CN106834324B - Recombinant expression vector capable of promoting soluble expression of protein and increasing expression quantity - Google Patents
Recombinant expression vector capable of promoting soluble expression of protein and increasing expression quantity Download PDFInfo
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Abstract
The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant expression vector capable of promoting soluble expression of protein and improving the expression level of the protein. The recombinant expression vector pET21b-HEMBP (pyr) is a prokaryotic expression vector, and is subjected to double enzyme digestion on the basis of pET21b, and is recombined and connected with a HE-MBP (pyr) -TEV sequence; the recombinant plasmid pUC57-HEMBP (pyr) -Nb is constructed and obtained by the steps of enzyme digestion, ligation, transformation, screening and the like. The recombinant expression vector can be used for expressing various proteins such as monoclonal antibody, antigen, polyprotein, c-di-GMP and c-GAMP synthetase. The application can better improve the protein expression quantity and the solubility of the protein by constructing, transforming and constructing a new recombinant expression vector, and has better practical value and popularization and application significance in gene engineering and protein engineering.
Description
Technical Field
The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant expression vector capable of promoting soluble expression of protein and improving the expression level of the protein.
Background
With the development of research in the fields of genomics, proteomics, bioinformatics and the like, recombinant DNA technology has been widely applied to the application research of various target proteins, such as the application research of biochemistry, structural biology, biotechnology and the like. As a product of the recombinant DNA technology, fusion proteins are a new class of biomolecules with multiple functional properties. The introduction of DNA recombination technology realizes the identification, modification, expression, separation and purification of target protein in various host biological systems, and the host biological systems which are applied more at present comprise: bacterial (e.coli), yeast, plant, insect and mammalian cell lines. In the host organisms, the escherichia coli has a dominant position due to the advantages of high propagation speed, low production cost, high protein yield and the like, and protein products expressed in the escherichia coli are also widely applied to industrial production, vaccine development, protein structure detection functional analysis and the like.
Coli, however, due to its limitations, such as lack of post-translational modifications to eukaryotic proteins, most foreign recombinant proteins are expressed in the form of inclusion bodies, it has been reported that 75% of human related proteins can be expressed in e.coli, but only 25% are active soluble expression (B ü ssow K, et al, Structural genes of human proteins-target selection and generation of a public availability expression complexes, micro Cell fact, 2005, 5: 4-21).
The fusion tag is generally a polypeptide or protein molecule which is stably and soluble expressed in escherichia coli, the use of the fusion tag can promote the target protein connected with the fusion tag to be correctly folded in the escherichia coli so as to improve the soluble expression and facilitate the protein purification, and in addition, the expressed target protein can also be used for identification and functional research. Fusion tags can be classified into two types according to their functions: one is a soluble tag that promotes soluble expression of recombinant proteins, such as glutathione S-transferase (GST), Maltose Binding Protein (MBP), thioredoxin A (TrxA), small ubiquitin-like modified protein (SUMO), steroid isomerase (KSI), and Trp LE; the other is an affinity tag which is convenient for recombinant protein purification, and the affinity tag comprises c-myc, HA, FLAG, 1D4, polyarginine, a streptavidin binding tag, calmodulin binding polypeptide and the like besides the polyhistidine tag (His 6) which is most applied at present. The affinity tag and the soluble tag are typically added simultaneously during the construction of the expression vector to facilitate soluble expression and subsequent purification of the recombinant protein. Since the fusion tag may interfere with the proper folding and functional activity of the target protein, a protease cleavage site needs to be designed between the fusion tag and the target protein when designing a recombinant expression vector, so that the target protein can be obtained after the fusion tag is cleaved by a corresponding protease.
The Tobacco Etch Virus (TEV) protease is one of the more currently used proteases, and the protease can specifically recognize the ENLYFQS/G amino acid sequence; no non-specific proteolysis on the target protein; TEV protease is still active under the conditions of low temperature, buffer solutions with different pH values and high ionic strength. In addition, TEV protease enzyme digestion is close to the C end of the recognition site, and the N end of the target protein is kept intact after enzyme digestion, so that the TEV protease enzyme digestion is a very good endoprotease.
