CN112646837A

CN112646837A - Recombinant human adiponectin expression vector, vector construction method and expression method

Info

Publication number: CN112646837A
Application number: CN201910966965.9A
Authority: CN
Inventors: 龚燕平; 李春霖; 刘敏燕; 卢艳慧; 闫双通; 孙般若; 李婷
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-04-13

Abstract

The invention relates to the technical field of biology, in particular to a recombinant human adiponectin expression vector, a vector construction method and an expression method. The recombinant human adiponectin expression vector comprises a nucleotide sequence shown in SEQ ID No.1 and an Fc-GS plasmid. The construction method of the recombinant human adiponectin expression vector comprises the following steps: obtaining a cDNA sequence of the human adiponectin through codon optimization according to the amino acid sequence of the human adiponectin; the cDNA sequence of the human adiponectin is shown as SEQ ID No. 1; designing a primer, amplifying the cDNA sequence, and recovering a target fragment after identification; and connecting the target fragment to an Fc-GS plasmid to obtain a recombinant human adiponectin expression vector. The recombinant human adiponectin expression vector is transfected into hamster ovary cells, recombinant human adiponectin-Fc fusion protein can be stably expressed, and the method is simple and efficient.

Description

Recombinant human adiponectin expression vector, vector construction method and expression method

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant human adiponectin expression vector, a vector construction method and an expression method.

Background

Adiponectin (ADIP) is an adipocyte-specific expression adipocytokine secreted into plasma, with a monomer molecular weight of about 30kD, and is named Acrp 30. Under normal conditions, the expression level of adiponectin in the body is stable, the concentration is about 3-30 mug/mL, and the adiponectin accounts for 0.01 percent of the total protein content in plasma.

Adiponectin is believed to cause RNA to be expressed 100-fold more tissue-specifically during adipocyte differentiation than other tissues. The human adiponectin gene is located on chromosome 3q27, has a full length of 16Kbp, consists of three exons and two introns, comprises 244 amino acids, and is divided according to functions from an amino terminal to a carboxyl terminal: a signal peptide domain (1-18AA), an adiponectin specific domain (19-41AA), a collagen repeat domain (42-107AA) and a globular domain sequence (108-244 AA). The signal peptide itself is cleaved off during secretion, the adiponectin specific domain sequence has no significant homology to most protein sequences, while the adiponectin globular domain sequence has significant homology to many proteins. At the beginning of the collagen repeating structural domain, 7 complete 'Gly-X-Pro' repeating units are arranged in an aggregation way; in the terminal sequence of the collagen repeat domain, there are 22 incomplete "Gly-X-Y" repeat units scattered in a distribution, forming a "stalk" of the collagen structure. In human plasma, there are two major classes of adiponectin: monomeric forms of adiponectin and multimeric forms of adiponectin. The monomeric adiponectin is divided into two types, namely full-length adiponectin and adiponectin which is obtained by hydrolyzing full-length adiponectin by protease and only has a portion with a globular domain of adiponectin, and is called globular adiponectin. The dimeric adiponectin refers primarily to the trimeric form of adiponectin, with minor amounts of hexamer and higher molecular weight dimeric adiponectin also present.

Adiponectin can regulate glucose and lipid metabolism in liver, muscle, and fat; can directly act on heart, vascular cell and macrophage to exert cardioprotective and anti-inflammatory effects; can improve the survival rate of pancreatic beta cells and participate in resisting malignant tumors caused by obesity and insulin resistance; is closely related to immune status; recent studies have shown that adiponectin may also have some effects on osteoarthritis and bone metabolism, and thus, studies on adiponectin are of great significance.

However, there are several problems with the current studies on adiponectin: 1. adiponectin in its various forms and polymers may differ in its role; 2. the assessment of adiponectin by different investigators is not consistent. Therefore, it is important to develop a large amount of stably expressed recombinant human adiponectin proteins.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provided are a recombinant human adiponectin expression vector, a vector construction method and an expression method, wherein the constructed expression vector stably expresses recombinant human adiponectin-Fc fusion protein in hamster ovary cells, and the method is simple and efficient.

