CN112358542A - Production method of recombinant CD36 protein in mammalian cells - Google Patents
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Abstract
The invention discloses a production method of recombinant CD36 protein in mammalian cells, which comprises the following steps: step one, recombinant plasmid construction, step two, transfection, step three, sample collection and purification, step four, and purity detection. According to the production method of the recombinant CD36 protein in the mammalian cells, His tag is added when a CD36 recombinant plasmid is constructed, so that the later purification process is simplified, the target protein can be quickly obtained through one-step affinity chromatography, the whole purification process is less than 1h, the whole process is operated at low temperature, the activity of the target protein is greatly ensured, when the recombinant plasmid is constructed, codons are optimized, a high-expression Expi293 system is used for expressing the target protein, the transfection condition is optimized, the expression quantity of the target protein is greatly improved, the purity of the target protein is detected by using an HPLC technology, and the high purity of the obtained recombinant CD36 protein is ensured.
Description
Technical Field
The invention relates to the technical field of recombinant CD36 protein production, in particular to a production method of a recombinant CD36 protein in mammalian cells.
Background
Adhesion of monocytes to endothelial cells and subsequent infiltration of monocytes play an important role in the early stage of atherosclerosis, monocytes gradually transform to macrophages with corresponding changes in their phenotype and function during infiltration into the arterial wall and turn into foam cells after phagocytosis of a large amount of modified low density lipoprotein, the expression of scavenger receptor CD36 antigen plays a very important role in the transformation of monocytes, CD36 is an important binding receptor of oxidized low density lipoprotein in vivo, the increased expression of CD36 antigen is related to differentiation of monocytes into macrophages and formation of foam cells, adhesion contact of monocytes to endothelial cells is not only simple but also simple physical contact, and cell-cell interaction often exists, i.e. during contact, corresponding signal transduction pathways are triggered, causing a change in the function of the cell.
The invention provides a production method of recombinant CD36 protein in mammalian cells, which effectively solves the problems of low activity of the recombinant CD36 protein and low purity of the recombinant CD36 protein.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a production method of recombinant CD36 protein in mammalian cells, which solves the problems of low activity of the recombinant CD36 protein and low purity of the recombinant CD36 protein.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for producing recombinant CD36 protein in mammalian cells specifically comprises the following steps:
step one, recombinant plasmid construction: carrying out codon optimization on the full-length gene of the recombinant CD36 by a gene synthesis technology, and subcloning the gene into a pcDNA3.1 vector to construct a CD36 recombinant plasmid;
step two, transfection: selecting Expi293 cells to inoculate a triangular flask, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi Fectamin 293 regents with Opti-MENI, adding the diluted Expi Fectamin 293 regents into the diluted sterile recombinant plasmids, uniformly mixing substances in the triangular flask, adding the mixture into the prepared cells, and culturing in an incubator;
step three, collecting and purifying: collecting a culture medium, directly purifying the culture medium by using an affinity chromatography technology, firstly activating agarose, then coupling protein to combine the protein with the activated agar, carrying out column selection, column filling and column washing on the coupling protein, slowly adding the culture medium into the coupling protein, carrying out elution treatment after uniform mixing, finally carrying out adsorption treatment on eluate, and neutralizing by using sodium bicarbonate and an adsorption substance to obtain final protein;
step four, detecting the purity: and (3) finishing detecting the purity of the target protein by using HPLC, preparing three groups of protein solutions with different concentrations by using a protein standard sample, then drawing a standard curve of the protein concentration and the peak height and the peak area, and then measuring the peak height and the peak area of the sample under the same condition to obtain the purity of the target protein.
Preferably, the recombinant CD36 protein expresses a sequence:
MYRMQLLSCIALSLALVTNSGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMFRSQVTGKINHHHHHH*。
preferably, His tag is added to the CD36 recombinant plasmid during the construction of the recombinant plasmid.
Preferably, the specific steps of transfection are as follows: taking the density as 3 multiplied by 106Inoculating the triangular flask with 95-99% survival rate Expi293 cells, diluting the prepared sterile recombinant plasmid with Opti-MENI, diluting the prepared Expi Fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent to the diluted sterile recombinant plasmid, gently mixing for 2-4 times, standing at room temperature for 30min, adding the mixture to the prepared cells, adding the mixture to the cells containing 8% CO2The culture was carried out in an incubator at 37 ℃ and 120rpm for 4 to 6 days.
Preferably, the purity detection comprises the following specific steps: and (3) finishing detecting the purity of the target protein by using HPLC (high performance liquid chromatography), preparing three groups of protein solutions with different concentrations by using a protein standard sample, drawing a standard curve relating the protein concentration to the peak height and the peak area, measuring the peak height and the peak area of the sample under the same condition to obtain the concentration of an unknown sample, and dividing the peak area and the peak height of the target protein by the sum of all the peak areas and the peak heights under the condition of ensuring that all the components in the sample to be detected have peaks.
