CN113336839A

CN113336839A - Method for expressing human fibroblast growth factor 21 through wheat germ cell-free protein synthesis system

Info

Publication number: CN113336839A
Application number: CN202110784254.7A
Authority: CN
Inventors: 李力; 马森; 李正哲; 李利民; 韩小贤; 鲍庆丹; 王凤成; 王晓曦; 孙冰华
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2021-09-03

Abstract

The invention establishes a wheat germ cell-free synthesis system for efficiently and quickly expressing human fibroblast growth factor 21 recombinant protein in a laboratory. The invention mainly uses wheat germ extract liquid which is cultured in a laboratory and is prepared by stripping wheat germ as a raw material to construct a wheat germ cell-free protein synthesis system, and uses mRNA which is transcribed in vitro from escherichia coli plasmid containing a human fibroblast growth factor 21 gene segment as a template to express human fibroblast growth factor 21 recombinant protein in vitro. The synthesized recombinant protein has a correct structure, the purification step is simple, the yield is suitable for subsequent experiments, and the overall operation of the synthesis method is simple, thereby laying a foundation for the next research on the human fibroblast growth factor 21.

Description

Method for expressing human fibroblast growth factor 21 through wheat germ cell-free protein synthesis system

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for establishing a wheat germ cell-free synthesis system for efficiently and quickly expressing human fibroblast growth factor 21 recombinant protein in a laboratory.

Background

FGF21 is a new member of the fibroblast growth factor family discovered in 2000 by Nishimura, human FGF21 has 209 amino acids and is 75% homologous to mouse FGF 21. Research shows that FGF21 is mainly secreted by liver and is also expressed in skeletal muscle, pancreas, thymus and cardiac muscle cells. FGF21 can activate glucose metabolism in fat cells, and the glucose metabolism process is independent of insulin, so that it is expected to become a new auxiliary medicine for treating diabetes. Meanwhile, correlation studies show that FGF21 plays an important regulatory role in the occurrence process of cardiovascular diseases and is a novel biological marker for predicting the risk of cardiovascular diseases. At present, an effective preparation means of FGF21 protein is lacked in the research, and the preparation of the natural FGF21 protein is mainly extracted from serum, so that the time and the labor are wasted, and the yield is low; the preparation of FGF21 recombinant protein mainly utilizes escherichia coli engineering bacteria as an expression vector for secretory expression, an expression process is easy to generate inclusion bodies in escherichia coli cells, the recombinant protein needs to be renatured to show a correct structure, and the expressed recombinant protein is complex in purification operation and has certain operation difficulty.

The cell-free protein expression system is a system for expressing protein in vitro by using exogenous DNA or mRNA as a template and supplementing substrate and energy substances into the system, organelles provided by cell extracts and combinations of various enzymes. The cell-free protein expression system does not need complete living cells, the constructed system can spontaneously complete the protein synthesis work in vitro, reduces the dependence on cells, and can be used for expressing and synthesizing proteins with cytotoxicity. Without the obstruction of the cell membrane of the living cell, the regulation substance can be freely added into the reaction system to regulate and promote the folding and modification of the protein. The sources of all substances in the cell-free protein expression system are clear, and the subsequent purification operation of protein synthesis is facilitated.

Disclosure of Invention

The invention aims to solve the problems of low yield, difficulty in purification, complex operation and the like of the preparation method for researching FGF21 at the present stage, exert the advantages of a wheat germ cell-free protein synthesis system for expressing recombinant protein, provide a method for expressing FGF21 recombinant protein by using the wheat germ cell-free protein synthesis system, provide a method for laboratory rapid synthesis of FGF21 and provide experimental materials for research of FGF 21.

Another object of the present invention is to provide a method for preparing an extract from wheat germ in the above system for synthesizing a cell-free protein from wheat germ.

