Fusion protein, recombinant vector, host cell and application thereof
By designing complementary fluorescent molecules in the proinsulin precursor, the problems of low conversion efficiency and spectral overlap of fluorescent timer proteins in existing technologies have been solved, achieving efficient and specific monitoring of insulin maturation.
Patent Information
- Application Number
- CN202411094054.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-09
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies for monitoring insulin maturation use fluorescent timer proteins have low fluorescence signal conversion efficiency and high background, and superfolded GFP and mCherry fluorescent proteins have problems with spectral overlap and cross-interference.
Design a fusion protein in which a complementary fluorescent molecule is composed of a first fragment and a second fragment, located between the B and C peptide chains and at the C-terminus of the proinsulin precursor, respectively. The fragments fold together via disulfide bonds to form a complete fluorescent molecule that emits a fluorescent signal. The fusion protein is expressed in a recombinant expression vector and in host cells to monitor insulin maturation.
It enables efficient and specific monitoring of the insulin maturation process, reduces background noise, and improves the spatial and temporal resolution of fluorescence signals, providing a direct indication of the amount of mature insulin in cells.
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Figure CN121494992A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a fusion protein, a recombinant vector, a host cell, and their applications. Background Technology
[0002] Insulin is the only hormone in the human body that can lower blood sugar and is irreplaceable for maintaining blood sugar levels. Insulin is synthesized in the form of proinsulin precursors, which include a signal peptide, a B-peptide chain, a C-peptide chain, and an A-peptide chain. The signal peptide on the proinsulin precursor is cleaved off shortly after synthesis, forming proinsulin. Proinsulin then enters the Golgi apparatus and forms secretory vesicles through budding. Within the vesicles, proinsulin is cleaved into mature insulin and a C-peptide chain by the proteases PC1, PC2, and CPE.
[0003] To monitor insulin synthesis, several solutions have been proposed: 1. Inserting a gene encoding a fluorescent timer protein (FT) into the sequence encoding the C-peptide chain of proinsulin. When the fusion protein of the proinsulin precursor is cleaved from the C-peptide chain, a change in fluorescence signal can be observed, indicating insulin maturation. 2. Inserting a gene encoding hyperfolded GFP (sfGFP) into the C-terminus of the proinsulin A-peptide chain, and simultaneously inserting a gene encoding mCherry into the middle of the C-peptide chain. Monitoring changes in these two fluorescence signals reveals the state of proinsulin. The first approach uses a fluorescent timer protein whose fluorescence changes over time, resulting in a long processing time and low conversion efficiency, leading to high background noise. It only reflects the duration of protein translation and cannot definitively indicate whether proinsulin has been cleaved and matured. The second approach suffers from spectral overlap, as the fluorescent protein, along with the cleaved C-peptide chain, disperses throughout the cell, causing cross-interference between different fluorescence signals. Summary of the Invention
[0004] The present invention aims to disclose a fusion protein, a recombinant vector, a host cell and their applications, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.
[0005] The first aspect of the present invention is to provide a fusion protein based on proinsulin precursor.
[0006] A second aspect of the present invention is to provide a recombinant expression vector incorporating the fusion protein described in the first aspect of the present invention.
[0007] A third aspect of the present invention is to provide a host cell capable of expressing the fusion protein described in the first aspect of the present invention.
[0008] A fourth aspect of the present invention is to provide a method for constructing the host cell described in the third aspect of the present invention.
[0009] The fifth aspect of the present invention is to provide the application directions of the fusion protein described in the first aspect of the present invention, the recombinant expression vector described in the second aspect of the present invention, or the host cell described in the third aspect of the present invention.
[0010] A sixth aspect of the present invention is to provide a method for monitoring intracellular insulin maturation.
[0011] The fusion protein described in the first aspect of this invention is a proinsulin precursor with a complementary fluorescent molecule inserted into it. The complementary fluorescent molecule consists of a first fragment and a second fragment. The first fragment is located between the B and C peptide chains of the proinsulin precursor, and the second fragment is attached to the C-terminus of the A peptide chain of the proinsulin precursor. A complementary fluorescent molecule refers to a fluorescent molecule divided into two non-fluorescent fragments, namely the first fragment and the second fragment. When the first and second fragments are spatially close enough, they can recombine to form a complete fluorescent molecule, i.e., it has fluorescent activity and can emit fluorescence. After undergoing signal peptide cleavage, the A and B peptide chains of the fusion protein fold under the action of disulfide bonds, and then the C peptide chain is cleaved by proteases PC1, PC2, and CPE to become mature insulin. At this time, the C-terminus of the B peptide chain and the C-terminus of the A peptide chain are close to each other and no longer obstructed by the C peptide chain, making the first and second fragments spatially close enough to assemble into a complete fluorescent protein that emits a fluorescent signal.
