Method for analyzing influence of variation on protein stability and application thereof
By modifying the lentiviral expression vector system, using resistant drug genes to replace fluorescent labels, and combining tetracycline-induced initiation, the problems of complex operation, high cost and limited applicability of high-throughput screening in the prior art are solved, and an efficient, economical and flexible mutant screening method is achieved.
Patent Information
- Application Number
- CN202411753738.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art has problems of complex operation, high cost and limited applicability when screening protein mutants at high throughput, especially in terms of fluorescent label dependence and background noise control.
The lentiviral expression vector system was modified, the fluorescent fusion gene was replaced with resistance drug genes, and the lentiviral vectors induced by tetracycline were combined to achieve efficient transduction and high-throughput screening.
Improves flexibility, efficiency and applicability of mutant screening, simplifies operational processes, reduces research costs, and applies to a wider range of cellular backgrounds and experimental conditions.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for high-throughput analysis of the effect of variation on protein stability and an application thereof. Background Art
[0002] Deep mutational scanning (DMS) is a powerful high-throughput technology used to systematically study the effects of mutations in proteins or other functional biomolecules on their function or stability. It can simultaneously analyze tens of thousands of mutants by combining the construction of mutant libraries, high-throughput screening, and deep sequencing, helping to understand how mutations affect the function, structure, and stability of proteins (Fowler, Douglas M, and Stanley Fields. "Deep mutational scanning: a new style of protein science." Nature methods vol. 11, 8 (2014): 801-7.).
[0003] VAMP-seq is a high-throughput method developed by Douglas M. Fowler's laboratory to assess the impact of protein variation on intracellular protein abundance. The technology was first published in Nature Genetics in 2018 (Matreyek, Kenneth A et al. "Multiplex assessment of protein variant abundance by massively parallel sequencing." Nature genetics vol. 50, 6 (2018): 874-882). At present, VAMP-seq has been applied to the study of variant abundance of many important disease genes such as PTEN, CYP2C9, CYP2C19, Parkin, etc. (Zhang, Lingxin et al. "CYP2C9 and CYP2C19: Deep Mutational Scanning and Functional Characterization of Genomic Missense Variants." Clinical and translational science vol.13,4(2020):727-742; Clausen, Lene et al. "A mutational atlas for Parkin proteostasis." Nature communications vol.15,11541.20Feb.2024.). VAMP-seq can be regarded as a specific application or variant of DMS, which focuses more on studying the effects of protein mutations on protein abundance and stability rather than direct functional changes, and has stronger universality. DMS is a broader technical platform that covers a wide range of studies from mutation to function to phenotype. It can not only be used to analyze protein stability, but also to study enzyme activity, ligand binding, gene regulation, etc. It has more advantages in studying the impact of gene variation on phenotype.
[0004] The core steps of VAMP-seq are similar to those of DMS, and its highlight is the use of a fluorescent reporter gene system: designing EGFP to fuse with the target gene as an observable signal to monitor changes in the abundance of variant proteins, and expressing mCherry through co-transcriptional independently translated internal ribosome entry site (IRES) elements as an internal reference control to correct expression differences in different cells and ensure the stability and repeatability of the experiment. The effect of mutations on protein stability is measured by the ratio of EGFP and mCherry fluorescence signals. The higher the abundance, the more stable the protein; the lower the abundance, the more likely the protein is to be degraded. VAMP-seq can very intuitively evaluate the effect of each mutant on protein stability. VAMP-seq screening is based on a simple fluorescence detection system, which can be expanded to large-scale high-throughput screening in combination with flow cytometry. Compared with the screening strategy of DMS, this method does not rely on specific phenotypic design, complex functional screening and conditional control, etc. This is a new high-throughput research strategy for genes that lack high-throughput functional screening methods. VAMP-seq can process a large number of mutants at one time, and is particularly suitable for screening thousands to tens of thousands of protein mutants. The operation is relatively simple and easy to automate. However, fluorescent proteins also have certain limitations, including that the signal may change over time, interference from nonspecific background fluorescence, and dependence on complex detection equipment. Therefore, it is necessary to find a method that is easy to operate, can achieve background noise control, and reduce research costs, which is more suitable for large-scale cell population screening.