The technical scheme adopted by the invention for realizing the aim comprises the following operation steps:

(1) introducing a pET28a-FGF21 plasmid containing a nucleotide sequence shown in Seq ID No.1 into a DH5 alpha escherichia coli competent cell to form a cloning vector, wherein the plasmid is provided with a T7 promoter-terminator system which can be used for subsequent mRNA in-vitro transcription operation, a His tag which can be used for subsequent recombinant protein purification operation, and a kanamycin resistance gene which can be used for resistance screening;

(2) cloning of FGF21 gene: screening an escherichia coli cloning vector into which a pET28a-FGF21 plasmid is successfully introduced by using an LB (lysogeny broth) resistance culture medium containing kanamycin (the final concentration of kanamycin is 50 mu g/mL), and extracting the plasmid after propagation culture to obtain plasmid DNA containing an FGF21 gene fragment;

(3) in vitro transcription of template mRNA: using the plasmid extracted in the step (2) as a template, designing a pair of primers for PCR amplification, and obtaining a large amount of linear fragments containing a T7 promoter-FGF 21 gene-T7 terminatorDNA. Using RiboMAX as a template for the linear DNA^TMPerforming mRNA in-vitro transcription operation by using a Large Scale RNA Production System-T7 kit (product number P1300 of PROMEGA biotech company in America), performing DNase digestion on a product after transcription is completed to purify mRNA, then diluting the transcription product by 100-300 times, measuring the absorbance value at 260 nm and calculating the mRNA content;

(4) preparing the wheat germ extract: the preparation of the extraction buffer comprises: the pH was adjusted to 7.6 with 2 mM calcium chloride, 4 mM dithiothreitol, 100 mM potassium acetate, 5mM magnesium acetate, 40 mM HEPES-KOH. Soaking and culturing a batch of wheat grains in a culture dish to a germinating state, manually stripping wheat germs, washing residual endosperm, dipping in dry water, adding liquid nitrogen to freeze and grind the wheat germs into powder, adding an extraction buffer solution into the powder, carrying out ultrasonic treatment, ultracentrifuging for 30 min at 26000 g for 30 min, taking supernatant, measuring the absorbance value at 260 nm to be 0.2-0.25, and freezing and storing the supernatant for later use;

(5) construction of wheat germ cell-free protein synthesis system and expression of FGF21 recombinant protein: and (3) uniformly mixing 20 mu L of the template mRNA in the step (3), 30 mu L of the wheat germ extraction liquid in the step (4), 747 mu L of the translation buffer solution and 3 mu L of the enzyme mixed solution to form a 800 mu L reaction system, and reacting at 37 ℃ for 24 hours to obtain the FGF21 recombinant protein. The translation buffer comprises: 25mM HEPES-KOH, 100 mM potassium acetate, 2.7 mM magnesium acetate, 5mM dithiothreitol, 16 mM creatine phosphate, 1.0 mM ATP, 0.25 mM MTTP, 0.4 mM spermidine, and 0.4 mM of a mixed solution of 20 amino acids (Beijing Soilebao Tech Co., Ltd.). The enzyme mixed solution contained 1. mu.L of creatine kinase, 1. mu.L of protease inhibitor and 1. mu.L of ribonuclease inhibitor;

(6) and (3) purifying the recombinant protein: purifying with His-tagged protein nickel ion chelating kit (Shanghai Biyuntian biotechnology Co., Ltd., product number P2226);

(7) identification of recombinant proteins: identifying the recombinant protein purified in the step (6) by using a human FGF21 ELISA detection kit (product number YX-060706H of Shanghai Hepeng Biotech Co., Ltd.), wherein if the ELISA reaction is positive, the FGF21 protein synthesized by the wheat germ cell-free protein synthesis system introduced by the invention has a correct structure;

(8) calculation of the yield of recombinant protein: determining the recombinant protein obtained in the step (6) by using the human FGF21 ELISA detection kit according to a standard curve drawn by the human FGF21 ELISA detection kit to obtain OD₄₅₀Substituting the absorbance value into a standard curve to calculate the synthetic yield of the FGF21 recombinant protein.

The invention has the following advantages:

(1) the time from the completion of the construction of the wheat germ cell-free protein synthesis system to the synthesis of the FGF21 recombinant protein is no longer than 2 days, which is far less than the existing preparation method of FGF21 protein;

(2) the cloning vector used in the invention has a His label and can be purified by a nickel ion metal chelating affinity column chromatography method, the purification operation is simple, the synthesized FGF21 recombinant protein has a correct structure, and the non-inclusion body does not need to carry out operations such as protein renaturation and the like;

(3) the final yield of the FGF21 recombinant protein synthesized by the method reaches 75.41 ng/mL, and the yield is far higher than that of a traditional serum extraction method, so that the subsequent research work on FGF21 is facilitated.