The invention provides a recombinant human adiponectin expression vector which comprises a nucleotide sequence shown in SEQ ID No.1 and Fc-GS plasmid.

The invention provides a construction method of a recombinant human adiponectin expression vector, which comprises the following steps:

step 1: obtaining a cDNA sequence of the human adiponectin through codon optimization according to the amino acid sequence of the human adiponectin; the cDNA sequence of the human adiponectin is shown as SEQ ID No. 1;

step 2: designing a primer, amplifying the cDNA sequence, and recovering a target fragment after identification;

and step 3: and connecting the target fragment to an Fc-GS plasmid to obtain a recombinant human adiponectin expression vector.

Preferably, the step 3 specifically comprises:

digesting the Fc-GS plasmid by using a first restriction enzyme, and then treating the Fc-GS plasmid for 20-40 minutes by using heat-sensitive phosphatase to inactivate the heat-sensitive phosphatase;

digesting the target fragment by using a second restriction enzyme; the first restriction enzyme and the second restriction enzyme are the same or are isocaudarner;

and connecting the Fc-GS plasmid subjected to restriction enzyme digestion and the target fragment overnight to obtain the recombinant human adiponectin expression vector.

Preferably, the first restriction enzyme is Bgl II and the second restriction enzyme is BamHI.

Preferably, after the step 3, the method further comprises:

digesting the recombinant human adiponectin expression vector by using Hind III and Xho I restriction endonucleases, and then carrying out agarose gel electrophoresis to identify the connection condition of target fragments;

and performing bidirectional sequencing on the recombinant human adiponectin expression vector to identify the inserted target fragment sequence.

The invention provides application of the recombinant human adiponectin expression vector in expressing human adiponectin-Fc fusion protein.

The invention provides an expression method of recombinant human adiponectin, which comprises the following steps:

step 1: transfecting the recombinant human adiponectin expression vector into hamster ovary cells by an electroporation transfection method;

step 2: screening hamster ovary cells transfected with the recombinant human adiponectin expression vector by using a MAX-containing culture medium;

and step 3: culturing the positive clone obtained after screening, extracting and separating protein to obtain the human adiponectin-Fc fusion protein.

Preferably, the hamster ovary cells have the endogenous glutamine synthetase gene knocked out of the cells by the CRISPR technique.

Preferably, the step 1 specifically comprises:

will be passedThe hamster ovary cells with endogenous glutamine synthetase gene knocked out by CRISPR technology are suspended in phosphate buffer to obtain cell suspension with the density of 1 × 10⁷～1×10⁸Cells/ml;

mixing the cell suspension with 20-30 μ g of the recombinant human adiponectin expression vector of claim 1, and performing electric shock.

Preferably, in step 3, after the protein is extracted and separated, the protein is identified and analyzed for concentration.

Compared with the prior art, the recombinant human adiponectin expression vector comprises the nucleotide sequence shown in SEQ ID No.1 and Fc-GS plasmid. The recombinant human adiponectin expression vector can transfect hamster ovary cells through electroporation, particularly positive clones can be screened out through a Glutamine Synthetase (GS) screening system in one turn, expression of human adiponectin-Fc fusion protein is rapidly and efficiently realized, and the recombinant human adiponectin expression vector is high in repeatability and good in universality. Experiments prove that the concentration of the fusion protein obtained by the method is at least 2mg/100ml, which is obviously higher than the concentration (0.2-1.35 mg/100ml) of the protein obtained by the existing eukaryotic cell cloning experiments.

The recombinant human adiponectin-Fc fusion protein is stably expressed in the CHO cell strain, a large amount of high-purity adiponectin protein can be prepared at one time, the method is simple and easy to implement, and the problems of complexity of biological amplification protein and corresponding high price are solved to a certain extent.