[0005] In DMS and VAMP-seq technology, integrating the mutant library into the cell genome and expressing it is a prerequisite for subsequent research. In the process of expressing the mutant library, choosing a suitable transfection method is the key to ensure that the mutant library is correctly expressed in the cell or system. Commonly used methods for expressing mutant libraries in cells include plasmid vector transient expression systems, Bxb1 recombinase systems, viral vector systems, CRISPR / Cas9 systems, etc. The Bxb1 recombinase system is a genome editing tool based on the serine recombinase Bxb1, which is widely used in gene insertion, regulation and recombination research. The Bxb1 recombinase is derived from bacteriophage Bxb1 and has the ability to mediate efficient, directional and irreversible gene recombination (Duportet, Xavier et al. "A platform for rapid prototyping of synthetic gene networks inmammalian cells." Nucleic acids research vol.42, 21 (2014): 13440-51.). The lentiviral vector system is a gene transduction tool designed based on a member of the retrovirus family, which can randomly integrate exogenous genes into the genome of host cells and provide stable gene expression. The system is widely used in gene function research, gene therapy, stem cell research and other fields (Park, Frank. "Lentiviral vectors: are they the future of animal transgenesis? "Physiological genomics vol.31,2 (2007): 159-73.). In terms of the comparison of the above two systems, the Bxb1 system is usually more complicated to operate for genome integration, and it is necessary to first establish a "landing pad" cell line in the experimental cell line, which involves monoclonal screening and verification, greatly increasing the workload in the early stage, especially for different cell lines, it takes a long time to establish the Bxb1 platform. Secondly, the integration efficiency of Bxb1 is usually low, and a large number of screenings are often required to obtain enough cell populations for high-throughput screening experiments. In addition, due to its dependence on specific integration sites, the Bxb1 system is not suitable for some difficult-to-edit cell lines or primary cells, which limits the versatility of VAMP-seq. Lentiviral vectors have efficient genome integration capabilities and are particularly suitable for various cell lines, including primary cells and stem cells that are difficult to transfect. They can also ensure persistent expression of transgenes, making them suitable for gene expression experiments that require long-term tracking in VAMP-seq, and can meet the needs of conducting VAMP-seq experiments in multiple cell lines simultaneously and quickly.Lentiviral vectors are relatively simple and routine to use. Such tools are always available in laboratories and can quickly insert genes without the need for complex preliminary preparations, which speeds up the startup process of VAMP-seq experiments. Summary of the invention
[0006] In the method for analyzing the effect of gene variation on protein function, the present invention transforms the traditional recombinase vector system into a lentiviral expression vector system, realizes efficient transduction of mutants and functional genes, and further replaces the fluorescent signal of mutant protein fusion with drug resistance protein as a detection signal of mutant protein abundance, evaluates the effect of mutant on its protein stability, and provides an economical, efficient and reliable method for high-throughput mutant screening. Based on this, the present invention is completed.
[0007] In a first aspect, the present invention provides a lentiviral expression vector with a gene mutant screening function, wherein the lentiviral expression vector is represented as ABGIM or AGBIM, wherein A is a lentiviral vector, B is a saturation mutation library, G is a functional gene, I is a co-transcribed and independently translated protein expression regulatory element, and M is an internal reference gene different from the functional gene G, wherein the functional gene G includes a fluorescent gene or a drug resistance gene, and the lentiviral expression vector is a lentiviral expression vector with high integration efficiency and wide applicability, which is formed by cloning an expression element (GIM or G-…-IM) containing a functional gene into a lentiviral vector A.
[0008] Furthermore, the fluorescent gene includes but is not limited to a green fluorescent gene (GFP), a yellow fluorescent gene (YFP) or a red fluorescent gene (RFP) and the like.
[0009] Preferably, the fluorescent gene is the enhanced green fluorescent gene (EGFP).
[0010] Furthermore, the drug resistance gene includes but is not limited to a neomycin resistance gene or a puromycin resistance gene.
[0011] Furthermore, the protein expression regulatory element I is IRES.
[0012] Furthermore, the internal reference gene M includes but is not limited to a red fluorescent gene (mCherry), a blue fluorescent gene (BFP) or a yellow fluorescent gene (YFP) and the like.
[0013] Preferably, the internal reference gene M is mCherry.
[0014] Furthermore, when the functional gene in the expression element is fused to the 3' end of the variant gene, the expression element containing the functional gene is GIM, and the lentiviral expression vector is expressed as ABGIM.
[0015] Furthermore, when the functional gene in the expression element is fused to the 5' end of the variant gene, the expression element containing the functional gene is G-...-IM, and the lentiviral expression vector is represented as AGBIM.
[0016] Further, in one embodiment of the present invention, when the functional gene G is the enhanced green fluorescent gene EGFP, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-EGFP-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0017] Further, in one embodiment of the present invention, when the functional gene G is the drug resistance gene BlastR, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-BlastR-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0018] In a second aspect, the present invention provides a method for high-throughput analysis of the effect of gene variation on protein stability, the method comprising the following steps:
[0019] S1. Design and synthesize a saturation mutation library of the target gene;
[0020] S2. Cloning the expression element (GIM or G-…-IM) containing the functional gene into a lentiviral vector to prepare a lentiviral expression vector with screening function, and cloning the mutant library synthesized in S1 into the lentiviral expression vector containing the functional gene, so that the mutant library is fused with the functional gene in the expression element, and obtaining the high-throughput universal screening lentiviral expression vector ABGIM or AGBIM containing the mutant library as described in the first aspect;
[0021] S3. Use the high-throughput universal screening lentiviral expression vector constructed in S2 to perform cell transfection;
[0022] S4. Enrich the positive cells transfected in S3 using resistance genes;
[0023] S5. Induce the expression of the expression frame (BGIM or GBIM) containing the functional gene in the positive cells enriched in S4, and perform high-throughput screening and deep targeted sequencing on the cell mutation library in which the internal reference gene M is successfully expressed;
[0024] S6. Measure the frequency of variants in S5 results by high-throughput sequencing and analyze the impact of mutations on protein stability.
[0025] Furthermore, in step S1, the saturation mutation library covers DNA sequences of all possible single amino acid variations of the target gene.