(III) advantageous effects
The invention provides a method for producing recombinant CD36 protein in mammalian cells. Compared with the prior art, the method has the following beneficial effects:
the recombinant CD36 protein is produced in mammal cell through codon optimization of recombinant CD36 full length gene and subcloning into pcDNA3.1 vector to constitute CD36 recombinant plasmid, adding His tag to CD36 recombinant plasmid in the construction of recombinant plasmid, and taking out recombinant CD36 protein with density of 3 x 106Inoculating the triangular flask with 95-99% survival rate Expi293 cells, diluting the prepared sterile recombinant plasmid with Opti-MENI, diluting the prepared Expi Fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent to the diluted sterile recombinant plasmid, gently mixing for 2-4 times, standing at room temperature for 30min, adding the mixture to the prepared fine recombinant plasmidIn the cells, the content of CO is 8%2The method comprises the steps of culturing for 4-6 days in an incubator at 37 ℃ under the condition that the rotating speed is 120rpm, adding His tag when constructing the CD36 recombinant plasmid, simplifying the purification process at the later stage, quickly obtaining the target protein through one-step affinity chromatography, wherein the whole purification process is less than 1h, performing low-temperature operation in the whole process, greatly ensuring the activity of the target protein, optimizing codons when constructing the recombinant plasmid, expressing the target protein by using a high-expression Expi293 system, optimizing transfection conditions, greatly improving the expression quantity of the target protein, detecting the purity of the target protein by using an HPLC technology, and ensuring that the purity of the obtained recombinant CD36 protein is higher.
Drawings
FIG. 1 is a schematic diagram of the HPLC detection result of recombinant CD36 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in figure 1 of the drawings,
example 1
A method for producing recombinant CD36 protein in mammalian cells specifically comprises the following steps:
step one, recombinant plasmid construction: carrying out codon optimization on the full-length gene of the recombinant CD36 by a gene synthesis technology, and subcloning the gene into a pcDNA3.1 vector to construct a CD36 recombinant plasmid;
step two, transfection: selecting Expi293 cells to inoculate a triangular flask, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi Fectamin 293 regents with Opti-MENI, adding the diluted Expi Fectamin 293 regents into the diluted sterile recombinant plasmids, uniformly mixing substances in the triangular flask, adding the mixture into the prepared cells, and culturing in an incubator;
step three, collecting and purifying: collecting a culture medium, directly purifying the culture medium by using an affinity chromatography technology, firstly activating agarose, then coupling protein to combine the protein with the activated agar, carrying out column selection, column filling and column washing on the coupling protein, slowly adding the culture medium into the coupling protein, carrying out elution treatment after uniform mixing, finally carrying out adsorption treatment on eluate, and neutralizing by using sodium bicarbonate and an adsorption substance to obtain the final recombinant CD36 protein;
step four, detecting the purity: and (3) finishing detecting the purity of the target protein by using HPLC, preparing three groups of protein solutions with different concentrations by using a protein standard sample, then drawing a standard curve of the protein concentration and the peak height and the peak area, and then measuring the peak height and the peak area of the sample under the same condition to obtain the purity of the target protein.
In the process of constructing the recombinant plasmid, His tag was added to the CD36 recombinant plasmid.
The specific steps of transfection are as follows: taking the density as 3 multiplied by 106The method comprises the steps of inoculating an Erlenmeyer flask with/mL Expi293 cells with the survival rate of 95%, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent into the diluted sterile recombinant plasmids, gently mixing the mixture for 2 times, standing at room temperature for 30min, adding the mixture into the prepared cells, and inoculating the cells with 8% CO in the presence of 8%2The culture was carried out in an incubator of (1) at a temperature of 37 ℃ and a rotation speed of 120rpm for 4 days.
The specific steps of purity detection are as follows: and (3) finishing detecting the purity of the target protein by using HPLC (high performance liquid chromatography), preparing three groups of protein solutions with different concentrations by using a protein standard sample, drawing a standard curve relating the protein concentration to the peak height and the peak area, measuring the peak height and the peak area of the sample under the same condition to obtain the concentration of an unknown sample, and dividing the peak area and the peak height of the target protein by the sum of all the peak areas and the peak heights under the condition of ensuring that all the components in the sample to be detected have peaks.