The His6 tag is one of the most widely used affinity tags at present. The molecular weight of the His6 label is small, and the His6 label does not interfere the function and the structure of the protein, so the His6 label is widely applied to crystallography research. In addition, the His6 tag can also be used to purify the fused target protein by immobilized metal ion affinity chromatography. Although the His6 tag does not interfere with the function and structure of the Protein and facilitates affinity purification, 30-50% of proteins that are prokaryotic expressed fused to the His6 tag are inclusion body expression (Smyth DR, et. al., Crystal structures of fusion proteins with large-affinity tags, Protein Sci., 2003, 12(7): 1313-22). In addition, the phenomenon of radioactive accumulation in the liver of the body in the use of His 6-tagged affibodies for molecular imaging of radionuclides in vivo is significantly higher than that of affibodies not tagged with His6 (Tolmachev, et al, HEHEHEHEHEHEHE-tagged affibody may of nucleic acids purified by IMAC, is connected laboratory labeled with [ [ solution ] ]99(m)Tc(CO)3](+)，and shows improvedbiodistribution with reduced hepatic radioactivity accumulation，BioconjugChem. 2010，21(11):2013-22）。
MBP (maltose binding protein), a maltose binding protein, is a protein encoded by the E.coli malIE gene, which is one of the members of the bacterial maltose transport system. MBP protein is strong in hydrophilicity, can be fused with various proteins, can be used as a molecular chaperone to help target protein to be folded correctly, and is commonly used for soluble expression of the target protein. In 1988, MBP is used as a fusion tag to be connected with a foreign gene for the first time and expresses a large amount of soluble recombinant protein in Escherichia coli, and is also a commonly used auxiliary protein for promoting the soluble expression of heterologous recombinant protein at present. Recombinant single chain antibodies (scFvs) are clinically useful proteins for therapy. However, inclusion bodies are easily formed during the process of expressing the protein, thereby causing the protein to be insoluble and inactive. When the MBP fusion tag was added at the C-terminus of scFvs, the protein was found to be highly soluble and active, and the fusion protein was very stable. In addition, the original proteins are insoluble, and the binding of MBP tags at the N-terminal of the proteins can also increase the solubility of the proteins.
However, in general, because the structural difference of proteins is large, when different proteins are targeted, a proper tag protein needs to be selected to better improve the expression amount and the soluble expression effect, so that a new tag protein or a recombinant vector needs to be searched and designed so as to adapt to the requirements of different protein expression.
Disclosure of Invention
Aiming at the defect that most of the recombinant protein in the escherichia coli is inclusion body at present, the invention provides a recombinant expression vector pET21b-HEMBP (pyr), which can better improve the protein expression quantity and the protein solubility compared with the traditional expression vector.
The technical solution of the present application is detailed as follows.
A can promote the recombinant expression vector of soluble expression of protein and expression level, the recombinant expression vector is a prokaryotic expression vector, is named pET21b-HEMBP (pyr), the recombinant expression vector is recombined and connected with HE-MBP (pyr) -TEV sequence after carrying on NdeI and BamHI double enzyme digestion on the basis of pET21 b; wherein HE is a modified histidine tag, MBP (Maltotriose-binding protein) is a protein tag derived from thermophilic archaea (Pyrococcus furiosus), and TEV is a tobacco etch virus protease cleavage sequence; the specific construction process comprises the following steps:
(1) construction of recombinant plasmid pUC57-HEMBP (pyr) -Nb
Artificially synthesizing a gene sequence of the HEMBP (pyr) -Nb, wherein the base sequence is shown as SEQ ID NO.1, and connecting the gene sequence with a plasmid pUC57 to construct a recombinant plasmid pUC57-HEMBP (pyr) -Nb;
(2) enzyme digestion and connection, the specific process is as follows:
carrying out double enzyme digestion on the recombinant plasmid pUC57-HEMBP (pyr) -Nb in the step (1) and the plasmid pET21b respectively by NdeI and XhoI
Carrying out 1% agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product;
connecting the enzyme digestion products by using T4 DNA ligase, and connecting overnight at 16 ℃;
(3) transformation and screening, and the specific process comprises the following steps:
the ligation product in step (2) is subjected to a chemical method (CaCl)2) Transformation into Escherichia coli (E.coli) BL21 (DE 3), then spread on LB solid medium (ampicillin, 100. mu.g/mL), and cultured overnight by inversion;
selecting positive single colony, inoculating into LB liquid culture medium containing ampicillin (100. mu.g/mL) resistance, culturing at 37 deg.C and 220 rpm for 6 h, and extracting plasmid according to the instructions of the SanPrep column type plasmid DNA small extraction kit;
carrying out NdeI and BamHI double enzyme digestion identification on the extracted plasmid;
and (4) storing the correctly identified escherichia coli containing the recombinant plasmid for later use.
The recombinant expression vector pET21b-HEMBP (pyr) capable of promoting the soluble expression and the expression level of the protein is applied to the protein expression, is used for a prokaryotic expression system of escherichia coli, can promote the soluble expression of the protein and improve the expression level of the protein, and the protein specifically comprises the following components: monoclonal antibodies (Nb, PCV2VHHC 3), antigens (H5 HA10, PCV2b (cap), NLSFMD, HAFnt, FMDV 98), polyprotein (Cago 60), and c-di-GMP and c-GAMP synthetases (CdIGMP 499 (caulobacterivitides), CdIP 026 (Phenylobacterium zucinatum), Prop acid OAS), and the like.