[0026] Further, in step S2, G in the expression element (GIM or G-...-IM) refers to a functional gene.
[0027] Furthermore, the functional gene includes a fluorescence gene or a drug resistance gene.
[0028] Furthermore, the fluorescent gene includes but is not limited to a green fluorescent gene (GFP), a yellow fluorescent gene (YFP) or a red fluorescent gene (RFP) and the like.
[0029] Preferably, the fluorescent gene is the enhanced green fluorescent gene (EGFP).
[0030] Furthermore, the drug resistance gene includes but is not limited to a neomycin resistance gene or a puromycin resistance gene.
[0031] Furthermore, in step S2, the lentiviral vector is a tetracycline-induced lentiviral vector.
[0032] Furthermore, in step S2, the GIM is to fuse the functional gene in the expression element to the 3' end of the variant gene, and the G-...-IM is to fuse the functional gene in the expression element to the 5' end of the variant gene.
[0033] Further, in one embodiment of the present invention, when the functional gene G in S2 is the enhanced green fluorescent gene EGFP, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-EGFP-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0034] Further, in one embodiment of the present invention, when the functional gene G in S2 is the drug resistance gene BlastR, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-BlastR-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0035] Furthermore, in step S3, during the cell transfection, a solution containing the lentivirus is added to the cell line to be studied. After the lentivirus enters the cell line to be studied, its RNA genome is reverse transcribed into DNA, and the exogenous gene is integrated into the genome of the host cell.
[0036] Further, in step S4, the resistance gene is puromycin.
[0037] Further, in step S5, the induction of positive cells refers to using DOX to induce variant expression.
[0038] Further, in step S5, the high-throughput screening is based on the protein abundance of the expression element containing the functional gene G, and the cell population carrying the gene mutation is high-throughput classified and screened by flow cytometry or resistance drugs.
[0039] Furthermore, when the functional gene G in the lentiviral expression vector of S2 is a fluorescent gene, the high-throughput screening is to perform high-throughput classification and screening of cell populations carrying gene mutations by flow cytometry.
[0040] Furthermore, when the functional gene G in the lentiviral expression vector of S2 is a drug-resistant gene, the high-throughput screening is a high-throughput classification and screening of cell populations carrying gene mutations by drug-resistant drugs.
[0041] Further, in step S5, the deep targeted sequencing refers to performing deep sequencing on the mutation library before and after screening, respectively.
[0042] Furthermore, in step S6, the analysis method for the effect on protein stability is to reasonably calculate the frequency change of the corresponding amino acids of the mutant gene before and after screening to obtain the mutant abundance score.
[0043] In a third aspect, the present invention provides a system for high-throughput analysis of the effects of variation on protein stability, the system comprising a saturated mutant building block, a lentiviral vector expression system building block, a cell transfection module, a high-throughput screening module, a deep sequencing module, and a mutant abundance score analysis module;
[0044] The saturation mutant building block is to design and synthesize a saturation mutation library of the target gene;
[0045] The construction module of the lentiviral vector expression system is to clone the expression element (GIM or G-…-IM) containing the functional gene into the lentiviral vector to prepare a lentiviral expression vector with screening function, and clone the synthesized mutation library into the lentiviral expression vector containing the functional gene, so that the mutation library is fused with the functional gene in the expression element to obtain the high-throughput universal screening lentiviral expression vector ABGIM or AGBIM containing the mutation library as described in the first aspect;
[0046] The cell transfection module uses a high-throughput universal screening lentiviral expression vector for cell transfection;
[0047] The high-throughput screening module is based on the protein abundance of the expression element containing the functional gene G, and performs high-throughput classification and screening of cell populations carrying gene mutations by flow cytometry or resistance drugs;
[0048] The deep targeted sequencing module performs high-throughput sequencing on the mutant library in cells that have been successfully transfected and have successfully expressed the internal reference gene;
[0049] The mutant abundance score analysis module analyzes the effect of mutations on protein stability based on the frequency of variants in high-throughput sequencing results.
[0050] Furthermore, in the saturation mutant construction module, the saturation mutation library covers the DNA sequences of all possible single amino acid variations of the target gene.
[0051] Furthermore, in the construction module of the lentiviral vector expression system, in the expression element (GIM or G-…-IM), G refers to a functional gene, I refers to a co-transcribed and independently translated protein expression regulatory element, and M refers to an internal reference gene.
[0052] Furthermore, the GIM is to fuse the functional gene in the expression element to the 3' end of the variant gene, and the G-...-IM is to fuse the functional gene in the expression element to the 5' end of the variant gene.
[0053] Furthermore, the functional gene includes a fluorescence gene or a drug resistance gene.
[0054] Furthermore, the fluorescent gene includes but is not limited to a green fluorescent gene (GFP), a yellow fluorescent gene (YFP) or a red fluorescent gene (RFP) and the like.
[0055] Preferably, the fluorescent gene is the enhanced green fluorescent gene (EGFP).
[0056] Furthermore, the drug resistance gene includes but is not limited to a neomycin resistance gene or a puromycin resistance gene.
[0057] Furthermore, the protein expression regulatory element I is IRES.
[0058] Furthermore, the internal reference gene M includes but is not limited to a red fluorescent gene (mCherry), a blue fluorescent gene (BFP) or a yellow fluorescent gene (YFP) and the like.
