RhFN recombinant human chemical fibronectin process of genetic engineering technology

Abstract

The invention discloses an rhFN recombinant human chemical fibronectin process of a genetic engineering technology, and particularly relates to the technical field of genetic engineering, and the rhFN recombinant human chemical fibronectin process comprises target gene acquisition, gene cloning and vector construction, host cell selection and transformation, cell culture and screening, protein expression and optimization, and protein separation and purification. According to the rhFN recombinant human chemical fibronectin process of the genetic engineering technology provided by the invention, high-quality target genes are accurately obtained through technologies such as new-generation sequencing and gene synthesis, and the gene expression efficiency and stability are enhanced by virtue of optimized vector construction and regulatory elements; protein expression ability and specificity are improved in host cell selection and transformation, efficient culture and accurate screening are realized in cell culture and screening links, total yield is increased in a protein expression optimization link, purity and efficiency are improved in a protein separation and purification link, and product quality and safety are ensured through comprehensive quality control. The requirements in the fields of medical treatment and scientific research are expected to be better met.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically, to the rhFN recombinant humanized fibronectin process of gene engineering technology. Background Art

[0002] rhFN recombinant humanized fibronectin is a protein artificially synthesized through genetic engineering technology. Fibronectin has many important physiological functions in the human body. It participates in the processes of cell adhesion, migration, proliferation and differentiation, and plays a key role in cell growth, tissue repair and maintaining the structural stability of the extracellular matrix. rhFN recombinant humanized fibronectin has broad application prospects in the medical field. In terms of wound healing, it can promote cell migration and proliferation and accelerate the wound repair process. In the medical and scientific research fields, the demand for rhFN recombinant humanized fibronectin is growing.

[0003] However, traditional related production processes have many limitations. In terms of host cell selection and transformation, there is a lack of effective comparative and precise gene editing methods, which limits the efficient expression of proteins. During cell culture and screening, improper selection of inducers, imprecise control of culture parameters and inefficient screening technology result in suboptimal cell growth and protein expression levels. The optimization of protein expression lacks systematic research and innovative models, and it is difficult to increase production. The single and inefficient protein separation and purification technology makes it difficult to obtain high-purity proteins. The imperfect quality control methods cannot fully guarantee the quality and safety of the products. These problems have seriously restricted the production and application of rhFN recombinant humanized fibronectin. Therefore, an innovative, efficient and comprehensively optimized process is urgently needed to solve them. Summary of the invention

[0004] In order to overcome the above defects of the prior art, the present invention provides a process for rhFN recombinant humanized fibronectin by genetic engineering technology to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a process for rhFN recombinant humanized fibronectin by genetic engineering technology, specifically comprising the following steps:

[0006] Step 1: Obtaining the target gene: Use the next-generation sequencing technology to perform deep sequencing on multiple healthy human tissue samples to obtain a large amount of sequence data of the rhFN gene. Use bioinformatics analysis tools to process and compare the sequencing results to determine the optimal rhFN gene fragment. Use gene synthesis technology or extract total RNA from the screened samples to obtain the complete target gene through reverse transcription polymerase chain reaction;

[0007] Step 2: Gene cloning and vector construction: select a suitable expression vector, insert the target gene into the vector, introduce enhancer and insulator regulatory elements into the vector, and use seamless cloning technology to complete the connection between the gene and the vector;

[0008] Step 3: Host cell selection and transformation: compare various mammalian cell lines, including CHO cells, HEK293 cells and NS0 cells, and select the cell line most suitable for expressing rhFN protein as the host cell. Use gene editing technology, use sgRNA concentration of 50-100nM and use CRISPR-Cas9 to specifically modify the host cell genome, optimize the transcription and translation mechanism in the cell, and accurately introduce the constructed vector into the host cell through microfluidics technology;

[0009] Step 4: Cell culture and screening: select an inducer with a concentration range of 0.1-5mM, perform large-scale cell culture in a bioreactor, monitor and control nutrient supply, metabolic waste discharge, pH value and dissolved oxygen parameters in real time, use high-throughput screening technology, and quickly screen out cell lines with high expression of rhFN protein based on fluorescence-activated cell sorting. Use cell metabolic engineering to optimize the cell's energy metabolism and material metabolism pathways;

[0010] Step 5: Protein expression and optimization: Use multi-factor experimental design to systematically study the effects of culture conditions on protein expression, explore continuous culture modes, and use metabolomics technology to analyze metabolite changes during cell culture to provide a basis for optimizing protein expression.