[0012] In some implementations of the first aspect of the present invention, the complementary fluorescent molecule is selected from Split-GFP, Split-YFP, Split-RFP, Split-mCherry, or Split-Luciferase. Further, the Split-GFP is composed of GFP1-10 and GFP11, the amino acid sequence of GFP1-10 is shown in SEQ ID No:7, and the amino acid sequence of GFP11 is shown in SEQ ID No:8; preferably, the Split-YFP is composed of YFP-N and YFP-C, the amino acid sequence of YFP-N is shown in SEQ ID No:9, and the amino acid sequence of YFP-C is shown in SEQ ID No:10; preferably, the Split-RFP is composed of RFP1-10 and RFP11, the amino acid sequence of RFP1-10 is shown in SEQ ID No:11, and the amino acid sequence of RFP11 is shown in SEQ ID No:12; preferably, the Split-mCherry is composed of mCherry1-10 and mCherry11, the amino acid sequence of mCherry1-10 is shown in SEQ ID No:13, and the amino acid sequence of mCherry11 is shown in SEQ ID No:8. As shown in No:14; preferably, the Split-Luciferase is composed of Luciferase-N and Luciferase-C, the amino acid sequence of Luciferase-N is shown in SEQ ID No:15, and the amino acid sequence of Luciferase-C is shown in SEQ ID No:16.
[0013] In some embodiments of the first aspect of the present invention, the fusion protein is composed, from the N-terminus to the C-terminus, a signal peptide, a B-peptide chain, a first fragment, a C-peptide chain, an A-peptide chain, a protein tag, and a second fragment. Adding the protein tag provides a certain spatial length to compensate for the length difference between the first and second fragments in the complementary fluorescent molecule, thus avoiding interference with the folding of the insulin protein; furthermore, it serves as a detection marker, facilitating the detection and tracing of the fusion protein during the construction process.
[0014] In a further application embodiment, the protein tag is selected from His tag, Flag tag, HA tag, or Myc tag. The amino acid sequence of the His tag is shown in SEQ ID No:17, the amino acid sequence of the Flag tag is shown in SEQ ID No:18, the amino acid sequence of the HA tag is shown in SEQ ID No:19, and the amino acid sequence of the Myc tag is shown in SEQ ID No:20.
[0015] In some embodiments of the first aspect of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID No:1 or SEQ ID No:3. The amino acid sequence shown in SEQ ID No:1 is a fusion protein with inserted GFP11 and GFP1-10, which, after sequentially cleaving the signal peptide and C peptide chain, splices together to form a complete GFP fluorescent group, causing mature insulin to emit a green fluorescent signal. The amino acid sequence shown in SEQ ID No:3 is a fusion protein with inserted mCherry11 and mCherry1-10, which, after sequentially cleaving the signal peptide and C peptide chain, splices together to form a complete mCherry fluorescent group, causing mature insulin to emit a red fluorescent signal.
[0016] The recombinant expression vector of the second aspect of the present invention contains a nucleotide sequence encoding the aforementioned fusion protein. By transferring the nucleotide sequence into the recombinant expression vector, host cells are able to express the fusion protein of the first aspect of the present invention.
[0017] In some embodiments of the second aspect of the invention, the nucleotide sequence encoding the fusion protein is shown in SEQ ID No:2 or SEQ ID No:4. The nucleotide sequence shown in SEQ ID No:2 is used to encode the Split-GFP fusion protein as shown in SEQ ID No:1; the nucleotide sequence shown in SEQ ID No:4 is used to encode the Split-mCherry fusion protein as shown in SEQ ID No:3.
[0018] In the third aspect of this invention, the recombinant expression vector described in the second aspect of this invention is transferred into the host cell, enabling it to express the fusion protein described in the first aspect of this invention. By continuously monitoring fluorescence signals and other methods, the changes in the amount of mature insulin in the host cell over a period of time can be determined.
[0019] The construction method according to the fourth aspect of the present invention includes the following steps: 1) The nucleotide fragments of the fusion protein were ligated into a lentiviral vector using double enzyme digestion to obtain a plasmid; 2) The plasmid and packaging plasmid were co-transfected into packaging cells to obtain viral particles; 3) Infect the target cells with the virus particles to obtain the host cells.