[0059] Preferably, the internal reference gene M is mCherry.
[0060] Furthermore, the lentiviral vector is a tetracycline-induced lentiviral vector.
[0061] In one embodiment of the present invention, when the functional gene G in S2 is the enhanced green fluorescent gene EGFP, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-EGFP-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0062] In one embodiment of the present invention, when the functional gene G in S2 is the drug resistance gene BlastR, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-BlastR-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
[0063] Furthermore, in the cell transfection module, during the cell transfection, a solution containing the lentivirus is added to the cell line to be studied. After the lentivirus enters the cell line to be studied, its RNA genome will be reverse transcribed into DNA, and the exogenous gene will be integrated into the genome of the host cell.
[0064] Furthermore, in the high-throughput screening module, when the functional gene G in the lentiviral expression vector is a fluorescent gene, the high-throughput screening is to perform high-throughput classification and screening of cell populations carrying gene mutations by flow cytometry.
[0065] Furthermore, in the high-throughput screening module, when the functional gene G in the lentiviral expression vector is a drug-resistant gene, the high-throughput screening is a high-throughput classification and screening of cell populations carrying gene mutations by drug-resistant drugs.
[0066] Furthermore, in the deep targeted sequencing module, the high-throughput sequencing refers to deep sequencing of the mutation library before and after screening, respectively.
[0067] Furthermore, in the mutant abundance score analysis module, the analysis method for the effect on protein stability is a method for obtaining the mutant abundance score by performing reasonable calculations based on the frequency changes of the corresponding amino acids of the mutant gene before and after screening.
[0068] In a fourth aspect, the present invention provides use of the lentiviral expression vector described in the first aspect, the method described in the second aspect, or the system described in the third aspect in screening gene variants that cause diseases, and potential key mutation sites of pathogenic genes can be screened out by analyzing the effects of mutant genes on protein stability.
[0069] In a fifth aspect, the present invention provides use of the lentiviral expression vector described in the first aspect, the method described in the second aspect, or the system described in the third aspect in screening gene variants that affect protein stability. By analyzing the effect of the variation on protein stability, the key amino acid sites of the target protein can be screened out.
[0070] Beneficial Effects
[0071] 1. After replacing the Bxb1 recombinase system in the VAMP-seq technology with a lentiviral vector, the flexibility, efficiency and applicability of the technology have been greatly improved. This modification scheme overcomes the limitations of the Bxb1 system in terms of integration site dependence and cell type applicability, and can efficiently construct a large-scale mutant library that is suitable for a wider range of cell backgrounds and experimental conditions. At the same time, through random integration and efficient infection, the improved technology can complete the screening and sequencing of mutants in a short time, significantly improving the high-throughput screening efficiency of VAMP-seq.
[0072] 2. The technical transformation of replacing the fluorescent fusion gene with the drug resistance gene combines the advantages of efficient screening, simple operation, low cost, and long-term application, and effectively solves the shortcomings of the VAMP-seq technology, such as fluorescent labeling relying on complex equipment, high background noise, and longer experimental cycles. This transformation makes large-scale screening applications simpler and faster, and may show significant advantages in in vivo screening applications, providing an economical, efficient and reliable technical solution for high-throughput mutant screening. BRIEF DESCRIPTION OF THE DRAWINGS
[0073] Figure 1 This is the overall technical roadmap of VAMP-seq. Note: A is the original VAMP-seq (Bxb1) technical route; B and C are the improved technical routes of this application.
[0074] Figure 2 This is the expression vector map of the Bxb1 recombinase system.
[0075] Figure 3 This is the "landing pad" vector map of the Bxb1 recombinase system.
[0076] Figure 4 Note: attB-HBZtile2-EGFP-IRES-mCherry expression library.
[0077] Figure 5 Heat map of abundance scores of variants in HBZtile2. Note: Measured by VAMP-seq original technology.
[0078] Figure 6 This is a map of lentiviral expression vectors.
[0079] Figure 7Note: pCW57-HBZtile2-EGFP-IRES-mCherry expression library.
[0080] Figure 8 , 9 is the abundance score map of HBZtile2 variants. Note: Figure 8 Measured using the lentiviral vector pCW57-HBZtile2-EGFP-IRES-mCherry, Fig. 9 Measured using the lentiviral vector pCW57-HBZtile2-BlastR-IRES-mCherry.
[0081] Fig.10 is hierarchical clustering of abundance scores.
[0082] Fig.11 Correlation analysis of variant abundance scores measured for the three experimental systems. DETAILED DESCRIPTION
[0083] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.
[0084] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.
[0085] Explanation of terms
[0086] Nonsense mutation: a form of gene mutation, specifically refers to a change in one or more nucleotides in the gene, causing the codon that originally encoded the amino acid to be converted into a stop codon (UAA, UAG or UGA). Due to the appearance of the stop codon, the translation process will be terminated prematurely and the complete protein cannot be formed.
[0087] Gene mutant: refers to a version of the gene sequence that may or may not affect gene function due to changes in the gene sequence, but the base sequence is different from the original sequence (generally called the wild type).