[0011] Step 6: Protein separation and purification, using affinity chromatography, ion exchange chromatography and gel filtration chromatography methods in sequence, or combining with two-phase aqueous extraction technology for protein separation and purification. The concentration of polymer or salt in two-phase aqueous extraction is 10%-30%. During the purification process, online monitoring technology is used to monitor protein purity and impurity content in real time, and an integrated purification platform is developed to realize automated and continuous separation and purification processes.

[0012] Preferably, the bioinformatics analysis tool in step 1 is BLAST, which is used to compare gene sequence similarities and help determine the relationship between the obtained rhFN gene fragment and known genes. The gene synthesis technology in step 1 is solid-phase phosphoramidite triester chemical synthesis, which synthesizes genes by gradually adding nucleotide monomers.

[0013] Preferably, the expression vector in step 2 is a viral vector or an artificial chromosome vector, and the seamless cloning technology in step 2 is used to ensure the accuracy and efficiency of the connection between the gene and the vector.

[0014] Preferably, the gene editing technology in step three is used to precisely modify a specific target gene in the genome of an organism. In step three, CRISPR-Cas9 combines with Cas9 protein through sgRNA to form a complex, and the complex accurately finds a specific position in the genome through complementary recognition of sgRNA and target DNA sequence.

[0015] Preferably, in the step 2, the combination and position of enhancer and insulator regulatory elements are determined through optimization simulation, and in the step 1, the sequencing depth used by the next-generation sequencing technology is at least 50X.

[0016] Preferably, in step 4, the time and frequency of adding the inducer are dynamically adjusted according to the cell growth stage, and in step 5, the multi-factor experimental design adopts response surface analysis.

[0017] Preferably, in step six, the ligand used in the affinity chromatography has been specifically screened and optimized.

[0018] Preferably, the culture adjustment in step 5 includes temperature, pH value, dissolved oxygen, culture medium components, inducer concentration and induction time, and the continuous culture mode in step 5 is perfusion culture to extend the cell production cycle and increase the total protein yield.

[0019] Preferably, the online monitoring technology in step six is ​​high performance liquid chromatography-mass spectrometry technology.

[0020] Technical effects and advantages of the present invention:

[0021] 1. Through deep sequencing using next-generation sequencing technology, combined with bioinformatics analysis tools and gene synthesis technology, high-quality and complete rhFN target genes can be obtained more accurately, laying the foundation for subsequent efficient expression. By selecting appropriate expression vectors, introducing enhancer and insulator regulatory elements, and using seamless cloning technology, the insertion accuracy and expression efficiency of the target gene in the vector are improved, ensuring stable gene expression. By comparing a variety of mammalian cell lines and performing gene editing, the ability and specificity of host cells to express rhFN protein are improved. The application of microfluidic technology also enhances the accuracy and efficiency of transformation. By selecting inducers with a suitable concentration range, combined with bioreactors and high-throughput screening technology, the culture conditions can be optimized in real time, and high-expressing cell lines can be quickly screened out. At the same time, the cell metabolic pathways are optimized to improve cell growth and protein expression levels.

[0022] 2. Through multi-factor experimental design and exploration of continuous culture mode, it is helpful to find the best culture conditions and increase the total protein yield. The application of metabolomics technology provides a deeper basis for optimizing expression. The comprehensive use of multiple chromatography methods and two-phase aqueous extraction technology, combined with online monitoring technology, can more efficiently obtain high-purity rhFN protein, while realizing the automation and continuity of the separation and purification process, improving production efficiency and consistency of product quality;

[0023] 3. In summary, through the mutual influence of the above-mentioned multiple effects, the rhFN recombinant humanized fibronectin process of genetic engineering technology can accurately obtain high-quality target genes through technologies such as next-generation sequencing and gene synthesis, enhance gene expression efficiency and stability with the help of optimized vector construction and regulatory elements, improve protein expression ability and specificity in host cell selection and transformation, realize efficient culture and precise screening in cell culture and screening, increase total output in protein expression optimization, improve purity and efficiency in protein separation and purification, and ensure product quality and safety through comprehensive quality control, which is expected to better meet the needs of medical and scientific research fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the process structure of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: The process of rhFN recombinant humanized fibronectin by genetic engineering technology specifically comprises the following steps:

[0027] Step 1: Obtaining the target gene: Use the next-generation sequencing technology to perform deep sequencing on multiple healthy human tissue samples to obtain a large amount of sequence data of the rhFN gene. Use bioinformatics analysis tools to process and compare the sequencing results to determine the optimal rhFN gene fragment. Use gene synthesis technology or extract total RNA from the screened samples to obtain the complete target gene through reverse transcription polymerase chain reaction;

[0028] Step 2: Gene cloning and vector construction: select a suitable expression vector, insert the target gene into the vector, introduce enhancer and insulator regulatory elements into the vector, and use seamless cloning technology to complete the connection between the gene and the vector;

[0029] Step 3: Host cell selection and transformation: compare various mammalian cell lines, including CHO cells, HEK293 cells, and NS0 cells, and select the cell line that is most suitable for expressing rhFN protein as the host cell. Use gene editing technology, use sgRNA concentration of 50nM and use CRISPR-Cas9 to specifically modify the host cell genome, optimize the transcription and translation mechanism in the cell, and accurately introduce the constructed vector into the host cell through microfluidics technology;

[0030] Step 4: Cell culture and screening: select an inducer with a concentration range of 0.1 mM, perform large-scale cell culture in a bioreactor, monitor and control nutrient supply, metabolic waste discharge, pH value and dissolved oxygen parameters in real time, use high-throughput screening technology, and quickly screen out cell lines with high expression of rhFN protein based on fluorescence-activated cell sorting. Use cell metabolic engineering to optimize the cell's energy metabolism and material metabolism pathways;

[0031] Step 5: Protein expression and optimization: Use multi-factor experimental design to systematically study the effects of culture conditions on protein expression, explore continuous culture modes, and use metabolomics technology to analyze metabolite changes during cell culture to provide a basis for optimizing protein expression.

[0032] Step 6: Protein separation and purification, using affinity chromatography, ion exchange chromatography and gel filtration chromatography methods in sequence, or combining with two-phase aqueous extraction technology for protein separation and purification. The concentration of polymer or salt in two-phase aqueous extraction is 30%. During the purification process, online monitoring technology is used to monitor protein purity and impurity content in real time, and an integrated purification platform is developed to realize automated and continuous separation and purification processes.

[0033] Example 2: The process of rhFN recombinant humanized fibronectin by genetic engineering technology specifically comprises the following steps:

[0034] Step 1: Obtaining the target gene: Use the next-generation sequencing technology to perform deep sequencing on multiple healthy human tissue samples to obtain a large amount of sequence data of the rhFN gene. Use bioinformatics analysis tools to process and compare the sequencing results to determine the optimal rhFN gene fragment. Use gene synthesis technology or extract total RNA from the screened samples to obtain the complete target gene through reverse transcription polymerase chain reaction;

[0035] Step 2: Gene cloning and vector construction: select a suitable expression vector, insert the target gene into the vector, introduce enhancer and insulator regulatory elements into the vector, and use seamless cloning technology to complete the connection between the gene and the vector;

[0036] Step 3: Host cell selection and transformation: compare various mammalian cell lines, including CHO cells, HEK293 cells, and NS0 cells, and select the cell line that is most suitable for expressing rhFN protein as the host cell. Use gene editing technology, use sgRNA concentration of 50nM and use CRISPR-Cas9 to specifically modify the host cell genome, optimize the transcription and translation mechanism in the cell, and accurately introduce the constructed vector into the host cell through microfluidics technology;

[0037] Step 4: Cell culture and screening: select an inducer with a concentration range of 0.5 mM, perform large-scale cell culture in a bioreactor, monitor and control nutrient supply, metabolic waste discharge, pH value and dissolved oxygen parameters in real time, use high-throughput screening technology, and quickly screen out cell lines with high expression of rhFN protein based on fluorescence-activated cell sorting. Use cell metabolic engineering to optimize the energy metabolism and material metabolism pathways of cells;

[0038] Step 5: Protein expression and optimization: Use multi-factor experimental design to systematically study the effects of culture conditions on protein expression, explore continuous culture modes, and use metabolomics technology to analyze metabolite changes during cell culture to provide a basis for optimizing protein expression.

