Preparation method and application of directionally evolved high-stability reverse transcriptase

Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of directed evolution high-stability reverse transcriptase, and the preparation method comprises the following steps: firstly, constructing an element library containing various reverse transcriptase elements; on the basis of computer-aided design, reverse transcriptase carriers with high expression potential and high activity are screened and combined from the constructed slave element library; carrying out directional modification on the preliminarily screened reverse transcriptase vector by utilizing a computer-aided molecular evolution platform; expressing the reverse transcriptase mutant obtained by transformation in a host cell; and finally, purifying the expressed reverse transcriptase by using a protein purification and quality inspection platform to obtain a high-purity enzyme product, so that the technical problem that the reverse transcriptase prepared in the prior art is insufficient in stability, catalytic efficiency, tolerance and the like and the application of the reverse transcriptase under complex experimental conditions is limited is solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing a directed evolution high-stability reverse transcriptase and its application. Background Art

[0002] Reverse transcriptase is an RNA-dependent DNA polymerase that was first discovered in RNA viruses in the 1970s. Its main function is to reverse transcribe RNA to generate complementary DNA chains (cDNA). This property makes reverse transcriptase play a key role in a variety of biotechnology applications, such as cDNA library construction, mRNA sequencing, RT-PCR quantification, etc., especially in scientific research and medical molecular diagnosis.

[0003] However, the reverse transcriptase prepared by the existing technology has deficiencies in stability, catalytic efficiency and tolerance, which limits its application under complex experimental conditions. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a highly stable reverse transcriptase by directed evolution and its application, aiming to solve the technical problem that the reverse transcriptase prepared in the prior art has deficiencies in stability, catalytic efficiency and tolerance, which limits its application under complex experimental conditions.

[0005] To achieve the above object, the present invention adopts a method for preparing a highly stable reverse transcriptase by directed evolution, comprising the following steps:

[0006] First, a component library containing various reverse transcriptase components is constructed;

[0007] Based on computer-aided design, reverse transcriptase vectors with high expression potential and high activity are screened and combined from the constructed sub-element library;

[0008] Using the computer-assisted molecular evolution platform, the reverse transcriptase vectors that were initially screened were modified in a targeted manner;

[0009] expressing the transformed reverse transcriptase mutant in a host cell;

[0010] Finally, the expressed reverse transcriptase is purified using a protein purification and quality inspection platform to obtain a high-purity enzyme product.

[0011] The specific method of constructing a component library containing multiple reverse transcriptase components is as follows:

[0012] Collect reverse transcriptase gene sequences from various biological sources (including but not limited to viruses, retrotransposons, etc.);

[0013] Compare and analyze the collected gene sequences to identify key enzyme activity and stability-related elements (such as active sites, structural domains, etc.);

[0014] The identified elements are modularly designed to construct an element library containing a variety of reverse transcriptase elements (providing a basis for subsequent screening and combination).

[0015] Among them, based on computer-aided design, the specific method of screening and combining a reverse transcriptase vector with high expression potential and high activity from the constructed component library is as follows:

[0016] Using computer-aided design tools, the constructed component library was virtually screened to predict the effects of different component combinations on reverse transcriptase expression and activity;

[0017] Based on the prediction results, the reverse transcriptase element combination with high expression potential and high activity was screened;

[0018] The screened elements are combined for gene synthesis to construct a reverse transcriptase vector.

[0019] Among them, the specific screening process is completed by the virtual screening model, and the construction method of the virtual screening model is as follows:

[0020] Collect a large amount of data about reverse transcriptase elements (including but not limited to gene sequences, structural information, known enzyme activity and stability data, etc.);

[0021] Preprocess the collected data, including data cleaning, format unification and standardization;

[0022] Extract features related to reverse transcriptase expression and activity (such as sequence similarity, domain characteristics, amino acid composition, etc.) from the preprocessed data;

[0023] Use feature engineering methods to select and optimize the extracted features;

[0024] Choose a machine learning algorithm or deep learning algorithm (such as random forest, support vector machine, neural network, etc.) as the model based on the characteristics of the data and the complexity of the problem;

[0025] The model was trained using the extracted features and known enzyme activity and stability data to construct a virtual screening model.