[0088] Nonsense mutant sequence: refers to a gene sequence generated by a nonsense mutation. The characteristic of this sequence is that at a certain position, the codon that originally encoded the amino acid is replaced by a stop codon, resulting in premature termination of the translation process. This sequence change may lead to loss of protein function or the production of abnormal protein products.
[0089] Example 1 Screening and evaluation of abundance changes of hemoglobin-HBZ mutants using the original VAMP-seq technology 1. Construction of mutant expression vector
[0090] A single amino acid mutant DNA fragment that completely covers the functional region of the HBZ gene was designed and synthesized. The mutation region selected for the library was 150-300bp in the CDS region of the HBZ gene, encoding amino acids 50-100 of the protein, namely the HBZtile2 mutation library. This mutation library was subsequently used consistently for high-throughput experimental tests.
[0091] Through homology arm design, HBZ (wild-type sequence), HBZ (TGA) (nonsense mutant sequence), and HBZtile2 mutant library were recombined into a promoter-less expression vector (attB-HBZ-EGFP-IRES-mCherry), so that the mutant library was fused with the fluorescent protein gene EGFP ( Figure 2 ). Each cell in the recombinant cell library expresses a single variant, and the fluorescent signal of the fusion allows the abundance of the variant to be assessed.
[0092] 2. Construction of tetracycline-induced “landing pad” cell line
[0093] HEK 293T cells were transfected with a lentiviral plasmid vector (LLP-BFP-Int-Blast) to construct a "landing pad" cell line with HEK 293T as the cell background. The lentiviral plasmid vector is induced by tetracycline, and the transcription of the rtTA3G tetracycline activator coding element is driven by the 3'CMV promoter. Upstream, the tetracycline-inducible promoter (Tet-induciblepromoter) drives the expression of the blue fluorescent protein BFP reporter gene, the recombinase Bxb1 gene (initiating the recombination reaction), and the resistance screening gene BlastR (enriching positive cells); the attP recombination site recognized by the Bxb1 recombinase is located between the tetracycline-inducible promoter and the BFP reporter gene, which is a position that allows accurate recombination of exogenous genes ( Figure 3 ). After recombination, the exogenous gene expression cassette will replace the BFP reporter gene and be driven by a tetracycline-inducible promoter.
[0094] 2.1 Lentiviral packaging and cell transfection
[0095] The lentiviral vector (LLP-BFP-Int-Blast) and packaging plasmid (pMD2G, psPAX2) were co-transfected into HEK 293T cells and packaged into viruses. The viral supernatant was collected and used to infect the target cells (HEK 293T); the viral titer was determined and the MOI was optimized to ensure appropriate infection efficiency and avoid interference of multiple copy integration sites on subsequent experiments.
[0096] 2.2 Monoclonal screening
[0097] Two days after cell infection, tetracycline was added to induce the expression of the cell fluorescent protein BFP. The BFP-positive cell population was sorted by flow cytometry and cultured as a single cell. Each cloned cell was a landing pad cell line. In theory, the genome of this cell line only has one and only one recombination site integrated.
[0098] The construction of the "landing pad" cell line, from virus packaging to target cell infection, sorting of positive monoclones, and clonal cell line culture, usually takes 3 to 4 weeks.
[0099] 2.3 Monoclonal verification
[0100] To ensure that only one recombination site is introduced into the screened clonal cell line. Two fluorescent plasmids (attB-EGFP, attB-mCherry) carrying attB recombination sites are transfected into monoclonal cells at the same time. If the monoclonal cell integrates only one recombination site, only one recombination occurs after transfection, and the clonal cell line only expresses a single fluorescent color (green or red); if the monoclonal cell integrates multiple recombination sites, random multi-site recombination may occur after transfection, and some cells in the cell population will express two fluorescent colors (green and red). The transfected clonal cells are verified by flow cytometry. The present invention screened and retained 6 ideal "landing pad" cell lines for subsequent mutation library integration.
[0101] This validation method, involving cell transfection and flow cytometry analysis, usually takes about 1 week.
[0102] 3. Transfection of mutant library using Bxb1 recombinase system
[0103] Cell transfection (recombination integration) Liposome transient transfection was used to transfect the mutant expression vectors (attB-HBZ, attB-HBZ(TGA), attB-HBZtile2) constructed in step 1 into the "landing pad" cell line screened in step 1, respectively, to obtain wild-type HBZ, HBZ(TGA) nonsense mutant, and mutant library in the HEK 293T cell background. Before transfection, DOX was added in advance to induce the expression of Bxb1 recombinase. When the mutant library was transferred in, the recombination reaction could be started immediately, and the target gene mutant and its fluorescent protein fusion gene were cloned into the specific recombination site recognized by Bxb1 recombinase.
[0104] 4. Fluorescence signal detection
[0105] Cell sorting: After transfection, cells begin to express the fused fluorescent protein. The abundance of different mutants is shown by the intensity of the fluorescent signal. The intensity of the fluorescent signal is directly related to the expression level of the target gene and the stability of the protein. The fluorescence intensity of each cell is detected by flow cytometry, and cells with different fluorescence intensities are sorted. Using the fluorescence distribution of wild-type HBZ and nonsense mutants as a reference, the cell population is divided into 4 different groups according to the intensity of the fluorescent signal ( Figure 4 ), sorted and collected, and then cultured to a sufficient number of cells for subsequent DNA extraction and sequencing analysis.
