CN115786397A - Transmembrane protein 176B recombinant vector and secretion expression method of transmembrane protein 176B - Google Patents
Transmembrane protein 176B recombinant vector and secretion expression method of transmembrane protein 176B Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a transmembrane protein 176B recombinant vector and a secretion expression method of transmembrane protein 176B. The transmembrane protein 176B recombinant vector comprises an expression vector and an insertion fragment, wherein the insertion fragment comprises a gene sequence for coding an IgG signal peptide, a gene sequence for coding the 142 th to 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding a mFC-6His protein label which are sequentially connected. According to the invention, a specific fragment of the transmembrane protein 176B is selected to be matched with an IgG signal peptide and a mFC-6His protein tag, and based on a eukaryotic cell expression system, the secretory expression of the transmembrane protein 176B is successfully realized, a high-concentration transmembrane protein 176B recombinant protein is obtained, and the obtaining of the high-concentration and high-purity transmembrane protein 176B provides possibility for deep research on the function of the transmembrane protein 176B and also provides convenience for developing a functional antibody thereof.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a transmembrane protein 176B recombinant vector and a secretion expression method of transmembrane protein 176B.
Background
Transmembrane protein (TMEM) is involved in intercellular and intracellular signal transmission, immune-related diseases, tumorigenesis and tumor development, and in various physiological processes, such as ion channel formation of plasma membrane, signal transduction pathway activation, cell chemotaxis, adhesion, apoptosis, autophagy, etc. Transmembrane protein 176B (TMEM 176B), previously known as TORID (tolerance-associated and inducible transcript), was first found in lung fibroblasts, belongs to the family of transmembrane 4A (Membrane-bridging 4-Domains, subset a, MS 4A) proteins, localized on human chromosome 7 (murine localized chromosome 6), which is differentially expressed in immune cells and certain cancers. It is shown that TMEM176B has an inhibitory effect on dendritic cell maturation, presumably inhibiting the anti-tumor immunity of the body. TMEM176B is up-regulated in antigen presenting cells of the allograft mouse model, but much remains to be understood about its role in intracellular processes. At present, few reports on the functions of transmembrane protein family members, particularly TMEM176B, indicate that the research on the transmembrane protein family members is still in the initial stage, and also provide wide space for the research in the field. This is because TMEM176B is localized to the nuclear membrane and it is difficult to isolate the purified protein by conventional methods. Researchers build some over-expression cell lines to realize the continuous over-expression of the TMEM176B gene, and perform verification through an antibody to research the properties of the TMEM176B gene, but the method can only preliminarily judge the functions of the TMEM176B gene, and cannot obtain high-quality purified protein for further research.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a transmembrane protein 176B recombinant vector and a secretion expression method based on the transmembrane protein 176B recombinant vector, so as to solve the technical problem that high-quality transmembrane protein 176B is difficult to obtain in the prior art.
The invention is realized by the following technical scheme:
the invention provides a transmembrane protein 176B recombinant vector, which comprises an expression vector and an insert fragment, wherein the insert fragment comprises a gene sequence for coding an IgG signal peptide, a gene sequence for coding the 142 th to the 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding a mFC-6His protein label which are sequentially connected; wherein the amino acid sequence of the 142 th to 196 th amino acids of the transmembrane protein 176B is shown as SEQ ID NO:4, respectively.
Further, the gene sequence encoding the 142 nd to 196 th amino acids of the transmembrane protein 176B is shown in SEQ ID NO:5, respectively.
Further, the amino acid sequence of the IgG signal peptide is shown as SEQ ID NO:2, and the gene sequence for coding the IgG signal peptide is shown as SEQ ID NO:3, respectively.
Further, the mFC-6His protein tag has an amino acid sequence shown in SEQ ID NO:6, the gene sequence for coding the mFC-6His protein tag is shown as SEQ ID NO: shown in fig. 7.
Further, the expression vector includes pcdna3.4.
In a second aspect, the invention provides a host cell comprising a recombinant vector for transmembrane protein 176B as described above.
Further, the host cell includes a HEK293F cell.
In a third aspect, the present invention provides a method for secretory expression of transmembrane protein 176B, comprising the steps of:
s1, transfecting a host cell by using the transmembrane protein 176B recombinant vector;
s2, culturing the transfected host cells, expressing the protein by using a eukaryotic expression system, collecting culture supernatant, and purifying to obtain the transmembrane protein 176B in a secretory form.