[0026] Among them, the specific method of using the computer-assisted molecular evolution platform to carry out directed modification of the reverse transcriptase vector initially screened is as follows:

[0027] The reverse transcriptase vectors initially screened were introduced into the computer-assisted molecular evolution platform;

[0028] By simulating the natural selection process, the reverse transcriptase vector was subjected to multiple rounds of iterative mutation and screening to further improve its stability and activity;

[0029] In each iteration, high-throughput sequencing technology is used to monitor the distribution and frequency of mutants to guide the next round of mutation strategy;

[0030] At the same time, in each round of iterative mutation, specific mutation sites are added, including positions 66, 69, 197, 204, 221, 223, 249, 259, 302, 313, 326, 334, 491 and 524.

[0031] Wherein, when the transformed reverse transcriptase mutant is expressed in a host cell:

[0032] Introducing the directed engineered reverse transcriptase mutant into a host cell;

[0033] The reverse transcriptase mutant is cultured in a host cell and expression is induced.

[0034] Among them, after obtaining high-purity enzyme products, quality testing should be carried out, and the testing items include enzyme activity determination and stability testing.

[0035] Among them, the constructed component library containing multiple reverse transcriptase components is a component library that can be dynamically updated. As new reverse transcriptase gene sequences are discovered and analyzed, new components are promptly incorporated into the component library.

[0036] Among them, when expressing the modified reverse transcriptase mutant in a host cell, after selecting the host cell, genetic modification should be performed (such as enhancing the promoter, optimizing codons, knocking out unnecessary genes, etc., to improve the expression level and stability of the reverse transcriptase).

[0037] Through genetic engineering technology, the modified host cells are fused or co-expressed with the reverse transcriptase gene to achieve efficient and stable reverse transcriptase production.

[0038] The reverse transcriptase prepared by the method for preparing a highly stable reverse transcriptase through directed evolution as described above is used in gene cloning, cDNA synthesis, expression analysis and mRNA sequencing.

[0039] The invention discloses a method for preparing a highly stable reverse transcriptase by directed evolution and its application, which comprises the following steps: firstly constructing an element library comprising a plurality of reverse transcriptase elements; based on computer-aided design, screening and combining reverse transcriptase vectors with high expression potential and high activity from the constructed element library; using a computer-aided molecular evolution platform, directingly transforming the reverse transcriptase vectors preliminarily screened; expressing the transformed reverse transcriptase mutants in host cells; and finally, purifying the expressed reverse transcriptase by using a protein purification and quality inspection platform to obtain a high-purity enzyme product, thereby solving the technical problem that the reverse transcriptase prepared in the prior art has deficiencies in stability, catalytic efficiency and tolerance, and limits its application under complex experimental conditions.

[0040] The present invention constructs a component library containing multiple reverse transcriptase components and uses computer-aided design and virtual screening models to efficiently screen out reverse transcriptase combinations with high expression potential and high activity from a large number of components. This method not only improves the screening efficiency, but also ensures that the screened reverse transcriptase has excellent performance.

[0041] At the same time, the present invention uses a computer-assisted molecular evolution platform to carry out directional transformation of the reverse transcriptase vector initially screened, and performs multiple rounds of iterative mutation and screening by simulating the natural selection process, which significantly improves the stability and activity of the reverse transcriptase. The prepared reverse transcriptase has excellent thermal stability and can react at 60°C while maintaining high enzyme activity, which is crucial for some experiments that require high temperature conditions.

[0042] Furthermore, the reverse transcriptase of the present invention has excellent synthesis ability and can efficiently reverse transcribe low-abundance or degraded RNA. This property makes the reverse transcriptase have unique advantages in applications such as cDNA library construction and mRNA sequencing that require processing of complex RNA samples.

[0043] Again, the reverse transcriptase of the present invention also has extremely high catalytic efficiency and can complete the synthesis of cDNA in a short time. Specifically, the reverse transcriptase can complete the synthesis of cDNA within 12 minutes, greatly shortening the experimental time and improving work efficiency. The reverse transcriptase of the present invention also performs well in amplification length and can amplify DNA sequences up to 16kb in length. This characteristic makes the reverse transcriptase have a wide range of application prospects in applications such as gene cloning, long-fragment cDNA synthesis, etc. that require the amplification of long-fragment DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 It is a flow chart of the method for preparing a highly stable reverse transcriptase by directed evolution of the present invention. DETAILED DESCRIPTION

[0046] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0047] See also Figure 1 , Figure 1 It is a flow chart of the method for preparing a highly stable reverse transcriptase by directed evolution of the present invention.