[0106] It usually takes about 1 to 2 weeks from library transfection to enrichment of enough positive cells for sorting experiments; the number of cells after sorting is usually not enough for subsequent experiments, and it takes about 1 to 2 weeks of culture before the cells can be collected for DNA extraction; in summary, the use of the Bxb1 recombinase system for mutant library integration and expression and variant screening takes a total of 2 to 3 months.
[0107] 5. High-throughput sequencing
[0108] Amplicon sequencing: Extract genomic DNA from each fluorescent signal group. Use specific primers to amplify the mutant region of the target gene, and the amplified product is deeply sequenced by the second-generation sequencing method (NGS). The sequencing data is used to determine the relative abundance of each mutant in different fluorescent signal groups. By analyzing the difference in fluorescence intensity, the effect of each mutant on protein stability is analyzed.
[0109] Amplicon library construction and sequencing usually takes about one week.
[0110] 6. Data Analysis
[0111] Variant abundance score calculation: Sequencing data is compared with the mutant sequences in the original mutant library to determine the relative abundance of each mutant in each fluorescent signal group, revealing which mutations have a positive or negative effect on protein stability. Figure 5 As shown, using the original VAMP-seq technology ( Figure 1 (A) It was found that the abundance of mutant proteins encoding positions 63, 67, 71, and 74 of the HBZ protein was significantly reduced, and when the amino acids at almost all positions mutated into nonsense variants, the fluorescence signal decreased (nonsense variants can be regarded as the internal reference control of this technology, and usually show no fluorescence).
[0112] Routine data analysis ~1 week.
[0113] Example 2 Replacing the gene recombination method in VAMP-seq technology (Bxb1 recombinase system replaced by lentiviral vector)
[0114] 1. Construction of mutant expression vector
[0115] The fluorescent gene element (EGFP-IRES-mCherry) originally used for variant abundance monitoring was cloned into a tetracycline-inducible lentiviral vector (pCW57-…EGFP-IRES-mCherry), and the HBZ (wild-type sequence), HBZ (TGA) (nonsense mutant sequence), and HBZtile2 mutant library used in Example 1 were cloned into the vector, and also fused with the fluorescent protein gene (e.g. Figure 6 ). The vector also integrates the resistance gene Puromycin driven by an independent promoter for enrichment of positive cells after transfection. The modified plasmid (or plasmid library) can be directly packaged into lentivirus for infection of any target cell. Each cell in the recombinant cell library expresses a single variant, and its fused fluorescent signal allows the abundance of the variant to be assessed.
[0116] 2. Transfection of mutant library using lentiviral system
[0117] Lentivirus packaging and cell transfection: Consistent with the method described in Example 1, the wild type (pCW57-HBZ-EGFP-IRES-mCherry), nonsense mutant (pCW57-HBZ(TGA)-EGFP-IRES-mCherry), and mutant expression vector library (pCW57-HBZtile2-EGFP-IRES-mCherry) constructed in step 1 were packaged into lentiviruses. The MOI was optimized to ensure the appropriate infection efficiency so that each variant only infects one cell statistically, and each variant is randomly integrated in the genome. In this embodiment, the functional gene for enriching positive cells is the puromycin resistance gene. After two days of viral infection of HEK293T, puromycin was added to screen out positive cells for subsequent experiments.
[0118] The library expression process from virus packaging to target cell infection and then to enrichment of enough positive cells for sorting experiments usually takes about 2 weeks. Compared with Example 1, there is no need to construct and verify the Landingpad cell line of the target cell in the early stage (also using the lentiviral system), which greatly simplifies the experimental process and shortens the experimental cycle by 1 to 2 months.
[0119] 3. Fluorescence signal detection
[0120] Cell sorting: Same as Example 1. The fluorescence signal distribution of the mutant library is as follows Figure 7 As shown:
[0121] As in Example 1, the number of cells after sorting is usually insufficient for subsequent experiments, and cells need to be cultured for another 1 to 2 weeks before they can be collected for DNA extraction.
[0122] 4. High-throughput sequencing
[0123] Amplicon sequencing: Same as Example 1. Same time consumption as Example 1.
[0124] 5. Data Analysis
[0125] Variant abundance score calculation: Same as Example 1. Figure 8 As shown, the Bxb1 recombinase system in VAMP-seq was replaced with a lentiviral vector system, and the HBZtile2 mutant library was tested by high-throughput detection. It was also found that the mutants and nonsense mutants encoding the HBZ protein at positions 63, 67, 71, 74 had the same trend in protein abundance changes. This result shows that the random integration of lentiviral vectors into the genome may cause expression differences caused by variant proteins, and the resulting background noise does not affect the evaluation of variant protein abundance changes in general. The experimental time consumption of this embodiment is consistent with that of Example 1.