Further, in step S1, constructing the transmembrane protein 176B recombinant vector comprises: connecting a gene sequence for coding an IgG signal peptide, a gene sequence for coding 142 th to 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding mFC-6his protein labels to an expression vector pCDNA3.4, then transforming the expression vector into an escherichia coli DH5 alpha competent cell, carrying out positive clone screening, and obtaining the transmembrane protein 176B recombinant vector through sequencing verification.
Further, in step S1, the transmembrane protein 176B recombinant vector is transfected into host cells, including HEK293F cells, using transient transfection techniques.
The fourth aspect of the invention provides a secreted protein prepared by the above-mentioned secretion expression method of the transmembrane protein 176B, wherein the secreted protein comprises the 142 th to 196 th amino acids of the transmembrane protein 176B and a mFC-6his protein tag, and the mFC-6his protein tag is closely attached to the C-terminal of the 142 th to 196 th amino acids of the transmembrane protein 176B.
The invention has the advantages and positive effects that:
according to the invention, a specific segment (142-196 aa) of the transmembrane protein 176B is selected to be matched with an IgG signal peptide and a mFC-6His protein tag to construct a transmembrane protein 176B recombinant vector, the recombinant vector is transferred into a host cell, secretion expression of the transmembrane protein 176B is successfully realized based on a eukaryotic cell expression system, the high-concentration TMEM176B recombinant protein is obtained, the expression amount is about 110mg/1000mL Cells, and the obtaining of the high-concentration and high-purity transmembrane protein 176B provides possibility for deep research of the function of the transmembrane protein 176B and also provides convenience for developing a functional antibody of the transmembrane protein 176B.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a diagram showing the structural analysis of the transmembrane domain of TMEM176B protein according to the embodiment of the present invention;
FIG. 2 is a tertiary structure analysis diagram of TMEM176B protein according to an embodiment of the present invention;
FIG. 3 is a diagram showing the amplification results of a target gene when plasmids 1 to 3 are constructed in accordance with an embodiment of the present invention;
FIG. 4 is a graph showing the cell culture concentration after eukaryotic cells have been transfected with plasmids 1 to 3 according to an embodiment of the present invention over time;
FIG. 5 is a graph of cell viability (Vabilty) over time after transfection of eukaryotic cells with plasmids 1-3 according to an embodiment of the present invention;
FIG. 6 is a diagram showing the protein expression verified by WB after eukaryotic cells were transfected with plasmids 1 to 3 according to an embodiment of the present invention;
FIG. 7 is a diagram showing protein purification after transfection of eukaryotic cells with plasmid 3 according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be included within the scope of the following claims.
For a better understanding of the invention, without limiting the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The embodiment of the invention provides a transmembrane protein 176B recombinant vector, which comprises an expression vector and an insert fragment, wherein the insert fragment comprises a gene sequence for coding IgG signal peptide, a gene sequence for coding 142 th to 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding mFC-6His protein label, which are sequentially connected; wherein the amino acid sequence of the 142 th to 196 th amino acids of the transmembrane protein 176B is shown as SEQ ID NO:4, respectively.
In the context of the present invention, a "signal peptide" is used to direct the secretory expression of a foreign protein. "secreted" means that a molecule of a protein or peptide is transported outside the cell after intracellular synthesis. The "mFC-6His protein tag" comprises mFC and 6 XHis, which are linked in sequence, and is named mFC-6His tag.
Transmembrane protein 176B (hereinafter referred to as TMEM176B protein) is a four-transmembrane protein belonging to transmembrane 4A (MS 4A) protein family, and comprises four transmembrane domains and a C-terminal Immunoreceptor Tyrosine Inhibition Motif (ITIM), and its amino acid sequence is described in NCBI accession No.: NM _001164207.1, nucleotide sequence see NCBI accession number: NP _001157679.1. The analysis of the protein structure and the transmembrane domain (see fig. 1-2) shows that the N end and the C end of the protein are both random linear structures and are irrelevant to the protein function, 61-81aa, 89-109aa, 121-141aa and 197-217aa are transmembrane domains, and the analysis of the tertiary structure of the TMEM176B protein shows that the reliability of the selected domains is high. The location of the transmembrane-spanning amino acid region, which should be avoided in selecting the expression region, in the nuclear membrane itself leads to difficult secretion of the protein, so that 55 amino acids in the 142-196 interval are selected as the final stable form in the present invention, and the 142-196 expression region (amino acid sequence see SEQ ID NO: 4) can cover the isoform region and can represent the protein function. The protein expressed by the segment can be used for developing functional antibodies.
