream GFP protein encoding gene.

[0070] Example 2

[0071] This embodiment illustrates the construction of construct II and its protein expression effect in HEK293 cells, specifically including:

[0072] 1. Construction of Construct II: Construct II uses the linker fragment-1 (SEQ ID NO:1) provided in Example 1 as the linker fragment for tandem connection between the luciferase LuxSit encoding gene, the luciferase Gluc encoding gene, the luciferase Rluc encoding gene, and the β-galactosidase encoding gene (the nucleotide sequences of each fragment are shown in Table 1). The structure of Construct II is as follows: Figure 4 As shown, construct II was constructed using the ClonExpress II One Step Cloning Kit and in accordance with the instructions. Construct II is DNA.

[0073] 2. Protein Expression of Construct II: Construct II was introduced into HEK293 cells using Lipofectamine 3000 according to the manufacturer's instructions. The cells were then incubated at 37°C with 5% CO2 for 72 hours. Cells were washed with PBS, and the cell pellet was collected. HEK293 cells were lysed using HEK-293 Whole Cell Lysate (Aladdin, catalog number cl155895, hereinafter the same) according to the manufacturer's instructions. The supernatant was collected by centrifugation and expressed using diphenyltetrazine substrate (Aladdin, catalog number D412592, luciferase LuxSit) and Pierce. TM Gaussia Luciferase Flash Detection Kit (Thermofisher, catalog number 16158, Luciferase Glucose), Renilla-Glo TM The activities of various enzyme molecules were detected using the Luciferase Assay Syste (Promega, catalog number E2810, luciferase Rluc) and the β-galactosidase assay kit (Thermofisher, catalog number K145501, β-galactosidase), with unintroduced primitive HEK293 cells as a blank control. Results are as follows: Figures 5-8 As shown.

[0074] Depend on Figures 5-8 The test results shown indicate that construct II can effectively achieve the co-expression of luciferase luxSit, luciferase Gluc, luciferase Rluc, and β-galactosidase in HEK293 cells.

[0075] Example 3

[0076] This embodiment illustrates the construction of construct III and the protein expression effect of its in vitro transcribed RNA in HEK293 cells, specifically including:

[0077] 1. Construction of Construct III: Construct III uses the linker fragment-1 (SEQ ID NO:1) provided in Example 1 as the linker fragment for tandem connection between the luciferase LuxSit encoding gene, the luciferase Gluc encoding gene, the luciferase Rluc encoding gene, and the β-galactosidase encoding gene (the nucleotide sequences of each fragment are shown in Table 1). The structure of Construct III is as follows: Figure 9 As shown in Table 1, the nucleotide sequences of each fragment were constructed using the ClonExpress II OneStep Cloning Kit and in accordance with the instructions. Construct III was a DNA fragment.

[0078] 2. In vitro transcription and RNA purification of construct III: (1) Using The T7 High-Efficiency RNA Synthesis Kit (NEB, catalog number E2040S) was used to perform in vitro transcription on construct III according to the instructions. The in vitro transcript was purified using 3M LiCl solution to obtain the transcript.

[0079] (2) After enriching the in vitro transcription products using an oligo(dT) column (BIA, catalog number 311 1219-2) and following the instructions, the oligo(dT) column was washed and eluted sequentially with 50 mM phosphate solution (pH=7) and DEPC water. The eluent was collected to obtain RNA.

[0080] 3. Protein Expression: RNA transcribed from construct III was introduced into HEK293 cells using Lipofectamine 3000 according to the manufacturer's instructions. The cells were then incubated at 37°C with 5% CO2 for 24 hours. Cells were washed with PBS, and the cell pellet was collected. HEK293 cells were lysed using HEK-293 Whole Cell Lysate according to the manufacturer's instructions. The expression was then performed using diphenyltetrazine and Pierce... TM Gaussia Luciferase Flash Detection Kit, Renilla-Glo TM The activities of various enzyme molecules were detected using the Luciferase Assay Système and the β-galactosidase assay kit, with unintroduced HEK293 cells serving as a blank control. The results are as follows: Figures 10-13 As shown.

[0081] Depend on Figures 10-13The test results shown indicate that, compared to the blank control, the RNA transcribed from construct III in vitro can effectively achieve co-expression of luciferase luxSit, luciferase Gluc, and luciferase Rluc in HEK293 cells. Although there was no statistically significant difference in β-galactosidase expression, it still showed an increasing trend, indicating that RNA tandem also has excellent co-expression effect.

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A construct for the co-expression of multiple proteins, characterized in that, The construct includes a target genome, which includes two or more coding genes, and adjacent coding genes are connected by a linker fragment; the linker fragment includes a stop codon and a start codon along the 5' to 3' direction, and the stop codon and start codon share a portion of the sequence.

2. The construct for co-expression of multiple proteins according to claim 1, characterized in that, The connection segment includes multiple independently configured stop codons, start codons, or multiple sets of stop codons and start codons sharing a common partial sequence.

3. The construct for co-expression of multiple proteins according to claim 1, characterized in that, The linker fragment includes one or more nucleotide fragments with sequences such as SEQ ID NO:1 to 4. Optionally, the nucleotide sequence of the linker fragment is as shown in SEQ ID NO:

1.

4. The construct for co-expression of multiple proteins according to claim 1, characterized in that, The construct is DNA and / or RNA.

5. The construct for co-expression of multiple proteins according to claim 1, characterized in that, The construct includes a promoter located upstream of the target genome.

6. The construct for co-expression of multiple proteins according to claim 5, characterized in that, The promoters include one or more of the following: CMV promoter, CAG promoter, T7 promoter, Lac promoter, Trp promoter, Tac promoter, SV40 promoter, CBA promoter, and EF1α promoter.

7. The construct for co-expression of multiple proteins according to claim 5, characterized in that, The promoter comprises a nucleotide fragment with a sequence as shown in SEQ ID NO:7 and / or a nucleotide fragment with a sequence as shown in SEQ ID NO:

10.

8. The application of the construct for co-expression of multiple proteins as described in any one of claims 1 to 7 in genetic engineering technology.

9. A method for co-expressing multiple proteins, characterized in that, The method includes: expressing the construct according to any one of claims 1 to 7 in a protein expression system to achieve the co-expression of multiple proteins.

10. The method for co-expressing multiple proteins according to claim 9, characterized in that, The protein expression system is an in vivo expression system and / or an in vitro expression system; Optionally, the in vitro expression system includes a cell expression system and / or a cell-free expression system.

Images