[0126] Example 3: Replacing the detection method of mutant abundance in VAMP-seq technology (fluorescent fusion gene EGFP is replaced by drug resistance gene BlastR)
[0127] 1. Construction of mutant expression vector
[0128] Construction of pCW57-…BlastR-IRES-mCherry vector: Based on the modification of Example 2, the fluorescent fusion gene used to detect the abundance of mutants was further replaced with a drug resistance gene to construct the pCW57-…BlastR-IRES-mCherry vector for high-throughput experiments. Similarly, the HBZ (wild-type sequence), HBZ (TGA) (nonsense mutant sequence), and HBZtile2 mutant libraries used in Examples 1 and 2 were cloned into the vector, respectively, so that they were fused with the drug resistance gene. Each cell in the recombinant cell library expresses a single variant, and its fused Blast resistance protein allows the abundance of the variant to be evaluated.
[0129] 2. Transfection of mutant library using lentiviral system
[0130] Lentivirus packaging and cell transfection: The same method as in Example 2 was used to package into lentivirus and infect HEK 293T cells. Consistent with the method described in Example 1, the wild type (pCW57-HBZ-BlastR-IRES-mCherry), nonsense mutant (pCW57-HBZ(TGA)-BlastR-IRES-mCherry), and mutant expression vector library (pCW57-HBZtile2-BlastR-IRES-mCherry) constructed in step 1 were packaged into lentivirus. The MOI was optimized to ensure appropriate infection efficiency, so that each variant theoretically only infected one cell, and each variant was randomly integrated in the genome. Two days after the virus infected HEK 293T, puromycin was added to screen out positive cells for subsequent experiments.
[0131] The time consumption is the same as that in Example 2. The library expression from virus packaging to target cell infection and then to the enrichment of sufficient positive cells for resistance screening experiments usually takes about 2 weeks.
[0132] 3. Blastisidin screening
[0133] Drug resistance screening: Collect positive cells after puromycin screening. Use DOX to induce variant expression two days in advance before treating cells with Blastisidin. The abundance of BlastR protein fused with the variant is affected by the abundance of the variant protein. Under drug screening pressure, the survival number of different variants will be different. Treat with drugs for 1 to 7 days, and continuously collect cells treated for different days for subsequent high-throughput sequencing.
[0134] Using drug resistance screening, the drug treatment time is usually stable within 1 week, and the treated cells can be directly collected for DNA extraction. Compared with Example 2, the number of cells after sorting determines the subsequent culture time (usually 1 to 2 weeks); and the use of fluorescent labeling for cell sorting relies on expensive flow cytometers, which take several hours to complete and have high instrument use costs, while drug treatment only takes ~0.5 hours, and the screening cost is low.
[0135] 4. High-throughput sequencing
[0136] Amplicon sequencing: Same as Example 1. Same time consumption as Examples 1 and 2.
[0137] 5. Data Analysis
[0138] Calculation of variant abundance score: Same as in Examples 1 and 2. Fig. 9As shown, based on the modification of Example 2, the fluorescent signal was replaced with the drug resistance gene BlastR, and the HBZtile2 mutant library was subjected to high-throughput detection. The results obtained were consistent with those of Examples 1 and 2.
[0139] The experimental time of this embodiment is consistent with that of Examples 1 and 2.
[0140] Example 4 Variant Cluster Analysis
[0141] Further cluster analysis of the variant abundance scores in HBZtile2 measured by the three experimental systems showed that the position information of low-abundance mutations detected by the three methods was highly consistent ( Fig.10 ), indicating that the three experimental systems have good reliability and complementarity in the study of discovering low-abundance protein variants.
[0142] Example 5 Correlation Analysis
[0143] The correlation analysis of the variant abundance scores in HBZtile2 measured by the three experimental systems showed that the correlation between the abundance scores of HBZ variants obtained by transfection using the Bxb1 recombinase system (using fluorescence detection signal) and the lentiviral transfection system (using fluorescence detection signal) and the lentiviral transfection system (using blastisidin screening) was 0.5 ( Fig.11 A) and 0.55( Fig.11 B). This indicates that the improved experimental system can achieve the same quality of testing results while saving costs and time.
Claims
1. A lentiviral expression vector with a gene mutant screening function, the lentiviral expression vector is represented by ABGIM or AGBIM, wherein: A is a lentiviral vector, B is a saturation mutation library, G is a functional gene, I is a co-transcriptional and independently translated protein expression regulatory element, M is an internal reference gene different from the functional gene G, the functional gene G includes a fluorescent gene or a drug resistance gene, and the lentiviral expression vector is a lentiviral expression vector with high integration efficiency and wide applicability, which is formed by cloning an expression element (GIM or G-…-IM) containing a functional gene into a lentiviral vector A.
2. According to the lentiviral expression vector of claim 1, the fluorescent gene includes but is not limited to a green fluorescent gene (GFP), a yellow fluorescent gene (YFP) or a red fluorescent gene (RFP), etc.; the drug resistance gene includes but is not limited to a neomycin resistance gene or a puromycin resistance gene; the protein expression regulatory element I is an IRES; the internal reference gene M includes but is not limited to a red fluorescent gene (mCherry), a blue fluorescent gene (BFP) or a yellow fluorescent gene (YFP), etc.
3. The lentiviral expression vector according to any one of claims 1 or 2, when the functional gene in the expression element is fused to the 3' end of the variant gene, the expression element containing the functional gene is GIM, and the lentiviral expression vector is expressed as ABGIM; when the functional gene in the expression element is fused to the 5' end of the variant gene, the expression element containing the functional gene is G-...-IM, and the lentiviral expression vector is expressed as AGBIM.