As is known, the secretion of the same expression cassette is greatly influenced by matching protein tags and signal peptides of different species, in the invention, a transmembrane protein expression cassette is constructed by selecting a specific fragment of TMEM176B protein and matching the specific signal peptide and the protein tags, specifically, an insertion fragment in an expression vector is connected with a signal peptide gene sequence, a TMEM176B protein 142-196aa gene sequence and a protein tag gene sequence in sequence from a 5 'end to a 3' end, the obtained coding protein is sequentially connected with an IgG signal peptide, the TMEM176B protein 142-196aa and a mFC-6His protein tag from an N end to a C end, the recombinant vector is transferred into a host cell, the secretion expression of the TMEM176B protein is successfully realized, high-concentration TMEM176B recombinant protein is obtained through WB verification and affinity purification, the expression amount is about 110mg/1000mL of Cells, and the subsequent transfection expression process can be optimized, so that higher expression amount is realized. The implementation of the invention provides reference for signal peptide and tag sequence of other transmembrane protein secretion expression.
Alternatively, the gene sequence encoding amino acids 142 to 196 of the transmembrane protein 176B is as shown in SEQ ID NO:5 for encoding TMEM176B protein with 61 amino acids from position 142 (inclusive) to position 196 (inclusive).
Alternatively, the amino acid sequence of the IgG signal peptide is as set forth in SEQ ID NO:2, and the gene sequence of the IgG signal peptide is shown as SEQ ID NO:3, respectively.
Alternatively, the mFC-6His protein tag has an amino acid sequence as set forth in SEQ ID NO:6, the gene sequence for coding the mFC-6His protein tag is shown as SEQ ID NO: shown at 7.
Optionally, the expression vector comprises a eukaryotic cell expression vector, which is pcdna3.4.
In another embodiment of the invention, a host cell is provided comprising a recombinant vector for transmembrane protein 176B as described above.
By culturing the above-described host cells by a conventional method in the art, the transmembrane protein 176B can be obtained in a high-purity, high-concentration secreted form. The advantages of the host cell comprising the transmembrane protein 176B recombinant vector as described above over the prior art are the same as the advantages of the transmembrane protein 176B recombinant vector as described above over the prior art and are not described in detail here.
Optionally, the host cell comprises a eukaryotic cell comprising a HEK293F cell.
In still another embodiment of the present invention, there is provided a use of the transmembrane protein 176B recombinant vector as described above or a host cell comprising the transmembrane protein 176B recombinant vector as described above in the field of producing a secreted protein. Specifically, the secretion expression method of the transmembrane protein 176B comprises the following steps:
s1, transfecting a host cell by using the transmembrane protein 176B recombinant vector;
s2, culturing the transfected host cells, expressing the protein by using a eukaryotic expression system, collecting culture supernatant, and purifying to obtain the transmembrane protein 176B in a secretion form.
The advantages of the secretion expression method of transmembrane protein 176B over the prior art are the same as the advantages of the recombinant vector of transmembrane protein 176B over the prior art, and are not described in detail here.
It should be noted that the secreted form of transmembrane protein 176B obtained in the culture supernatant only includes TMEM176B protein 142-196aa and mFC-6His protein tag, mFC-6His protein tag is immediately C-terminal to said TMEM176B protein, while IgG signal peptide is anchored to the cell membrane during its secretion process and is cleaved off, which is not involved in the function of the protein nor in the protein structure of the secreted form.