[0039] Step 6: Protein separation and purification, using affinity chromatography, ion exchange chromatography and gel filtration chromatography methods in sequence, or combining with two-phase aqueous extraction technology for protein separation and purification. The concentration of polymer or salt in two-phase aqueous extraction is 30%. During the purification process, online monitoring technology is used to monitor protein purity and impurity content in real time, and an integrated purification platform is developed to realize automated and continuous separation and purification processes.

[0040] Example 3: The process of rhFN recombinant humanized fibronectin by genetic engineering technology specifically comprises the following steps:

[0041] Step 1: Obtaining the target gene: Use the next-generation sequencing technology to perform deep sequencing on multiple healthy human tissue samples to obtain a large amount of sequence data of the rhFN gene. Use bioinformatics analysis tools to process and compare the sequencing results to determine the optimal rhFN gene fragment. Use gene synthesis technology or extract total RNA from the screened samples to obtain the complete target gene through reverse transcription polymerase chain reaction;

[0042] Step 2: Gene cloning and vector construction: select a suitable expression vector, insert the target gene into the vector, introduce enhancer and insulator regulatory elements into the vector, and use seamless cloning technology to complete the connection between the gene and the vector;

[0043] Step 3: Host cell selection and transformation: compare various mammalian cell lines, including CHO cells, HEK293 cells, and NS0 cells, and select the cell line that is most suitable for expressing rhFN protein as the host cell. Use gene editing technology, use sgRNA concentration of 75nM and use CRISPR-Cas9 to specifically modify the host cell genome, optimize the transcription and translation mechanism in the cell, and accurately introduce the constructed vector into the host cell through microfluidics technology;

[0044] Step 4: Cell culture and screening: select an inducer with a concentration range of 0.5 mM, perform large-scale cell culture in a bioreactor, monitor and control nutrient supply, metabolic waste discharge, pH value and dissolved oxygen parameters in real time, use high-throughput screening technology, and quickly screen out cell lines with high expression of rhFN protein based on fluorescence-activated cell sorting. Use cell metabolic engineering to optimize the energy metabolism and material metabolism pathways of cells;

[0045] Step 5: Protein expression and optimization: Use multi-factor experimental design to systematically study the effects of culture conditions on protein expression, explore continuous culture modes, and use metabolomics technology to analyze metabolite changes during cell culture to provide a basis for optimizing protein expression.

[0046] Step 6: Protein separation and purification, using affinity chromatography, ion exchange chromatography and gel filtration chromatography methods in sequence, or combining with two-phase aqueous extraction technology for protein separation and purification. The concentration of polymer or salt in two-phase aqueous extraction is 30%. During the purification process, online monitoring technology is used to monitor protein purity and impurity content in real time, and an integrated purification platform is developed to realize automated and continuous separation and purification processes.

[0048] The proteins prepared by the rhFN recombinant humanized fibronectin process of the genetic engineering technology of the above Examples 1-3 were tested respectively, and the following data were obtained:

[0049]

[0050] As can be seen from the above table, in Example 3, by increasing the concentration of sgRNA in the CRISPR-Cas9 system to 75 nM and maintaining the concentration of the inducer (IPTG) at 0.5 mM, the purity of rhFN recombinant humanized fibronectin can be significantly improved, which is of great significance for obtaining high-quality target proteins and improving their application value in medical and biological research. High-purity proteins help reduce potential side effects caused by impurities, enhance the stability and biological activity of proteins, and provide more reliable guarantees for related drug development and clinical applications.

[0051] Finally, it should be noted that the drawings of the embodiments disclosed in the present invention only involve structures related to the embodiments disclosed in the present invention, and other structures can refer to common designs. In the absence of conflicts, the same embodiment and different embodiments of the present invention can be combined with each other;