Drawings

FIG. 1 is a map of the cloning plasmid pET28a-FGF21 used in the present invention.

FIG. 2 shows a linear DNA of plasmid DNA after single digestion, and its length was verified by electrophoresis on a 1% agarose gel. In the figure, lane 1 is a DNA marker,

lanes

2 and 3 are samples, and lane 4 is a negative control.

FIG. 3 is an agarose gel electrophoresis of the amplified product after PCR amplification using the extracted plasmid DNA as a template before mRNA transcription. In the figure, lane 1 is a DNA marker, lanes 2 to 5 are PCR amplification products, and lane 6 is a negative control.

Fig. 4 is a comparison graph of germination states of wheat grains cultured in a laboratory, wherein the number 1 is that the wheat grains are not germinated and are still in a dormant state, the numbers 2-4 are that the wheat grains in the germination state can be used for preparing a wheat embryo extract, and the number 5 is that the wheat grains which have already started to germinate cannot be used.

FIG. 5 is a standard curve of the ELISA detection kit for human FGF 21.

Detailed Description

Example 1

Step (1), pET28a-FGF21 plasmid containing nucleotide sequence shown in Seq ID No.1 is introduced into DH5 alpha colibacillus competent cells to form a cloning vector, the plasmid has a T7 promoter-terminator system which can be used for subsequent mRNA in vitro transcription operation, a His tag which can be used for subsequent recombinant protein purification operation, a kanamycin resistance gene which can be used for resistance screening, and the DH5 alpha colibacillus competent cells can be prepared by a chemical method or an electrical stimulation method.

Step (2) cloning of FGF21 Gene: an Escherichia coli cloning vector into which pET28a-FGF21 plasmid was successfully introduced was selected using LB resistance medium containing kanamycin (final concentration of kanamycin is 50. mu.g/mL), plasmid DNA containing FGF21 gene fragment was extracted after propagation culture, and the extracted plasmid DNA was subjected to single-restriction with BglII restriction endonuclease and analyzed by electrophoresis on 1% agarose gel, as shown in FIG. 2, wherein lane 1 is DNA marker,

lanes

2 and 3 are samples having a length of about 6000bp corresponding to the length of pET28a-FGF21, and lane 4 is negative control. Screening an escherichia coli cloning vector into which a pET28a-FGF21 plasmid is successfully introduced by using an LB (lysogeny broth) resistance culture medium containing kanamycin (the final concentration of kanamycin is 50 mu g/mL), and extracting the plasmid after propagation culture to obtain plasmid DNA containing an FGF21 gene fragment; .

Step (3) in vitro transcription of template mRNA: and (3) designing a pair of primers to carry out PCR amplification by taking the plasmid extracted in the step (2) as a template to obtain a large amount of linear DNA containing a T7 promoter-FGF 21 gene-T7 terminator fragment. Using RiboMAX as a template for the linear DNA^TMThe Large Scale RNA Production System-T7 kit (product number P1300 from PROMEGA Biotechnology corporation, USA) performs in vitro transcription of mRNA, after transcription is completed, DNase digestion is performed on the product to purify mRNA, then the transcription product is diluted 100 to 300 times, the absorbance value at 260 nm is measured, and the mRNA content is calculated.

A pair of primers was designed as follows:

a forward primer: 5'-CACCATACCCACGCCGAAAC-3'

Reverse primer: 5'-CGAGAAAGGAAGGGAAGAAAGC-3'

The PCR amplification procedure adopts pre-denaturation at 94 ℃ for 5 min, then denaturation at 94 ℃ for 1 min, annealing at 59 ℃ for 1 min, extension at 72 ℃ for 1.5 min, and finally extension at 72 ℃ for 10 min to complete amplification. Then, mRNA in vitro transcription operation is performed using the amplification product as a template. The intent of this step is to expand the template linear DNA concentration to transcribe higher concentrations of mRNA. Before the subsequent transcription operation, the PCR amplification product needs to be analyzed by 1% agarose gel electrophoresis, and whether the length of the PCR amplification product meets the design expectation is judged, as shown in FIG. 3, lane 1 in the figure is a DNA marker, lanes 2-5 are the PCR amplification product, the length of which is about 1500bp and meets the expected result of primer design, and lane 6 is a negative control.