Specifically, in step S1, the gene sequence of the IgG signal peptide, the gene sequence of the TMEM176B protein 142-196aa and the mFC-6his protein tag gene sequence are connected to the multiple cloning sites on the expression vector pCDNA3.4, so that the transmembrane protein 176B recombinant vector can be obtained. For simplicity of operation, an expression vector with a signal peptide and a mFC-6his protein tag can be used as an initial vector, so that the construction of the recombinant vector of the present invention can be omitted. For example, the gene sequence (shown as SEQ ID NO: 3) for encoding IgG signal peptide and the gene sequence (shown as SEQ ID NO: 5) for encoding 142 th to 196 th amino acids of transmembrane protein 176B are connected to an expression vector pCDNA3.4 with IgG signal peptide and mFC-6his protein labels, then the expression vector is transformed into Escherichia coli DH5 alpha competent cells, positive clone screening is carried out, and sequencing verification is carried out to obtain the transmembrane protein 176B recombinant vector.
Specifically, in step S1, the transmembrane protein 176B recombinant vector is transfected into host cells, including HEK293F cells, using transient transfection techniques.
Specifically, in step S2, the purification method is affinity purification.
The invention further provides a secretory protein which is prepared by the secretory expression method of the transmembrane protein 176B, and the secretory protein comprises amino acids 142 to 196 of the transmembrane protein 176B and a mFC-6his protein tag, wherein the mFC-6his protein tag is closely attached to the C end of the amino acids 142 to 196 of the transmembrane protein 176B.
The advantages of the secreted protein over the prior art are the same as the advantages of the secretory expression method of transmembrane protein 176B over the prior art as described above, and are not described in detail here.
The invention will be further illustrated with reference to the following specific examples. The following examples are examples of experimental procedures not specified under specific conditions, generally according to the conditions recommended by the manufacturer.
1. Recombinant vector construction
TMEM176B protein full length 263aa, wherein 61-81aa, 89-109aa, 121-141aa and 197-217aa are transmembrane regions, combining mouse sequence homology analysis, selecting 142-196aa as a final stable form, constructing a mammalian expression vector, and expressing the protein by using an Expi-293F mammalian cell eukaryotic system. The techniques employed for the insertion of the signal peptide and protein tag into the expression vector pcdna3.4 are conventional in the art and will not be described further herein. This section only describes the insertion process of transmembrane protein 176B. This example constructs the following recombinant vectors:
| numbering | Name of gene | Signal peptide | Region(s) | Label (R) |
| |
|
1 | 142-196aa | C-mFC- |
| Plasmid | ||||
| 2 | |
2 | 142-196aa | C-AVI- |
| Plasmid | ||||
| 3 | |
2 | 142-196aa | C-mFC-6His |
The sequence information referred to above is as follows:
primer design was performed using Primer 5 with the Primer sequence:
PCR amplification was performed with the above primers using RK20715 kit (purchased from ABClonal) in a 50. Mu.L PCR system, where: gloria Nova HS 2X HF Master Mix 25 μ L, ddH 2 O22. Mu.L, template 1. Mu.L, and upstream/downstream primer 1+1. Mu.L. Setting a PCR amplification reaction program: denaturation at 94 deg.C (high temperature disruption of bacterial solution) for 4min, (94 deg.C denaturation melting 40s, 58 deg.C annealing primer and template binding 30s, 72 deg.C extension 1 min) x 30cycLe,72 deg.C extension 10min. The template is a plasmid containing a 142-196aa region of TMEM176B protein and is named as: pUC57-TMEM176B.
After the completion of the PCR reaction, a single band of 165bp was observed by agarose gel electrophoresis (see FIG. 3), indicating that the pure target gene was obtained, and then the pure target gene was obtained by recovering the band using Kangji agarose gel DNA recovery kit (cat # CW 2302).
The purified target gene is connected and transformed with an expression vector pCDNA3.4 with a signal peptide and a protein tag by adopting a homologous recombination mode, and a connecting system is as follows: 2 μ L of target gene, 3 μ L of pCDNA3.4 vector, and 5 μ L of 2 XMultiF Seamless Assembly Mix (purchased from ABClonal, cat # RM 20523), wherein the ratio of the target gene to the expression vector pCDNA3.4 is determined according to the concentration after recovery, and the molar ratio range is 3:1-10, and the target gene and the expression vector are ligated at 50 ℃ for 30min. Then 10 mu L of the ligation product is completely transferred into DH5 alpha competent cells, colony PCR identification is carried out by adopting a universal primer after ice bath, heat shock, resuscitation and overnight culture, and the PCR system is 20uL, wherein: 4 μ L of bacterial liquid, 1+1 μ L of upstream and downstream universal sequencing primers, 10 μ L, ddH of hot start Taq 2X PCR premix (containing Dye, available from ABClonal, cat # RK 20605) 2 O4. Mu.L. PCR amplification reaction program: 95 ℃/5 min: (95℃/30S，58℃/30S，68℃/1min)×35cycle，68℃/5min→4℃∞。
Positive clones in colony PCR identification are subjected to sequencing verification by adopting a vector universal primer (sequencing company: wuhan Jin Kairui Biotechnology Co., ltd.), expression plasmids with correct sequencing are used for subsequent protein expression, the concentration of the obtained plasmid 1 is 1000ng/uL, the concentration of the obtained plasmid 2 is 1122ng/uL, and the concentration of the obtained plasmid 3 is 1200ng/uL.