Drawings

FIG. 1 is an Fc-GS plasmid map;

FIG. 2 is a diagram showing the results of agarose gel electrophoresis after PCR amplification of cDNA;

FIG. 3 is a diagram showing the results of agarose gel electrophoresis after double digestion of ADIP-FC-GS plasmid;

FIG. 4 shows the sequencing results of the ADIP-FC-GS plasmid;

FIG. 5 is a graph showing SDS-PAGE electrophoresis results of proteins produced after transfection of hamster ovary cells with the ADIP-FC-GS plasmid;

FIG. 6 is a standard curve diagram of protein concentration for enzyme-linked immunoassay.

Detailed Description

For a further understanding of the invention, alternative embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are included merely to further illustrate the features and advantages of the invention, and are not intended to limit the invention.

The embodiment of the invention discloses a recombinant human adiponectin expression vector, which comprises a nucleotide sequence shown in SEQ ID No.1 and Fc-GS plasmid.

The Fc-GS plasmid map is shown in FIG. 1.

The embodiment of the invention discloses a construction method of a recombinant human adiponectin expression vector, which comprises the following steps:

the amino acid sequence of HUMAN adiponectin was obtained at NCBI and has the sequence number UniProtKB-Q15848(ADIPO _ HUMAN) as shown in SEQ ID No. 2. For sample purification and detection, His-tag and Flag-tag can be fused at the N terminal, and the amino acid sequence is shown as SEQ ID No.3, or the amino acid sequence after IgG-Fc fragment is fused at the C terminal, and the amino acid sequence is shown as SEQ ID No. 4.

Obtaining cDNA sequence after codon optimization.

the primers were designed as follows:

a forward primer: CTGGGATCCCAGGAGACCACAACCCAGG, respectively;

reverse primer: CGC GGATCCTCAATTGGTATCATGGTA, respectively;

the polymerase chain reaction total reaction system for amplifying the cDNA sequence is as follows: template cDNA 1. mu.L, forward primer 3. mu.L, reverse primer 3. mu.L, 5 XPrimeStar Buffer 6. mu.L, dNTPS (2.5mM each) 0.5. mu.L, Taq DNA Polymerase 0.5. mu.L, plus ddH₂O to 30. mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 25 cycles; extension at 72 ℃ for 5 min. The materials were purchased from Takara Bio, Beijing, China, Inc. (Beijing, Takara Bio)). After 10g/L agarose gel electrophoresis analysis, the PCR product is cut from about 748bp band.

The 748bp fragment is the target fragment.

Optionally, step 3 specifically includes:

digesting the Fc-GS plasmid by using a first restriction enzyme, and then treating the Fc-GS plasmid for 20-40 minutes by using heat-sensitive phosphatase to inactivate the heat-sensitive phosphatase; the inactivation temperature is preferably 80-90 ℃, and the inactivation time is preferably 20-30 min;

Optionally, the first restriction enzyme is Bgl II and the second restriction enzyme is BamHI.

The linker system of the Fc-GS plasmid and the target fragment after restriction enzyme digestion is:

6ul of the BamHI enzyme digested fragment of interest, 2ul of the treated Fc-GS plasmid, 1ul of Buffer, 1ul of T4 DNA ligase in total volume of 10ul, and ligated overnight at 16 ℃. Conventionally using CaCl₂Method TOP10 competent bacteria were prepared, then the bacteria were transformed with the ligation mixture and the broth was plated on LB plates containing 100ug/ml AMP and incubated overnight. Miniprep plasmid using the centrifugal column extraction kit as usual. The above materials were purchased from major biotechnology companies, Shanghai, China.

Optionally, after the step 3, the method further includes:

The embodiment of the invention discloses application of the recombinant human adiponectin expression vector in the technical scheme in expression of human adiponectin-Fc fusion protein.

The embodiment of the invention also discloses an expression method of the recombinant human adiponectin, which comprises the following steps:

optionally, the hamster ovary cells have the endogenous glutamine synthetase gene knocked out of the cells by CRISPR technology.