(9) Step (4), preparation of the wheat germ extract: the preparation of the extraction buffer comprises: the pH was adjusted to 7.6 with 2 mM calcium chloride, 4 mM dithiothreitol, 100 mM potassium acetate, 5mM magnesium acetate, 40 mM HEPES-KOH. Soaking and culturing a batch of wheat grains in a culture dish to a germinating state, and carefully stripping the wheat germs by using a sterile scalpel when preparing the wheat germ extract, wherein the wheat germ extract does not contain endosperm as much as possible. Collecting wheat germ, washing off residual endosperm in clean gauze, and drying moisture on the surface of wheat germ with absorbent paper. Freezing wheat germ with liquid nitrogen, grinding into powder, and adding extraction buffer. Treating with 30% ultrasonic power, centrifuging at 26000 g for 30 min to obtain supernatant, freeze preserving wheat germ extractive solution at-20 deg.C, determining absorbance value at 260 nm to be 0.2-0.25, and freeze preserving the supernatant; the culture state of wheat grains is shown in figure 4.

Step (5), construction of a wheat germ cell-free protein synthesis system and expression of FGF21 recombinant protein: and (3) uniformly mixing 20 mu L of the template mRNA in the step (3), 30 mu L of the wheat germ extraction liquid in the step (4), 747 mu L of the translation buffer solution and 3 mu L of the enzyme mixed solution to form a 800 mu L reaction system, and reacting at 37 ℃ for 24 hours to obtain the FGF21 recombinant protein. The translation buffer comprises: 25mM HEPES-KOH, 100 mM potassium acetate, 2.7 mM magnesium acetate, 5mM dithiothreitol, 16 mM creatine phosphate, 1.0 mM ATP, 0.25 mM MTTP, 0.4 mM spermidine, and 0.4 mM of a mixed solution of 20 amino acids (Beijing Soilebao Tech Co., Ltd.). The enzyme mixed solution contained 1. mu.L of creatine kinase, 1. mu.L of protease inhibitor and 1. mu.L of ribonuclease inhibitor.

And (6) purifying the recombinant protein: purification was performed using a His-tag protein purification nickel ion chelating kit (Shanghai Biyuntian Biotechnology Co., Ltd., product No. P2226). As shown in the attached figure 1, the pET28a-FGF21 plasmid has a 6 XHis tag at the N-terminal, histidine has strong affinity with nickel ions and can be used for purifying recombinant proteins, and the protein purification time can be greatly shortened by using a His-tag protein purification kit.

Step (7) identification of recombinant protein: the recombinant protein purified in the step (6) is identified by using a human FGF21 ELISA detection kit (product number YX-060706H of Shanghai Hepeng Biotech Co., Ltd.), and the ELISA reaction is positive, which indicates that the FGF21 protein synthesized by the wheat germ cell-free protein synthesis system introduced by the invention has a correct structure;

calculating the yield of the recombinant protein in the step (8): determining the recombinant protein obtained in the step (6) by using the human FGF21 ELISA detection kit according to a standard curve drawn by the human FGF21 ELISA detection kit to obtain OD₄₅₀Substituting the absorbance value into a standard curve to calculate the synthetic yield of the FGF21 recombinant protein to be 75.41 ng/mL.

The human FGF21 ELISA detection kit used in the steps (7) and (8) adopts a double-antibody one-step sandwich method enzyme-linked immunosorbent assay, and can complete the identification of the recombinant protein and the calculation of the yield at the same time.

Although the invention has been described in detail in the foregoing text with reference to specific embodiments and examples, it will be apparent to one skilled in the art that there is room for improvement in the present invention and that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention and, therefore, the scope of the invention is to be determined by the appended claims.