[0020] In some implementations of the fourth aspect of the present invention, the lentivirus vector is pCDH-CMV-MCS-EF1-Puro.
[0021] In some implementations of the fourth aspect of the present invention, the enzyme digestion sites of the double digestion are EcoRI and BAMHRI, respectively.
[0022] In some implementations of the fourth aspect of the present invention, the packaging plasmids are pLP / VSVG and pSPAX2.
[0023] In some embodiments of the fourth aspect of the present invention, the packaging cells are 293T cells.
[0024] In some embodiments of the fourth aspect of the present invention, the target cell is an INS1-E cell.
[0025] In some embodiments of the fourth aspect of the invention, the infected target cells are screened as positive host cells by puromycin.
[0026] The fifth aspect of this invention provides applications including using, for example, the host cells as a drug screening model. Further, the drug screening model is used to screen drugs that promote insulin maturation. Even further, the screening method involves monitoring fluorescence signals in host cells treated with the drug to obtain information such as the proinsulin cleavage efficiency within the host cells, thereby determining whether the drug has the effect of promoting insulin maturation.
[0027] The monitoring method provided in the sixth aspect of this invention includes the following steps: A) Obtain the host cells as described in the third aspect of the present invention, or transfer the recombinant expression vector as described in the second aspect of the present invention into living cells; B) Acquire real-time cell images and obtain fluorescence signals indicating insulin maturation by scanning the wavelengths adapted to the complementary fluorescent molecules with a laser.
[0028] Recombinant cells that express the fusion protein can be used to directly monitor insulin maturation in cells using the methods described above, which helps to further study the mechanism of insulin maturation.
[0029] The beneficial effects of this invention are as follows: The fusion protein can fold into fluorescent insulin after being cleaved by the signal peptide and C peptide chain, which can be used to indicate the content of mature insulin in cells.
[0030] The recombinant expression vector or the host cell can be used to construct a cell model. This cell model is used to screen compounds that can promote proinsulin maturation and increase insulin levels. High-throughput compound library screening methods are used to identify lead compounds to assess their potential therapeutic effects or side effects, serving as candidate molecules for subsequent drug development and commercialization. Attached Figure Description
[0031] Figure 1 This is a schematic diagram illustrating the technical principle of the fusion protein; Figure 2This is an immunofluorescence detection image of host cells expressing the Split-GFP fusion protein as described in Example 1, with a scale bar of 5 µm; Figure 3 This is an immunofluorescence image of host cells expressing the Split-mCherry fusion protein as described in Example 2, with a scale bar of 5 µm; Figure 4 This is an immunofluorescence assay of the Myc tag fusion protein in Comparative Example 1, with a scale bar of 5 µm. Figure 5 This is an immunofluorescence detection image of the fusion protein without additional protein tags in Comparative Example 1, with a scale bar of 5 µm. Figure 6 This is an immunofluorescence assay of the fusion protein in Comparative Example 1 where the Flag tag was replaced with the Myc tag. The scale bar is 5 µm. Detailed Implementation
[0032] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.
[0033] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0034] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the kit and product instructions. Unless otherwise specified, the biological materials used in the kits are commercially available.
[0035] The technical principles of this application are as follows: Figure 1As shown in the diagram, taking the Split-GFP fusion protein as an example, the Split-GFP fusion protein is composed of a signal peptide, a B peptide chain, GFP11, a C peptide chain, an A peptide chain, a myc tag, and GFP1-10 linked sequentially, with the amino acid sequence shown in SEQ ID No:1. The Split-GFP fusion protein is synthesized in the rough endoplasmic reticulum in the form of a proinsulin precursor within the cell. Subsequently, the N-terminal signal peptide is cleaved to form proinsulin, which then enters the endoplasmic reticulum. Proinsulin is then transported to the Golgi apparatus for further processing and encapsulation, forming vesicles from the trans-Golgi apparatus in the form of budding. These vesicles are then transported to the vicinity of the cell membrane, where they fuse with the cell membrane to release their contents extracellularly. During transport, proton pumps on the vesicles transport protons from the cell into the vesicles, causing the pH value inside the vesicles to drop from the initial 6.5 to 5.2. This activates the activity of proteolytic enzymes PC1 and PC2, as well as carboxypeptidase E (CPE), which cleaves the C-peptide chain in the middle of proinsulin. The B-peptide chain and the A-peptide chain are linked by disulfide bonds to form a fold, which causes GFP11 at the C-terminus of the B-peptide chain to come closer to GFP1-10 at the C-terminus of the A-peptide chain, combining to form a complete GFP fluorescent group, thereby indicating that the Split-GFP fusion protein develops into mature insulin.