Optionally, step 1 specifically includes:

suspending hamster ovary cells (CHO-K1) with endogenous glutamine synthetase gene knocked out by CRISPR technology in phosphate buffer to obtain cell suspension with density of 1 × 10⁷～1×10⁸Cells/ml;

and mixing the cell suspension with 20-30 mu g of the recombinant human adiponectin expression vector in the technical scheme, and performing electric shock.

Knocking out the endogenous glutamine synthetase gene of the cell, avoiding the endogenous influence of the host cell, reducing the false positive rate of cloning, reducing the dosage of MSX and shortening the time of cloning. Namely: the hamster ovary cells with the knocked-out endogenous glutamine synthetase gene are used as host cells for heterologous protein expression, and have the advantages of small screening pressure, short screening period, high cloning positive rate and the like. Tests show that more than 95% of cells in the cell culture screened by the system are positive clones.

In the electroporation transfection method, the voltage is preferably 270-280 v, the time is 15-20 ms, and the total discharge time is 18-25 ms.

transfer hamster ovary cells transfected with recombinant human adiponectin expression vector to a culture dish, and add DMEM complete medium. Then, the cells were placed in a carbon dioxide incubator overnight for culture. After 2 to 3 days, the medium was replaced with a screening medium containing 50uM MSX (M5379, SIGMA, USA), and the culture was continued for about 10 to 15 days. After significant clones had formed, all cells in the dish were digested with pancreatin (C0201, bi yun day biotechnology), mixed and then used for scale-up culture to produce protein.

Specifically, positive clones obtained after screening were inoculated in a CD011 serum-free medium. The culture medium contained 1mg/L of insulin, and the concentration of carbon dioxide was 5%. Then placing the mixture on a carbon dioxide shaking bed, and carrying out shaking culture at 110-120 rpm for 10-15 days. The supernatant was collected by centrifugation. And extracting and separating protein from the supernatant by using a protein A chromatographic column.

Optionally, after the protein is extracted and separated, the protein is identified and analyzed for concentration.

Alternatively, the molecular weight of the isolated protein is identified using SDS-PAGE electrophoresis.

The molecular weight of the human adiponectin-Fc fusion protein is about 60 KD. The human adiponectin contains 226 amino acids after cutting off the secretory signal peptide and has the molecular weight of about 30 KD; the Fc protein consists of 229 amino acids and has a molecular weight of about 30KD, and the overall molecular weight of the two proteins is about 60KD after fusion.

Alternatively, the concentration of the isolated human adiponectin-Fc fusion protein is detected by enzyme-linked immunosorbent assay (ELISA).

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of recombinant human adiponectin expression vector

Synthesizing a cDNA sequence shown as SEQ ID No. 1;

and carrying out PCR amplification on the cDNA sequence by taking the cDNA sequence as a template. The polymerase chain reaction total reaction system is as follows: template cDNA 1. mu.L, forward primer 3. mu.L, reverse primer 3. mu.L, 5 XPrimeStar Buffer 6. mu.L, dNTPS (2.5mM each) 0.5. mu.L, Taq DNA Polymerase 0.5. mu.L, plus ddH₂O to 30. mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 95 deg.CDenaturation for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 25 cycles; extension at 72 ℃ for 5 min.

After 10g/L agarose gel electrophoresis analysis, the PCR product is cut from about 748bp band. Referring to FIG. 2, the electrophoresis result shows that a band is evident at 748bp, which is the target fragment. After the target fragment is recovered by agarose electrophoresis and recovered, the restriction enzyme digestion is carried out according to the following reaction system: 10ul of PCR fragment; k Buffer 2 ul; 1ul of BamHI enzyme; 7ul of water; the reaction was carried out at 37 ℃ for 2h in a total volume of 20 ul.