4. The lentiviral expression vector according to any one of claims 1 to 3, when the functional gene G is the enhanced green fluorescent gene EGFP, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-EGFP-IRES-mCherry, wherein: A is a lentiviral vector, and B is a saturation mutation library; when the functional gene G is the drug resistance gene BlastR, the protein expression regulatory element I is IRES, and the internal reference gene is mCherry, the high-throughput universal screening lentiviral expression vector is AB-BlastR-IRES-mCherry, wherein A is a lentiviral vector and B is a saturation mutation library.
5. A method for high-throughput analysis of the effect of gene variation on protein stability, the method comprising the following steps: S1. Design and synthesize a saturation mutation library of the target gene; S2. The expression element (GIM or G-…-IM) containing the functional gene is cloned into a lentiviral vector to prepare a lentiviral expression vector with screening function, and the mutant library synthesized in S1 is cloned into the lentiviral expression vector containing the functional gene, so that the mutant library is fused with the functional gene in the expression element to obtain the high-throughput universal screening lentiviral expression vector ABGIM or AGBIM containing the mutant library as claimed in claim 1; S3. Use the high-throughput universal screening lentiviral expression vector constructed in S2 to perform cell transfection; S4. Enrich the positive cells transfected in S3 using resistance genes; S5. Induce the expression of the expression frame (BGIM or GBIM) containing the functional gene in the positive cells enriched in S4, and perform high-throughput screening and deep targeted sequencing on the cell mutation library in which the internal reference gene M is successfully expressed; S6. The frequency of variants in S5 results was measured by high-throughput sequencing to analyze the effect of mutations on protein stability.
6. The method according to claim 5, wherein in step S1, the saturation mutation library covers the DNA sequences of all possible single amino acid variations of the target gene; In step S2, G in the expression element (GIM or G-…-IM) refers to a functional gene; the lentiviral vector is a tetracycline-induced lentiviral vector; the GIM is to fuse the functional gene in the expression element to the 3' end of the variant gene, and the G-…-IM is to fuse the functional gene in the expression element to the 5' end of the variant gene; In step S3, during the cell transfection, a solution containing the lentivirus is added to the cell line to be studied. After the lentivirus enters the cell line to be studied, its RNA genome is reverse transcribed into DNA, and the exogenous gene is integrated into the genome of the host cell; In step S4, the resistance gene is puromycin; In step S5, the induction of positive cells refers to the use of DOX to induce variant expression; the high-throughput screening is based on the protein abundance of the expression element containing the functional gene G, and the cell population carrying the gene mutation is subjected to high-throughput classification and screening by flow cytometry or resistance drugs; the deep targeted sequencing refers to deep sequencing of the mutant library before and after screening respectively; In step S6, the analysis method for the effect on protein stability is to reasonably calculate the frequency change of the corresponding amino acids of the mutant gene before and after screening to obtain the mutant abundance score.
7. The method according to claim 5 or 6, wherein in step S5, when the functional gene G in the lentiviral expression vector described in S2 is a fluorescent gene, the high-throughput screening is high-throughput classification and screening of cell populations carrying gene mutations by flow cytometry; when the functional gene G in the lentiviral expression vector described in S2 is a drug-resistant gene, the high-throughput screening is high-throughput classification and screening of cell populations carrying gene mutations by resistance drugs.
8. A system for high-throughput analysis of the effects of variation on protein stability, the system comprising a saturation mutant construction module, a lentiviral vector expression system construction module, a cell transfection module, a high-throughput screening module, a deep sequencing module, and a mutant abundance score analysis module; The saturation mutant building block is to design and synthesize a saturation mutation library of the target gene; The construction module of the lentiviral vector expression system is to clone the expression element (GIM or G-…-IM) containing the functional gene into the lentiviral vector to prepare a lentiviral expression vector with screening function, and clone the synthesized mutation library into the lentiviral expression vector containing the functional gene, so that the mutation library is fused with the functional gene in the expression element to obtain the high-throughput universal screening lentiviral expression vector ABGIM or AGBIM containing the mutation library as claimed in claim 1; The cell transfection module uses a high-throughput universal screening lentiviral expression vector for cell transfection; The high-throughput screening module is based on the protein abundance of the expression element containing the functional gene G, and performs high-throughput classification and screening of cell populations carrying gene mutations by flow cytometry or resistance drugs; The deep targeted sequencing module performs high-throughput sequencing on the mutant library in cells that have been successfully transfected and have successfully expressed the internal reference gene; The mutant abundance score analysis module analyzes the effect of mutations on protein stability based on the frequency of variants in high-throughput sequencing results.
9. Use of the lentiviral expression vector according to claims 1-4, the method according to claims 5-7 or the system according to claim 8 in screening gene variants that cause diseases, and screening out potential key mutation sites of pathogenic genes by analyzing the effect of mutant genes on protein stability.
10. Use of the lentiviral expression vector according to claims 1-4, the method according to claims 5-7 or the system according to claim 8 in screening gene variants that affect protein stability, wherein the key amino acid sites of the target protein can be screened out by analyzing the effect of the variation on protein stability.