[0036] Example 1: Constructing host cells stably expressing the Split-GFP fusion protein
[0037] 2) The pCDH-CMV-MCS-EF1-Puro plasmid was double-digested with NheⅠ and BamHI to obtain a linearized plasmid.
[0038] 3) The amplification product from step 1) is ligated with the linearized plasmid from step 2) using a ligase to obtain the recombinant plasmid.
[0039] 4) The recombinant plasmid and packaging plasmids pLP / VSVG and pSPAX2 were co-transfected into Hek293T cells in a ratio of 4:1:3 to package lentivirus.
[0040] 5) 48 hours after transfection, the culture supernatant of Hek293T cells was collected, filtered, and then used to infect INS1-E cells.
[0041] 6) After 12 hours of infection, replace the medium with one containing puromycin and continue culturing to finally screen out host cells that stably express the Split-GFP fusion protein.
[0042] Host cells were fixed and immunofluorescence assays were used to confirm whether the Split-GFP fusion protein could specifically label mature insulin. A TRITC-labeled polyclonal antibody against insulin was used to label proinsulin and insulin; an Alexa647-labeled monoclonal antibody against proinsulin was used to specifically detect proinsulin; and DAPI dye was used to label cell nuclei. Fluorescence images were acquired using a Zeiss 980 fluorescence microscope. Figure 2 As shown, the purple proinsulin is mainly concentrated around the blue nucleus (i.e., the anti-Golgi apparatus), while recombinant insulin, which matures from the Split-GFP fusion protein and exhibits green fluorescence, mainly appears near the cell membrane, with the fluorescence being stronger closer to the cell membrane. Furthermore, the green fluorescence signal in the host cell co-localizes very well with the insulin antibody signal, especially near the cell membrane. These results indicate that the Split-GFP fusion protein can effectively indicate mature insulin.
[0043] Example 2: Constructing host cells stably expressing the Split-mCherry fusion protein
[0044] Host cells were fixed and immunofluorescence assays were used to confirm whether the Split-mCherry fusion protein could specifically label mature insulin. An Alexa488-labeled insulin polyclonal antibody was used to label proinsulin and insulin; an Alexa647-labeled proinsulin monoclonal antibody was used to specifically detect proinsulin; and DAPI dye was used to label cell nuclei. Fluorescence images were acquired using a Zeiss 980 fluorescence microscope. Figure 3 As shown, the red mCherry signal mainly appears near the cell membrane, largely overlapping with the green insulin polyclonal antibody signal. Furthermore, the closer to the cell membrane, the stronger the fluorescence. This demonstrates that the designed Split-mCherry fusion protein can also indicate the presence of mature insulin.
[0045] Comparative Example 1: The effect of protein tags on fusion proteins Because the A peptide chain of the original insulin contains 21 amino acid residues and the B peptide chain contains 30 amino acid residues, there is steric hindrance at the C-terminus of both peptide chains after folding. If complementary fluorescent molecules are added directly between the B and C peptide chains and at the C-terminus of the A peptide chain, the fluorescence signal may not be emitted due to the steric hindrance.
[0046]
[0047]
[0048] Immunofluorescence was used to detect fluorescence images acquired using a Zeiss 980 fluorescence microscope, as shown below. Figures 4 to 6 As shown. Figure 4 The host cells obtained in Example 1 were labeled with a TRITC-labeled Myc antibody, and the cell nuclei were labeled with Alexa 647 and DAPI dye. Figure 4 As shown, in host cells that stably express the Split-GFP fusion protein, the positions of the red and green signals highly overlap. Figure 5 TRITC-labeled polyclonal antibodies were used to label proinsulin and insulin, and DAPI dye was used to label the cell nucleus. For example... Figure 5 As shown, compared to host cell 1, red signals distributed around the cell nucleus and near the cell membrane, as well as a very weak green signal, can be detected. Figure 6 TRITC-labeled polyclonal antibodies were used to label proinsulin and insulin, and DAPI dye was used to label the cell nucleus. For example... Figure 6 As shown, the fusion protein expressed in host cell 2 is mainly localized in the trans-Golgi apparatus and vesicles, while the GFP signal in the reporter cell is mainly concentrated near the cell membrane, showing good co-localization with the signal labeled by the Flag antibody. This indicates that protein tag insertion is necessary, providing flexibility to allow the Split-GFP fragment to bind fully, thus exhibiting a sufficiently strong detection signal; replacing the Myc tag with a Flag tag of similar length can achieve the same purpose.