[0048] The present invention provides a method for preparing a highly stable reverse transcriptase by directed evolution, comprising the following steps:

[0049] S1. First, construct a component library containing multiple reverse transcriptase components;

[0050] For this specific embodiment, the specific method of constructing a component library containing multiple reverse transcriptase components is as follows:

[0051] The gene sequences of reverse transcriptase were collected from various biological sources;

[0052] Compare and analyze the collected gene sequences to identify key enzyme activity and stability-related elements;

[0053] The identified elements were modularly designed to construct an element library containing a variety of reverse transcriptase elements.

[0054] Furthermore, the constructed component library containing a variety of reverse transcriptase components is a component library that can be dynamically updated. As new reverse transcriptase gene sequences are discovered and analyzed, new components can be incorporated into the component library in a timely manner.

[0055] In addition to collecting reverse transcriptase gene sequences from biological sources such as viruses and retrotransposons, it can also be expanded to other biological groups, such as extreme environment microorganisms, which may contain reverse transcriptase elements with special stability and activity.

[0056] S2. Based on computer-aided design, a reverse transcriptase vector with high expression potential and high activity is screened and combined from the constructed component library;

[0057] For this specific embodiment, based on computer-aided design, the specific method of screening and combining reverse transcriptase vectors with high expression potential and high activity from the constructed component library is as follows:

[0058] Using computer-aided design tools, the constructed component library was virtually screened to predict the effects of different component combinations on reverse transcriptase expression and activity;

[0059] Based on the prediction results, the reverse transcriptase element combination with high expression potential and high activity was screened;

[0060] The screened elements are combined for gene synthesis to construct a reverse transcriptase vector.

[0061] In this process, the specific screening process is completed by the virtual screening model, and the construction method of the virtual screening model is as follows:

[0062] Collect a large amount of data about reverse transcriptase elements (including but not limited to gene sequences, structural information, known enzyme activity and stability data, etc.);

[0063] Preprocess the collected data, including data cleaning, format unification and standardization;

[0064] Extract features related to reverse transcriptase expression and activity (such as sequence similarity, domain characteristics, amino acid composition, etc.) from the preprocessed data;

[0065] Use feature engineering methods to select and optimize the extracted features;

[0066] Choose a machine learning algorithm or deep learning algorithm (such as random forest, support vector machine, neural network, etc.) as the model based on the characteristics of the data and the complexity of the problem;

[0067] The model was trained using the extracted features and known enzyme activity and stability data to construct a virtual screening model.

[0068] At the same time, after the model is built, the model is optimized through cross-validation, parameter tuning and other methods to improve its prediction accuracy and generalization ability.

[0069] The model is validated using an independent test dataset to ensure its performance in real-world applications.

[0070] S3. Use computer-assisted molecular evolution platform to carry out directed transformation of the reverse transcriptase vectors initially screened;

[0071] According to this specific implementation method, the specific method of using the computer-assisted molecular evolution platform to carry out directed transformation of the reverse transcriptase vector initially screened is as follows:

[0072] The reverse transcriptase vectors initially screened were introduced into the computer-assisted molecular evolution platform;

[0073] By simulating the natural selection process, the reverse transcriptase vector was subjected to multiple rounds of iterative mutation and screening to further improve its stability and activity;

[0074] In each iteration, high-throughput sequencing technology is used to monitor the distribution and frequency of mutants to guide the next round of mutation strategy;

[0075] At the same time, in each round of iterative mutation, specific mutation sites are added, including positions 66, 69, 197, 204, 221, 223, 249, 259, 302, 313, 326, 334, 491 and 524.

[0076] S4, expressing the transformed reverse transcriptase mutant in a host cell;

[0077] According to this specific embodiment, the reverse transcriptase mutant that has undergone targeted modification is introduced into the host cell;

[0078] The reverse transcriptase mutant is cultured in a host cell and expression is induced.