Example 2
A method for producing recombinant CD36 protein in mammalian cells specifically comprises the following steps:
step one, recombinant plasmid construction: carrying out codon optimization on the full-length gene of the recombinant CD36 by a gene synthesis technology, and subcloning the gene into a pcDNA3.1 vector to construct a CD36 recombinant plasmid;
step two, transfection: selecting Expi293 cells to inoculate a triangular flask, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi Fectamin 293 regents with Opti-MENI, adding the diluted Expi Fectamin 293 regents into the diluted sterile recombinant plasmids, uniformly mixing substances in the triangular flask, adding the mixture into the prepared cells, and culturing in an incubator;
step three, collecting and purifying: collecting a culture medium, directly purifying the culture medium by using an affinity chromatography technology, firstly activating agarose, then coupling protein to combine the protein with the activated agar, carrying out column selection, column filling and column washing on the coupling protein, slowly adding the culture medium into the coupling protein, carrying out elution treatment after uniform mixing, finally carrying out adsorption treatment on eluate, and neutralizing by using sodium bicarbonate and an adsorption substance to obtain the final recombinant CD36 protein;
step four, detecting the purity: and (3) finishing detecting the purity of the target protein by using HPLC, preparing three groups of protein solutions with different concentrations by using a protein standard sample, then drawing a standard curve of the protein concentration and the peak height and the peak area, and then measuring the peak height and the peak area of the sample under the same condition to obtain the purity of the target protein.
In the process of constructing the recombinant plasmid, His tag was added to the CD36 recombinant plasmid.
The specific steps of transfection are as follows: taking the density as 3 multiplied by 106The method comprises the steps of inoculating an Erlenmeyer flask with 99% survival rate Expi293 cells, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent into the diluted sterile recombinant plasmids, gently mixing the diluted Expi293 Regent and the diluted sterile recombinant plasmids for 4 times, and then inoculating into a rear chamberStanding at room temperature for 30min, adding the mixture to the prepared cells in a solution containing 8% CO2The culture was carried out in an incubator at 37 ℃ and 120rpm for 6 days.
The specific steps of purity detection are as follows: and (3) finishing detecting the purity of the target protein by using HPLC (high performance liquid chromatography), preparing three groups of protein solutions with different concentrations by using a protein standard sample, drawing a standard curve relating the protein concentration to the peak height and the peak area, measuring the peak height and the peak area of the sample under the same condition to obtain the concentration of an unknown sample, and dividing the peak area and the peak height of the target protein by the sum of all the peak areas and the peak heights under the condition of ensuring that all the components in the sample to be detected have peaks.
Example 3
A method for producing recombinant CD36 protein in mammalian cells specifically comprises the following steps:
step one, recombinant plasmid construction: carrying out codon optimization on the full-length gene of the recombinant CD36 by a gene synthesis technology, and subcloning the gene into a pcDNA3.1 vector to construct a CD36 recombinant plasmid;
step two, transfection: selecting Expi293 cells to inoculate a triangular flask, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi Fectamin 293 regents with Opti-MENI, adding the diluted Expi Fectamin 293 regents into the diluted sterile recombinant plasmids, uniformly mixing substances in the triangular flask, adding the mixture into the prepared cells, and culturing in an incubator;
step three, collecting and purifying: collecting a culture medium, directly purifying the culture medium by using an affinity chromatography technology, firstly activating agarose, then coupling protein to combine the protein with the activated agar, carrying out column selection, column filling and column washing on the coupling protein, slowly adding the culture medium into the coupling protein, carrying out elution treatment after uniform mixing, finally carrying out adsorption treatment on eluate, and neutralizing by using sodium bicarbonate and an adsorption substance to obtain the final recombinant CD36 protein;
step four, detecting the purity: and (3) finishing detecting the purity of the target protein by using HPLC, preparing three groups of protein solutions with different concentrations by using a protein standard sample, then drawing a standard curve of the protein concentration and the peak height and the peak area, and then measuring the peak height and the peak area of the sample under the same condition to obtain the purity of the target protein.
In the process of constructing the recombinant plasmid, His tag was added to the CD36 recombinant plasmid.
The specific steps of transfection are as follows: taking the density as 3 multiplied by 106The method comprises the steps of inoculating an Erlenmeyer flask with/mL Expi293 cells with the survival rate of 97%, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent into the diluted sterile recombinant plasmids, gently mixing the mixture for 3 times, standing at room temperature for 30min, adding the mixture into the prepared cells, and inoculating the cells with 8% CO in the presence of 8%2The culture was carried out in an incubator of (1) at a temperature of 37 ℃ and a rotation speed of 120rpm for 5 days.