10ul of BglII digested Fc-GS plasmid was taken, treated with heat-sensitive phosphatase for 30min and then the phosphatase was inactivated at 80 ℃ for 20 min. 6ul of digested target fragment, 2ul of phosphatase-treated plasmid fragment, 1ul of Buffer, 1ul of T4 DNA ligase, total volume of 10ul, and ligation overnight at 16 ℃. Conventionally using CaCl₂Method TOP10 competent bacteria were prepared, then the bacteria were transformed with the ligation mixture and the broth was plated on LB plates containing 100ug/ml AMP and incubated overnight. The ADIP-FC-GS plasmid was formed.

Taking 5ul of ADIP-FC-GS plasmid, 2ul of enzyme digestion reaction buffer solution, 1ul of Hind III restriction enzyme, 1ul of Xho I restriction enzyme and 11ul of deionized water, and placing the mixture in a water bath kettle at 37 ℃ for digestion for 2 hours after uniform mixing, wherein the total volume is 20 ul.

The expected size of the gene fragment for human adiponectin-FC is approximately 1434 bp. After double digestion, agarose gel electrophoresis was performed, and the results are shown in FIG. 3. In FIG. 3, there are actually 3 samples, in which the sizes of the cleaved fragments of the 1# and 2# samples are consistent with the expected values, and the 3# sample is a negative control.

The results of the two-way sequencing showed that the inserted gene sequence was identical to the human adiponectin gene sequence, and the sequencing map is shown in FIG. 4.

Example 2 expression of recombinant human adiponectin fusion protein in hamster ovary cells

The CRISPR technique knocked out the endogenous glutamine synthetase gene of CHO-K1 cells, and CHO-K1 cells (Invitrogen) in logarithmic growth phase were taken, trypsinized, and then resuspended in PBS solution to adjust the density to about 1X 10⁷Cells/ml. The cell suspension was mixed with 20ug ADIP-FC-GS plasmid,standing on ice for 5 min. The cell suspension was transferred to a pre-cooled electroporation cuvette (165- & 2081, Biorad, 0.4ml capacity, 0.4cm gap) and the electroporator was adjusted to square wave mode with voltage 280v, time 20ms, one electroporation, total discharge time 20 ms. The cells were transferred to a 10cm dish and 10ml of DMEM complete medium was added. Then, the cells were placed in a carbon dioxide incubator overnight for culture. On day 2, the medium was changed to a selection medium containing 50uM MSX (M5379, SIGMA, USA), and the culture was continued for about 14 days. After significant clones had formed, all cells in the dish were digested with pancreatin (C0201, bi yun day biotechnology) and mixed. A portion was frozen in liquid nitrogen. The other part is used for producing the human adiponectin-FC protein by amplification culture.

Cells were seeded in 300ml of CD011 serum-free medium. The culture medium contained 1mg/L of insulin, and the concentration of carbon dioxide was 5%. Then, the cells were placed on a carbon dioxide shaker and cultured with shaking at 110rpm for 10 days. The supernatant was collected by centrifugation at 10000 rpm. A Protein A column (11-0034-94, general electric GE Healthcare, 10X 50mm) was equilibrated with 15ml PBS. The supernatant was applied to a centrifugal column for chromatography. After the sample was run, the column was washed with 15ml PBS. The protein was eluted with 5ml of glycine buffer pH 3.5 and collected stepwise at 1 ml/tube. The fractions eluted containing the protein were pooled and the buffer was changed to PBS using an ultrafiltration tube. The protein concentration was diluted to 100ug/ml with PBS solution. Filtering with 0.2um filter, and aseptically packaging.

40ul of the purified protein solution was mixed with 10ul of Load Buffer and boiled at 100 ℃ for 2 minutes. SDS-PAGE was then routinely performed. Referring to FIG. 5, M is Marker, 1 is the experimental sample, and 2 is the recombinant human adiponectin protein purchased from the outside. Electrophoresis results show that the molecular weight of the synthesized human adiponectin-Fc fusion protein is about 60KD, and is the same as the molecular weight of the recombinant human adiponectin globular protein purchased in the market.