[0079] When expressing the modified reverse transcriptase mutant in a host cell, the most suitable host cell is selected for expression according to the characteristics of the reverse transcriptase, such as protein size, post-translational modification requirements, etc. For example, for reverse transcriptases that require complex post-translational modifications, mammalian cells can be selected; for those with higher expression level requirements, Escherichia coli or yeast can be selected. After selecting the host cell, genetic modification should be performed (such as enhancing the promoter, optimizing codons, knocking out unnecessary genes, etc., to improve the expression level and stability of the reverse transcriptase). The culture conditions of the modified host cells are optimized, such as adjusting parameters such as temperature, pH, dissolved oxygen, etc., to increase the yield and activity of the reverse transcriptase.

[0080] Through genetic engineering technology, the modified host cells are fused or co-expressed with the reverse transcriptase gene to achieve efficient and stable reverse transcriptase production.

[0081] S5. Finally, the expressed reverse transcriptase is purified using a protein purification and quality inspection platform to obtain a high-purity enzyme product.

[0082] According to this specific embodiment, after obtaining the high-purity enzyme product, a quality test should be carried out, and the test items include enzyme activity determination and stability test.

[0083] The method for preparing a highly stable reverse transcriptase by directed evolution of the present invention comprises the following steps: firstly, a component library comprising a plurality of reverse transcriptase components is constructed; based on computer-aided design, a reverse transcriptase vector with high expression potential and high activity is screened and combined from the constructed component library; a computer-aided molecular evolution platform is used to perform directed transformation on the reverse transcriptase vector preliminarily screened; the transformed reverse transcriptase mutant is expressed in a host cell; and finally, a protein purification and quality inspection platform is used to purify the expressed reverse transcriptase to obtain a high-purity enzyme product, thereby solving the technical problem that the reverse transcriptase prepared in the prior art has deficiencies in stability, catalytic efficiency and tolerance, which limits its application under complex experimental conditions.

[0084] The present invention constructs a component library containing multiple reverse transcriptase components and uses computer-aided design and virtual screening models to efficiently screen out reverse transcriptase combinations with high expression potential and high activity from a large number of components. This method not only improves the screening efficiency, but also ensures that the screened reverse transcriptase has excellent performance.

[0085] At the same time, the present invention uses a computer-assisted molecular evolution platform to carry out directional transformation of the reverse transcriptase vector initially screened, and performs multiple rounds of iterative mutation and screening by simulating the natural selection process, which significantly improves the stability and activity of the reverse transcriptase. The prepared reverse transcriptase has excellent thermal stability and can react at 60°C while maintaining high enzyme activity, which is crucial for some experiments that require high temperature conditions.

[0086] Furthermore, the reverse transcriptase of the present invention has excellent synthesis ability and can efficiently reverse transcribe low-abundance or degraded RNA. This property makes the reverse transcriptase have unique advantages in applications such as cDNA library construction and mRNA sequencing that require processing of complex RNA samples.

[0087] Again, the reverse transcriptase of the present invention also has extremely high catalytic efficiency and can complete the synthesis of cDNA in a short time. Specifically, the reverse transcriptase can complete the synthesis of cDNA within 12 minutes, greatly shortening the experimental time and improving work efficiency. The reverse transcriptase of the present invention also performs well in amplification length and can amplify DNA sequences up to 16kb in length. This characteristic makes the reverse transcriptase have a wide range of application prospects in applications such as gene cloning, long-fragment cDNA synthesis, etc. that require the amplification of long-fragment DNA.

[0088] The reverse transcriptase prepared by the method for preparing a highly stable reverse transcriptase through directed evolution as described above is used in gene cloning, cDNA synthesis, expression analysis and mRNA sequencing.

[0089] In the field of gene cloning, highly stable reverse transcriptase can ensure the accurate transcription of full-length cDNA from RNA templates, thereby improving the success rate and accuracy of cloning and providing a solid foundation for subsequent gene function research and disease diagnosis.

[0090] In terms of cDNA synthesis, the efficient catalytic ability of this reverse transcriptase makes the synthesis of full-length cDNA faster and more reliable. This provides a high-quality template for mRNA sequencing, ensuring the accuracy and reliability of the sequencing results.

[0091] In expression analysis, highly stable reverse transcriptase can accurately quantify gene expression levels, providing reliable data support for biological and medical research. This helps to reveal gene regulation mechanisms and understand changes in gene expression in organisms under different physiological or pathological conditions.