[0052] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The rhFN recombinant humanized fibronectin process using genetic engineering technology is characterized by: The specific steps include: Step 1: Obtaining the target gene: Use the next-generation sequencing technology to perform deep sequencing on multiple healthy human tissue samples to obtain a large amount of sequence data of the rhFN gene. Use bioinformatics analysis tools to process and compare the sequencing results to determine the optimal rhFN gene fragment. Use gene synthesis technology or extract total RNA from the screened samples to obtain the complete target gene through reverse transcription polymerase chain reaction; Step 2: Gene cloning and vector construction: select a suitable expression vector, insert the target gene into the vector, introduce enhancer and insulator regulatory elements into the vector, and use seamless cloning technology to complete the connection between the gene and the vector; Step 3: Host cell selection and transformation: compare various mammalian cell lines, including CHO cells, HEK293 cells and NS0 cells, and select the cell line most suitable for expressing rhFN protein as the host cell. Use gene editing technology, use sgRNA concentration of 50-100nM and use CRISPR-Cas9 to specifically modify the host cell genome, optimize the transcription and translation mechanism in the cell, and accurately introduce the constructed vector into the host cell through microfluidics technology; Step 4: Cell culture and screening: select an inducer with a concentration range of 0.1-5mM, perform large-scale cell culture in a bioreactor, monitor and control nutrient supply, metabolic waste discharge, pH value and dissolved oxygen parameters in real time, use high-throughput screening technology, and quickly screen out cell lines with high expression of rhFN protein based on fluorescence-activated cell sorting. Use cell metabolic engineering to optimize the cell's energy metabolism and material metabolism pathways; Step 5: Protein expression and optimization: Use multi-factor experimental design to systematically study the effects of culture conditions on protein expression, explore continuous culture modes, and use metabolomics technology to analyze metabolite changes during cell culture to provide a basis for optimizing protein expression. Step 6: Protein separation and purification, using affinity chromatography, ion exchange chromatography and gel filtration chromatography methods in sequence, or combining with two-phase aqueous extraction technology for protein separation and purification. The concentration of polymer or salt in two-phase aqueous extraction is 10%-30%. During the purification process, online monitoring technology is used to monitor protein purity and impurity content in real time, and an integrated purification platform is developed to realize automated and continuous separation and purification processes.

2. The rhFN recombinant humanized fibronectin process according to the genetic engineering technology of claim 1, characterized in that: The bioinformatics analysis tool in step 1 is BLAST, which is used to compare gene sequence similarities and help determine the relationship between the obtained rhFN gene fragment and known genes. The gene synthesis technology in step 1 is solid-phase phosphoramidite triester chemical synthesis, which synthesizes genes by gradually adding nucleotide monomers.

3. The rhFN recombinant humanized fibronectin process according to the genetic engineering technology of claim 1, characterized in that: The expression vector in step 2 is a viral vector or an artificial chromosome vector, and the seamless cloning technology in step 2 is used to ensure the accuracy and efficiency of the connection between the gene and the vector.

4. The rhFN recombinant humanized fibronectin process according to the genetic engineering technology of claim 1, characterized in that: The gene editing technology in step three is used to precisely modify a specific target gene in the genome of an organism. In step three, CRISPR-Cas9 combines with Cas9 protein through sgRNA to form a complex, and the complex accurately finds a specific position in the genome through complementary recognition of sgRNA and target DNA sequence.

5. The process for rhFN recombinant humanized fibronectin by genetic engineering technology according to claim 1, characterized in that: In the step 2, the combination and position of enhancer and insulator regulatory elements are determined through optimization simulation. In the step 1, the sequencing depth used by the new generation sequencing technology is at least 50X.

6. The process for rhFN recombinant humanized fibronectin by genetic engineering technology according to claim 1, characterized in that: In the step 4, the time and frequency of adding the inducer are dynamically adjusted according to the cell growth stage. In the step 5, the multi-factor experimental design adopts the response surface analysis method.

7. The process for rhFN recombinant humanized fibronectin by genetic engineering technology according to claim 1, characterized in that: In step six, the ligand used in affinity chromatography is specifically screened and optimized.

8. The process for rhFN recombinant humanized fibronectin by genetic engineering technology according to claim 1, characterized in that: The culture adjustment in step 5 includes temperature, pH value, dissolved oxygen, culture medium components, inducer concentration and induction time. The continuous culture mode in step 5 is perfusion culture to extend the cell production cycle and increase the total protein yield.

9. The process for rhFN recombinant humanized fibronectin by genetic engineering technology according to claim 1, characterized in that: The online monitoring technology in step six is ​​high performance liquid chromatography-mass spectrometry technology.

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