The specific steps of purity detection are as follows: and (3) finishing detecting the purity of the target protein by using HPLC (high performance liquid chromatography), preparing three groups of protein solutions with different concentrations by using a protein standard sample, drawing a standard curve relating the protein concentration to the peak height and the peak area, measuring the peak height and the peak area of the sample under the same condition to obtain the concentration of an unknown sample, and dividing the peak area and the peak height of the target protein by the sum of all the peak areas and the peak heights under the condition of ensuring that all the components in the sample to be detected have peaks.
Sequence for recombinant CD36 protein expression:
MYRMQLLSCIALSLALVTNSGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMFRSQVTGKINHHHHHH*。
and those not described in detail in this specification are well within the skill of those in the art.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A method for producing a recombinant CD36 protein in a mammalian cell, comprising: the method specifically comprises the following steps:
step one, recombinant plasmid construction: carrying out codon optimization on the full-length gene of the recombinant CD36 by a gene synthesis technology, and subcloning the gene into a pcDNA3.1 vector to construct a CD36 recombinant plasmid;
step two, transfection: selecting Expi293 cells to inoculate a triangular flask, diluting prepared sterile recombinant plasmids with Opti-MENI, diluting prepared Expi Fectamin 293 regents with Opti-MENI, adding the diluted Expi Fectamin 293 regents into the diluted sterile recombinant plasmids, uniformly mixing substances in the triangular flask, adding the mixture into the prepared cells, and culturing in an incubator;
step three, collecting and purifying: collecting a culture medium, directly purifying the culture medium by using an affinity chromatography technology, firstly activating agarose, then coupling protein to combine the protein with the activated agar, carrying out column selection, column filling and column washing on the coupling protein, slowly adding the culture medium into the coupling protein, carrying out elution treatment after uniform mixing, finally carrying out adsorption treatment on eluate, and neutralizing by using sodium bicarbonate and an adsorption substance to obtain the final recombinant CD36 protein;
step four, detecting the purity: and (3) finishing detecting the purity of the target protein by using HPLC, preparing three groups of protein solutions with different concentrations by using a protein standard sample, then drawing a standard curve of the protein concentration and the peak height and the peak area, and then measuring the peak height and the peak area of the sample under the same condition to obtain the purity of the target protein.
2. A method of producing a recombinant CD36 protein in mammalian cells according to claim 1, wherein: the expressed sequence of the recombinant CD36 protein is as follows:
MYRMQLLSCIALSLALVTNSGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMFRSQVTGKINHHHHHH*。
3. a method of producing a recombinant CD36 protein in mammalian cells according to claim 1, wherein: in the process of constructing the recombinant plasmid, Histag is added into the CD36 recombinant plasmid.
4. A method of producing a recombinant CD36 protein in mammalian cells according to claim 1, wherein: the specific steps of the transfection are as follows: taking the density as 3 multiplied by 106Inoculating Expi293 cells with a survival rate of 95-99% into a triangular flask, diluting the prepared sterile recombinant plasmid with Opti-MENI, diluting the prepared Expi Fectamin 293Regent with Opti-MENI, standing at room temperature for 5min, adding the diluted Expi Fectamin 293Regent to the mixtureAdding into diluted sterile recombinant plasmid, mixing gently for 2-4 times, standing at room temperature for 30min, adding the mixture into prepared cells, adding into the cells containing 8% CO2The culture was carried out in an incubator at 37 ℃ and 120rpm for 4 to 6 days.
5. A method of producing a recombinant CD36 protein in mammalian cells according to claim 1, wherein: the purity detection method comprises the following specific steps: and (3) finishing detecting the purity of the target protein by using HPLC (high performance liquid chromatography), preparing three groups of protein solutions with different concentrations by using a protein standard sample, drawing a standard curve relating the protein concentration to the peak height and the peak area, measuring the peak height and the peak area of the sample under the same condition to obtain the concentration of an unknown sample, and dividing the peak area and the peak height of the target protein by the sum of all the peak areas and the peak heights under the condition of ensuring that all the components in the sample to be detected have peaks.
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2020
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| CN1868546A (en) * | 2005-05-27 | 2006-11-29 | 中国医学科学院医药生物技术研究所 | Model for screening human scavenger donee CD36 antagonist and its application |
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| CN106434564A (en) * | 2016-11-01 | 2017-02-22 | 南宁输血医学研究所 | Establishing method of CD36 mutant gene stable eukaryotic expression cell line causing CD36 deletion |
| US20200102370A1 (en) * | 2018-09-28 | 2020-04-02 | Massachusetts Institute Of Technology | Collagen-localized immunomodulatory molecules and methods thereof |
| CN109734793A (en) * | 2019-03-14 | 2019-05-10 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | A kind of ZnT8 recombinant protein and its preparation method and application |
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Application publication date: 20210212 |