[0092] In the field of mRNA sequencing, highly stable reverse transcriptases are the key to ensuring successful sequencing. Due to the characteristics of mRNA being easily degraded and having low abundance, traditional reverse transcriptases are often difficult to meet the high requirements of mRNA sequencing. The highly stable reverse transcriptases of directed evolution provided by the present invention can overcome these challenges, ensure that the target mRNA is accurately transcribed from a complex RNA mixture, and provide high-quality templates for subsequent sequencing and analysis. This makes the results of mRNA sequencing more accurate and reliable, and helps to reveal subtle changes and regulatory mechanisms of gene expression in organisms.

[0093] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing a highly stable reverse transcriptase by directed evolution, characterized in that: The steps include: First, a component library containing various reverse transcriptase components is constructed; Based on computer-aided design, reverse transcriptase vectors with high expression potential and high activity are screened and combined from the constructed sub-element library; Using the computer-assisted molecular evolution platform, the reverse transcriptase vectors that were initially screened were modified in a targeted manner; expressing the transformed reverse transcriptase mutant in a host cell; Finally, the expressed reverse transcriptase is purified using a protein purification and quality inspection platform to obtain a high-purity enzyme product.

2. The method for preparing a directed-evolution high-stability reverse transcriptase according to claim 1, characterized in that: The specific method of constructing a component library containing multiple reverse transcriptase components is as follows: The gene sequences of reverse transcriptase were collected from various biological sources; Compare and analyze the collected gene sequences to identify key enzyme activity and stability-related elements; The identified elements were modularly designed to construct an element library containing a variety of reverse transcriptase elements.

3. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 2, characterized in that: Based on computer-aided design, the specific method of screening and combining reverse transcriptase vectors with high expression potential and high activity from the constructed component library is as follows: Using computer-aided design tools, the constructed component library was virtually screened to predict the effects of different component combinations on reverse transcriptase expression and activity; Based on the prediction results, the reverse transcriptase element combination with high expression potential and high activity was screened; The screened elements are combined for gene synthesis to construct a reverse transcriptase vector.

4. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 3, characterized in that: The specific screening process is completed by the virtual screening model, and the construction method of the virtual screening model is as follows: Collect a large amount of data on reverse transcriptase elements; Preprocess the collected data, including data cleaning, format unification and standardization; Extract features related to reverse transcriptase expression and activity from the preprocessed data; Use feature engineering methods to select and optimize the extracted features; Choose a machine learning algorithm or a deep learning algorithm as the model based on the characteristics of the data and the complexity of the problem; The model was trained using the extracted features and known enzyme activity and stability data to construct a virtual screening model.

5. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 4, characterized in that: The specific method of using the computer-assisted molecular evolution platform to carry out directed modification of the reverse transcriptase vector initially screened is as follows: The reverse transcriptase vectors initially screened were introduced into the computer-assisted molecular evolution platform; By simulating the natural selection process, the reverse transcriptase vector was subjected to multiple rounds of iterative mutation and screening to further improve its stability and activity; In each iteration, high-throughput sequencing technology is used to monitor the distribution and frequency of mutants to guide the next round of mutation strategy; At the same time, in each round of iterative mutation, specific mutation sites are added, including positions 66, 69, 197, 204, 221, 223, 249, 259, 302, 313, 326, 334, 491 and 524.

6. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 5, characterized in that: When expressing the modified reverse transcriptase mutant in a host cell: Introducing the directed engineered reverse transcriptase mutant into a host cell; The reverse transcriptase mutant is cultured in a host cell and expression is induced.

7. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 6, characterized in that: After obtaining high-purity enzyme products, quality testing should be carried out. Testing items include enzyme activity determination and stability testing.

8. The method for preparing a directed-evolution high-stability reverse transcriptase according to claim 7, characterized in that: The constructed component library containing multiple reverse transcriptase components is a component library that can be dynamically updated. As new reverse transcriptase gene sequences are discovered and analyzed, new components are promptly incorporated into the component library.

9. The method for preparing a directed-evolved high-stability reverse transcriptase according to claim 8, characterized in that: When expressing the modified reverse transcriptase mutant in a host cell, genetic modification should be performed after selecting the host cell.

10. A reverse transcriptase prepared by the method for preparing a highly stable reverse transcriptase by directed evolution according to claim 9, characterized in that: Applications in gene cloning, cDNA synthesis, expression analysis and mRNA sequencing.

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