The ELISA kit (enzyme-linked Biotechnology Co., Ltd., Shanghai, China) was used for the measurement according to the kit instructions. A log-log standard curve was plotted for the series of adiponectin concentrations.

TABLE 1 determination of OD values of samples

The system can obtain about 200ml of fermentation liquor. The OD values of each standard and diluted sample determined by ELISA are shown in table 1. The standard curve of protein concentration for ELISA assay and the fitting equation are shown in FIG. 6. Total protein concentration-concentration x dilution fold calculated from the standard curve. The total protein concentration was calculated to be 20ug/ml and the amount of protein was calculated to be 2mg/100 ml.

In the prior art, the concentration of protein obtained by eukaryotic cell cloning experiments is 0.2-1.35 mg/100ml more. Therefore, the preparation method for stably expressing the recombinant human adiponectin-Fc fusion protein in the CHO cell strain can prepare a large amount of adiponectin protein with high purity at one time, is simple and easy to implement, solves the problems of complexity and corresponding high price of biological amplification protein to a certain extent, and is a test preparation means with good prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant human adiponectin expression vector, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 1 and an Fc-GS plasmid.

2. a construction method of recombinant human adiponectin expression vector, is characterized in that, comprises the following steps:

Step 1: According to the amino acid sequence of human adiponectin, the cDNA sequence of human adiponectin is obtained through codon optimization; the cDNA sequence of human adiponectin is shown in SEQ ID No.1;

Step 2: Design primers, amplify the cDNA sequence, and recover the target fragment after identification;

Step 3: The target fragment is ligated into the Fc-GS plasmid to obtain a recombinant human adiponectin expression vector.

3. construction method according to claim 2, is characterized in that, described step 3 is specifically:

Use the first restriction enzyme to digest the Fc-GS plasmid, and then treat it with thermosensitive phosphatase for 20-40 minutes to inactivate the thermosensitive phosphatase;

Utilize the second restriction endonuclease to digest the target fragment; the first restriction endonuclease and the second restriction endonuclease are the same or are isocaudal enzymes;

The Fc-GS plasmid digested with restriction endonuclease and the target fragment were ligated overnight to obtain a recombinant human adiponectin expression vector.

4. The construction method according to claim 3, wherein the first restriction endonuclease is Bgl II, and the second restriction endonuclease is BamHI.

5. construction method according to claim 2, is characterized in that, after described step 3, also comprises:

Digest the recombinant human adiponectin expression vector with Hind III and Xho I restriction enzymes, and then perform agarose gel electrophoresis to identify the connection of the target fragment;

Bidirectional sequencing of the recombinant human adiponectin expression vector was performed to identify the inserted target fragment sequence.

6. The application of the recombinant human adiponectin expression vector of claim 1 in expressing human adiponectin-Fc fusion protein.

7. an expression method of recombinant human adiponectin, is characterized in that, comprises the following steps:

Step 1: Transfect the recombinant human adiponectin expression vector of claim 1 into hamster ovary cells by electroporation transfection method;

Step 2: Screen the hamster ovary cells transfected with the recombinant human adiponectin expression vector using the medium containing MAX;

Step 3: Culture the positive clones obtained after screening, extract and isolate the protein, and obtain a human adiponectin-Fc fusion protein.

8. The expression method according to claim 7, wherein the endogenous glutamine synthase gene of the hamster ovary cells is knocked out by CRISPR technology.

9. The expression method according to claim 8, wherein the step 1 is specifically:

The hamster ovary cells in which the endogenous glutamine synthetase gene has been knocked out by CRISPR technology were suspended in phosphate buffer to obtain a cell suspension with a density of 1×10 ⁷ to 1×10 ⁸ cells/ml;

The cell suspension is mixed with 20-30 μg of the recombinant human adiponectin expression vector according to claim 1, and electric shock is performed.

10 . The expression method according to claim 7 , wherein in the step 3, after extracting and separating the protein, the protein is identified and concentration analyzed. 11 .