The sequences of the universal primers are shown as follows:
2. transfection
Eukaryotic cells HEK293F are divided into transient transfection and stable transfection, transient transfection plasmid DNA is not integrated into chromosomes after entering cells, and exogenous genes are expressed in an episomal state; the transient transfection period is short, the target protein can be quickly obtained, and the method can be applied to groping and large-scale high-throughput protein screening in the research and development stage. In this embodiment, the transient transfection technique is adopted to transfect the plasmids 1 to 3 into the eukaryotic cell HEK293F, respectively, to obtain a host cell expressing a secretory protein, which specifically includes the following steps:
(1) Eukaryotic cells HEK293F were isolated at 1.3E6 cells/mL one day prior to transfection in medium, cell volume 27mL;
(2) Prior to transfection, all reagents were left at room temperature and cell density was adjusted to 2.6E6/mL;
(3) Diluting 30. Mu.g of plasmid DNA (plasmid 1 or plasmid 2 or plasmid 3) with 1.5mL of Opti-MEM in a sterile tube, adding 90. Mu.L of PEI (1 mg/mL, pH 7.1) to 1.5mL of Opti-MEM, mixing well, and standing for 5min;
(4) Adding the mixed solution of PEI into the mixed solution of the plasmids, turning or pipetting and mixing (the process of uniformly mixing is required to be slowly carried out), and then incubating for less than 20 minutes at room temperature;
(5) Adding the plasmid/PEI mixture into a eukaryotic cell culture solution, and fully mixing the plasmid/PEI mixture by gentle rotation, wherein the total volume of the mixed cells is 30mL;
(6) 1.5mL is fed for the first time 16-20 hours after transfection, and the cell survival rate and the cell density are measured after 96h; expressing for 4 days on day 0 of transfection, detecting viable cell number and cell survival rate every day, stopping expression and collecting HEK293F cell and cell culture supernatant when cell survival rate is reduced to below 70%, and determining results as shown in FIGS. 4-5;
(7) The supernatant was collected and affinity purified by adding 10mM AEBSF.
3. Expression and purification
3.1 expression verification by Western Blot (WB)
Sample preparation: taking 100 mu L of transfected HEK293F cells, centrifuging at 3000r/min for 10min, collecting 20 mu L of centrifuged supernatant, adding 20 mu L of 2 × loading buffer for sample preparation, heating at 97 ℃ for 10min, and naming the mixture as supernatant (supernatant); meanwhile, collecting the precipitate, suspending the precipitate by using 100 mu L PBS, taking 20 mu L, adding 20 mu L to obtain a 2 × loading buffer sample, heating the sample at 97 ℃ for 10min, and naming the sample as a cell (cell); the WB detection process is as follows:
(1) Loading 5 mu L of the prepared sample to carry out SDS-PAGE electrophoresis, wherein the electrophoresis adopts a constant pressure mode, 5% concentrated gel is 80V, when marker begins to be separated for about 25min and is adjusted to 120V, and when bromophenol blue reaches the bottom of the separation gel, the electrophoresis is stopped;
(2) Film transfer: the assembly sequence is as follows: the method comprises the following steps of (1) rotating a film and clamping a black surface (a negative electrode), a spongy cushion, 3 layers of filter paper, glue, a film, 3 layers of filter paper, the spongy cushion and a red surface (a positive electrode); film transfer time: 200mA, 90-180min;
(3) And (3) sealing: marking and washing the membrane transfer liquid after the membrane transfer is finished (TBST, 5min multiplied by 2 times); putting the cleaned membrane into a container containing 3% skimmed milk (prepared by TBST), and sealing at room temperature for 60-90min;