[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
Claims
1. A proinsulin fusion protein, characterized in that, The fusion protein is a precursor insulin with a complementary fluorescent molecule inserted into it. The complementary fluorescent molecule consists of a first fragment and a second fragment. The first fragment is located between the B peptide chain and the C peptide chain of the precursor insulin, and the second fragment is attached to the C-terminus of the A peptide chain of the precursor insulin.
2. The fusion protein according to claim 1, characterized in that, The complementary fluorescent molecule is selected from Split-GFP, Split-YFP, Split-RFP, Split-mCherry, or Split-Luciferase.
3. The fusion protein according to claim 2, characterized in that, The Split-GFP is composed of GFP1-10 and GFP11, the amino acid sequences of GFP1-10 are shown in SEQ ID No:7, and the amino acid sequence of GFP11 is shown in SEQ ID No:8; preferably, the Split-YFP is composed of YFP-N and YFP-C, the amino acid sequence of YFP-N is shown in SEQ ID No:9, and the amino acid sequence of YFP-C is shown in SEQ ID No:10; preferably, the Split-RFP is composed of RFP1-10 and RFP11, the amino acid sequences of RFP1-10 are shown in SEQ ID No:11, and the amino acid sequence of RFP11 is shown in SEQ ID No:12; preferably, the Split-mCherry is composed of mCherry1-10 and mCherry11, the amino acid sequences of mCherry1-10 are shown in SEQ ID No:13, and the amino acid sequence of mCherry11 is shown in SEQ ID No:
8. As shown in No:14; preferably, the Split-Luciferase is composed of Luciferase-N and Luciferase-C, the amino acid sequence of Luciferase-N is shown in SEQ ID No:15, and the amino acid sequence of Luciferase-C is shown in SEQ ID No:
16.
4. The fusion protein according to claim 1, characterized in that, The fusion protein consists of a signal peptide, a B peptide chain, a first fragment, a C peptide chain, an A peptide chain, a protein tag, and a second fragment, sequentially from the N-terminus to the C-terminus.
5. The fusion protein according to claim 4, characterized in that, The protein tag is selected from His tag, Flag tag, HA tag or Myc tag.
6. The fusion protein according to claim 5, characterized in that, The amino acid sequence of the His tag is shown in SEQ ID No:17; preferably, the amino acid sequence of the Flag tag is shown in SEQ ID No:18; preferably, the amino acid sequence of the HA tag is shown in SEQ ID No:19; preferably, the amino acid sequence of the Myc tag is shown in SEQ ID No:
20.
7. A recombinant expression vector, characterized in that, It comprises a nucleotide sequence encoding the fusion protein of any one of claims 1 to 6.
8. The recombinant expression vector according to claim 7, characterized in that, The nucleotide sequence is shown in SEQ ID No:2 or SEQ ID No:
4.
9. A host cell, characterized in that, Contains the recombinant expression vector as described in claim 7 or 8.
10. The method for constructing the host cell according to claim 9, characterized in that, Including the following steps: 1) The nucleotide fragments of the fusion protein were ligated into a lentiviral vector using double enzyme digestion to obtain a plasmid; 2) The plasmid and packaging plasmid were co-transfected into packaging cells to obtain viral particles; 3) Infect the target cells with the virus particles to obtain the host cells.
11. The application of the fusion protein according to any one of claims 1 to 6, the recombinant expression vector according to any one of claims 7 to 8, or the host cell according to claim 9 in a method for real-time detection of proinsulin cleavage efficiency.
12. The use of the fusion protein of any one of claims 1 to 6, the recombinant expression vector of any one of claims 7 to 8, or the host cell of claim 9 in constructing a drug screening model.
13. The application according to claim 12, characterized in that, The drug in question is one that promotes insulin maturation.
14. A method for monitoring intracellular insulin maturation, characterized in that, Including the following steps: A) Obtain the host cell as described in claim 9, or transfer the recombinant expression vector as described in any one of claims 7 to 8 into living cells; B) Acquire real-time cell images and obtain fluorescence signals indicating insulin maturation by scanning the wavelengths adapted to the complementary fluorescent molecules with a laser.