SEQUENCE LISTING

<110> industrial university of Henan

<120> a method for expressing human fibroblast growth factor 21 by a wheat germ cell-free protein synthesis system

<130> 2021.7.1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 630

<212> DNA

<213> Artificial Synthesis

<400> 1

atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60

cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120

gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180

ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240

ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300

ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360

tgcagcttcc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420

ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480

ccagctcgct tcctgccact accaggcctg ccccccgcac tcccggagcc acccggaatc 540

ctggcccccc agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 600

cagggccgaa gccccagcta cgcttcctga 630

Claims

1. A method for expressing human fibroblast growth factor 21 via a wheat germ cell-free protein synthesis system, comprising: 1) constructing a cloning plasmid which takes Escherichia coli pET28a as a vector and contains an FGF21 gene, wherein the nucleotide sequence of FGF21 is shown as Seq ID No. 1; 2) transcribing by an mRNA in-vitro transcription kit to obtain an mRNA template; 3) and (3) constructing a wheat germ cell-free protein synthesis system, and adding an mRNA template into the wheat germ cell-free protein synthesis system to express the FGF21 recombinant protein.

2. The method of claim 1, wherein the cloning plasmid in step 1) is constructed by introducing pET28a-FGF21 plasmid containing the nucleotide sequence shown in Seq ID No.1 into DH 5. alpha. E.coli competent cells to construct a cloning vector, selecting the E.coli cloning vector into which pET28a-FGF21 plasmid has been successfully introduced using LB resistance medium containing kanamycin, and extracting the plasmid after propagation culture to obtain a cloning plasmid containing FGF21 gene fragment.

3. The method of claim 1, wherein the in vitro transcription method of the middle mRNA in the step 2) is: carrying out PCR amplification by taking the plasmid extracted in the step 1) as a template to obtain a large amount of linear DNA containing a T7 promoter-FGF 21 gene-T7 terminator fragment, carrying out mRNA in-vitro transcription operation by taking the linear DNA as the template through a T7 mRNA in-vitro transcription kit, carrying out DNase digestion on a product after the transcription is finished to purify the mRNA, and then diluting a transcription product by 100-300 times.

4. The method of claim 3, wherein the primers for PCR amplification are as follows:

a forward primer: 5'-CACCATACCCACGCCGAAAC-3'

Reverse primer: 5'-CGAGAAAGGAAGGGAAGAAAGC-3'

The PCR amplification procedure adopts pre-denaturation at 94 ℃ for 5 min, then denaturation at 94 ℃ for 1 min, annealing at 59 ℃ for 1 min, extension at 72 ℃ for 1.5 min, and finally extension at 72 ℃ for 10 min to complete amplification.

5. The method as claimed in claim 1, wherein the step 3) comprises mixing 20 μ L of template mRNA, 30 μ L of wheat germ extract, 747 μ L of translation buffer solution and 3 μ L of enzyme mixed solution uniformly to form 800 μ L of reaction system, and reacting at 37 ℃ for 24 h to obtain FGF21 recombinant protein.

6. The method of claim 5, wherein the translation buffer comprises: HEPES-KOH, potassium acetate, magnesium 2 acetate, dithiothreitol, creatine phosphate, ATP, GTP, spermidine, and an amino acid mixed solution.

7. The method according to claim 5, wherein the enzyme mixture solution contains 1 μ L of creatine kinase, 1 μ L of protease inhibitor and 1 μ L of ribonuclease inhibitor.

8. The method of claim 5, wherein the FGF21 recombinant protein is purified using a His-tag protein purification kit.

9. A preparation method of wheat germ extraction liquid is characterized by comprising the following steps: soaking wheat grains to be in a germinating state, stripping wheat germs, washing residual endosperm, dipping water, adding liquid nitrogen to freeze and grind the wheat germs into powder, adding an extraction buffer solution into the powder, carrying out ultrasonic treatment, carrying out ultracentrifugation for 30 min at 26000 g, taking supernatant, keeping the absorbance value of the supernatant at 260 nm of 0.2-0.25, and freezing and storing the supernatant for later use.

10. The method of claim 9, wherein the preparing extraction buffer comprises calcium chloride, dithiothreitol, potassium acetate, magnesium acetate, HEPES-KOH, and adjusting the pH to 7.6.