(4) 6His-tag primary antibody incubation: after the sealing is completed, the sealing liquid is poured off. Primary antibody solution diluted with 3% skim milk (TBST formulation) 1 at 7000 was added, gently shaken on a shaker, and incubated at room temperature for 2h or overnight at 4 deg.C (4 deg.C followed by incubation for 15-30min at room temperature). After the primary antibody incubation is finished, pouring out the primary antibody solution; rinsing the membrane with TBST for 5min 4 times;
(5) And (3) secondary antibody incubation: before the primary antibody incubation is completed, an enzyme-labeled secondary antibody 1 corresponding to the primary antibody species: 5000 dilutions were made to the amount required for the experiment (TBST dilution). And (3) putting the washed membrane into a container containing the secondary antibody solution, slowly shaking on a shaking table, and incubating at room temperature for 60-80min. After the secondary antibody incubation is finished, pouring out the secondary antibody solution; rinsing the membrane with TBST for 5min 4 times;
(6) Exposure: the membrane was removed from the TBST with forceps, drained appropriately, and placed on a gel tray. Mixing with ECL Solution I and Solution II in equal volume, adding onto the membrane uniformly, and covering completely. The substrate reacts with the membrane for about 30 seconds and is placed in a chemiluminescent imaging system. Setting the exposure time to be 3s;10s;30s;60s;120s.
The harvested cells and the supernatant were subjected to WB assay, and the results are shown in FIG. 6. The target band is detected in HEK293F cells transfected by plasmids 1-3, but only the target band is detected in the culture supernatant of the HEK293F cells transfected by plasmid 3, the TMEM176B protein 142-196aa is secreted and expressed extracellularly, and the target band is not detected in the culture supernatants of plasmid 1 and plasmid 2, and the protein is expressed in the cells. It should be noted that, since plasmid 2 has no large tag such as FC, the theoretical size of the inserted protein sequence is only calculated to be 8.74kD, and the reason for the formation of the large inserted protein sequence may be a nonspecific band, which is obtained by exposure to light under conditions of long-term and enhanced color developing solution, and it is considered that no expression is observed. According to the WB assay, the protein of plasmid 3 was secreted extracellularly, and therefore the supernatant could be purified.
3.2 His purification
WB-verified plasmid 3 secretory expression cell supernatant for affinity purification, purification column using Polv-Prep @ chromatography Columns (from BIO-RAD, cat # 731-1550) and Ni Bestarose FF (from Shanghai Bogelong), the operation flow is as follows:
(1) And (3) incubation: the sterilized 10mL purification cartridge was removed and the cartridge was rinsed 1-2 times with endotoxin-free water. The Ni-IDA affinity purification matrix was removed from the 4 ℃ freezer and 0.5mL of matrix was pipetted into the purification column and after ethanol run-off (commercial matrix was stored in 20% ethanol) 6 column volumes were washed with endotoxin-free water. Column volume refers to the volume of matrix in the purification column, not the volume of the column tube. The packing was equilibrated for 6 column volumes with Binding Buffer. Adding the balanced matrix into the supernatant tube, sealing with sealing membrane, and placing on a rotary culture device at 20rpm and 4 deg.C for 4 hr-overnight.
(2) Flow-through: after the incubation was completed, the tube was trimmed and centrifuged at 600rpm,10min,4 ℃. And pouring the centrifuged supernatant into a new centrifuge tube to obtain the flow-through. While leaving about 5mL of supernatant for suspending the matrix, the matrix was transferred to a purification cartridge and allowed to run through (this run through was not collected).
(3) And (3) eluting and impurity washing:
a. 3mL Washing Buffer was added to the purification column tube to wash off the contaminating proteins in the matrix. Gravity flow, the effluent was collected with a sterilized 5mL EP tube that needed to be inserted on ice to maintain a low temperature. After the elution is finished, the column is detected by G250 (100 mu L G is put into a 96-well plate, 10 mu L of the eluent which is dripping is added), if the G250 turns blue, 3mL of Washing Buffer is added to wash the column continuously until the G250 does not turn blue, and the Washing Buffer pre-elution is finished.
b. The column was washed with 0.5mL of Elution Buffer 1, and the matrix-bound protein was washed off. Gravity flow, the effluent was collected in 1.5mL EP tube without endotoxin, the collection tube was kept cold on an ice box. After the Elution is finished, the Elution is carried out by G250 (100 mu L G is put into a 96-well plate, 10 mu L of the eluent which is dripping is added), if G250 turns blue, the Elution is continued until G250 does not turn blue, and the Elution Buffer 1 pre-Elution is finished.
c. Add 0.5mL Elution Buffer 2 column wash, wash down the matrix binding protein. Gravity flow, the effluent was collected in 1.5mL EP tube without endotoxin, the collection tube was kept cold on an ice box. After the Elution is finished, the Elution is carried out by using G250 (100 mu L G is taken in a 96-well plate, 10 mu L of the eluent which is dripping out is added), if the G250 turns blue, the Elution is continued until the G250 does not turn blue, and the Elution of the Elution Buffer 2 is finished.
d. The column was washed with 0.5mL of Elution Buffer 3, and the protein bound to the substrate was washed off. Gravity flow, the effluent was collected with 1.5mL endotoxin-free EP tubes that were kept cold on an ice-box. After the Elution is finished, the Elution is carried out by using G250 (100 mu L G is taken in a 96-well plate, 10 mu L of the eluent which is dripping out is added), if the G250 turns blue, the Elution is continued until the G250 does not turn blue, and the Elution of the Elution Buffer 3 is finished.
e. Add 0.5mL of Elution Buffer 4 column wash, wash down the matrix-bound protein. Gravity flow, the effluent was collected in 1.5mL EP tube without endotoxin, the collection tube was kept cold on an ice box. After the Elution was completed, the Elution was carried out with G250 (100. Mu. L G was placed in a 96-well plate, and 10. Mu.L of the eluting solution that was being dripped was added), and if G250 changed to blue, the Elution was continued until G250 did not change to blue, and the Elution with Elution Buffer 4 was completed.
f. The flow-through collected above was subjected to SDS-PAGE using an eluent (Elution Buffer), typically 6 samples, i.e.20. Mu.L protein + 5. Mu.L 6 Loading Buffer.
The formulation of the above-mentioned Buffer (Binding Buffer) is specifically as follows:
| buffer name | Composition (I) |
| Binding Buffer | 20mM Tris-HCL,250mM NaCl,10mM Imidazole,10％glycero pH 8.0 |
| |
20mM Tris-HCL,250mM NaCl,40mM Imidazole,10％glycero pH 8.0 |
| |
20mM Tris-HCL,250mM NaCl,80mM Imidazole,10％glycero pH 8.0 |
| |
20mM Tris-HCL,250mM NaCl,250mM Imidazole,10％glycero pH 8.0 |
| |
20mM Tris-HCL,250mM NaCl,500mM Imidazole,10％glycero pH 8.0 |
According to mFC-6His label carried by plasmid 3, selecting to respectively carry out His label purification on the harvested supernatant, carrying out gradient Elution by using Imidazole (Imidazole) with different concentrations, carrying out electrophoresis on BSA, marker (MK), supernatant (supernatant), purified flow-through liquid (FT), wash Buffer (W) in the purification process and Elution Buffer with different gradients, and judging the amount of protein in the eluate, wherein the result is shown in figure 7.
From the gel chart of FIG. 7, it can be seen that TMEM176B protein in the form of plasmid 3 secretion is eluted at a concentration of 80mM and 250mM imidazole, a small amount of protein is eluted at 40mM imidazole, and the eluates are collected in several gradients of 80-1, 80-2, 80-3, 250-1 and 250-2, and the protein concentration is determined to be 110mg/1000mL Cells.
In conclusion, the invention selects a specific sequence (142-196 aa) of the TMEM176B protein, designs the combination and matching of 2 signal peptides and 2 labels for the TMEM176B protein, finally realizes the secretory expression of the TMEM176B protein under the coexistence condition of the signal peptide 2 (mouse IgG signal peptide) and mFC-6His, successfully obtains the TMEM176B recombinant protein through WB verification and affinity purification, has the expression amount of about 110mg/1000mL Cells, and can optimize the transfection expression process subsequently so as to realize higher expression amount. The obtained TMEM176B recombinant protein with high concentration and purity can be used for research of specific antibodies, such as development of functional antibodies against the TMEM176B protein.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The transmembrane protein 176B recombinant vector is characterized by comprising an expression vector and an insertion fragment, wherein the insertion fragment comprises a gene sequence for coding an IgG signal peptide, a gene sequence for coding the 142 th to the 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding a mFC-6His protein label which are sequentially connected; wherein the amino acid sequence of the 142 th to 196 th amino acids of the transmembrane protein 176B is shown as SEQ ID NO:4, respectively.
2. The transmembrane protein 176B recombinant vector according to claim 1, wherein the gene sequence encoding the 142 th to 196 th amino acids of the transmembrane protein 176B is as shown in SEQ ID NO:5, respectively.
3. The transmembrane protein 176B recombinant vector of claim 1, wherein the amino acid sequence of said IgG signal peptide is as set forth in SEQ ID NO:2, and the gene sequence of the IgG signal peptide is shown as SEQ ID NO:3, respectively.
4. The transmembrane protein 176B recombinant vector according to claim 1, wherein the mFC-6His protein tag has an amino acid sequence as set forth in SEQ ID NO:6, and the gene sequence for coding the mFC-6His protein tag is shown as SEQ ID NO: shown at 7.
5. The transmembrane protein 176B recombinant vector of claim 1, wherein the expression vector comprises pcdna3.4.
6. A host cell comprising the transmembrane protein 176B recombinant vector of any one of claims 1-5, wherein the host cell comprises a HEK293F cell.
7. A method for secretory expression of transmembrane protein 176B, comprising the steps of:
s1, transfecting a host cell with the transmembrane protein 176B recombinant vector of any one of claims 1-5;
s2, culturing the transfected host cells, expressing the protein by using a eukaryotic expression system, collecting culture supernatant, and purifying to obtain the transmembrane protein 176B in a secretory form.
8. The method for secretory expression of transmembrane protein 176B according to claim 7, wherein the step S1 of constructing the transmembrane protein 176B recombinant vector comprises: connecting a gene sequence for coding an IgG signal peptide, a gene sequence for coding 142 th to 196 th amino acids of the transmembrane protein 176B and a gene sequence for coding mFC-6his protein labels to an expression vector pCDNA3.4, then transforming the expression vector into an escherichia coli DH5 alpha competent cell, carrying out positive clone screening, and obtaining the transmembrane protein 176B recombinant vector through sequencing verification.
9. The method for secretory expression of transmembrane protein 176B according to claim 7, wherein in step S1, the transmembrane protein 176B recombinant vector is transfected into a host cell comprising HEK293F cell using transient transfection technique.
10. A secreted protein produced by the method for secretory expression of the transmembrane protein 176B according to any one of claims 8 to 9, wherein the secreted protein comprises amino acids 142 to 196 of the transmembrane protein 176B and a mFC-6his protein tag, and the mFC-6his protein tag is positioned immediately C-terminal to amino acids 142 to 196 of the transmembrane protein 176B.
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| JP2012100629A (en) * | 2010-11-12 | 2012-05-31 | Dainippon Sumitomo Pharma Co Ltd | Tumor angiogenesis inhibitor |
| CN114262718A (en) * | 2021-12-28 | 2022-04-01 | 武汉爱博泰克生物科技有限公司 | Cell secretion expression method and application of human Ly6d recombinant protein |
| CN115052617A (en) * | 2019-11-07 | 2022-09-13 | 淀粉样蛋白解决方案株式会社 | A composition for preventing or treating degenerative brain disease comprising TMEM176B, its expression or activity regulator as active ingredient |
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| JP2012100629A (en) * | 2010-11-12 | 2012-05-31 | Dainippon Sumitomo Pharma Co Ltd | Tumor angiogenesis inhibitor |
| CN115052617A (en) * | 2019-11-07 | 2022-09-13 | 淀粉样蛋白解决方案株式会社 | A composition for preventing or treating degenerative brain disease comprising TMEM176B, its expression or activity regulator as active ingredient |
| CN114262718A (en) * | 2021-12-28 | 2022-04-01 | 武汉爱博泰克生物科技有限公司 | Cell secretion expression method and application of human Ly6d recombinant protein |
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