Compared with the label sequence commonly used in the prior art, the recombinant expression vector pET21b-HEMBP (pyr) provided by the application contains an HE affinity label, an MBP (pyr) soluble label and a TEV internal cutting site, and the technical advantages are embodied in the following aspects:
(1) coli, which is the most widely used host bacterium for expressing exogenous recombinant protein at present, has certain self limitations, such as lack of post-translational modification of eukaryotic protein, and expression of most exogenous recombinant protein in the form of inclusion body, so that the solubility of the exogenous protein after expression is not high;
to overcome this drawback, a novel MBP (Maltotriose-binding protein) soluble tag derived from Pyrococcus furiosus (WP _ 011013078), which originally functions in vivo as an adenosine triphosphate conjugate transport (abctranport system) that is involved in Maltotriose, belonging to the solute-binding protein family i of bacteria, is used in the present application, unlike the conventional MBP tag; based on the novel MBP (pyr) soluble label, the solubility of the recombinant protein is promoted and improved;
(2) the His6 label is the most widely used affinity chromatography purification label at present, has the advantages of small molecular weight, capability of purifying protein under a denaturation condition, no immunogenicity, no influence on immunological analysis and the like, but still has certain defects, such as easy formation of inclusion bodies, difficult dissolution and the like of recombinant protein expressed based on the His6 label;
in order to solve the defect that the expressed recombinant protein is easy to form an inclusion body by the His6 label, the application adopts a novel HE affinity label (HEHEHEHEHEHEHE) on the premise of ensuring that the recombinant protein can still be well affinity purified; the HE tag is formed by replacing histidine at even number positions with glutamic acid (HEHEHEHE-tag) with higher hydrophilicity, and the replacement ensures that an affinity body marked by the HEHEHEHEHEHEHE-tag can better improve the soluble expression effect of the recombinant protein while the protein can be purified by immobilized metal affinity chromatography, so that the HE tag can be used for substituting a His6 tag for affinity purification of the fusion protein;
(3) in the prior art, when a prokaryotic expression system is adopted to express fusion protein, the adopted prokaryotic expression vector does not contain TEV protease enzyme cutting sites generally, so that target protein can not be obtained by cutting off a fusion tag; according to the invention, the TEV protease enzyme cutting site is integrated in the recombinant expression vector, so that the purpose of cutting the fusion tag to obtain the target protein can be conveniently realized.
In general, the recombinant expression vector can better improve the protein expression quantity and the solubility of the protein by constructing, modifying and constructing a new recombinant expression vector, and has better practical value and popularization and application significance in genetic engineering and protein engineering.
Drawings
FIG. 1 is a schematic diagram of recombinant plasmid construction, wherein A is a schematic diagram of a construction process of a recombinant expression vector pET21b-HEMBP (pyr);
b is a schematic construction flow diagram of a recombinant plasmid expression vector pET21B-HEMBP (pyr) -H5HA10, pET21B-HEMBP (pyr) -PCV2B, pET21B-HEMBP (pyr) -NLSFMD, pET21B-HEMBP (pyr) -HAFnt;
FIG. 2 is a schematic diagram showing the construction process of FIG. 1, in which C is a recombinant plasmid expression vector containing 10 genes of interest (Nb, PCV2VHHC3, H5HA10, PCV2b, HAFnt, FMDV98, Cago60, CdIGMP499 (Caulobacter viruses), CdIGMP026 (Phenylobacterium zucinum), Prop acid OAS);
d is a construction flow chart of a recombinant expression plasmid pET21b-His6-MBP (pyr) -gene (the gene represents a target gene PCV2VHHC3, H5HA10, NLSFMD, HAFnt, FMDV98, Cago60, CdiGMP499 or Prop acid OAS);
FIG. 3 shows the electrophoresis (left) and restriction enzyme identification (right) of the constructed recombinant expression plasmid pET21b-HEMBP (pyr) -Nb, in which: m is DL5000 DNA Marker; 1: pUC57-HEMBP-Nb (NdeI/XhoI, target fragment length 1635 bp); 2, pET21b original plasmid; pET21b-HEMBP-Nb (NdeI/BamHI, small fragment length 1242 bp);
FIG. 4 shows the electrophoresis (A) and the restriction enzyme identification (B1, B2) of the constructed recombinant plasmids pET21B-HEMBP (pyr) -gene and pET21B-His6 MBP-gene;
in the A picture: m is DL5000 DNA Marker; 1, pET21b-His6MBP vector; pET21b-HEMBP (pyr) -Nb (387 bp); 3, a plasmid containing FMDV98 (639 bp) gene; 4, a plasmid containing PCV2b (705 bp) gene; 5, plasmid containing CdIGMP499 (714 bp) gene; 6, plasmid containing CdIGMP026 (648 bp) gene; 7, a plasmid containing the Propacid OAS (1035 bp) gene; 8, plasmid containing NLSFMD (672 bp) gene; 9: a plasmid containing the HAFnt (951 bp) gene; 10, a plasmid containing H5HA10 (522 bp) gene; 11 plasmid containing PCV2VHHC3 (414 bp) gene; 12 Cago60 (801 bp): a plasmid of the gene;
b1 is a diagram of pET21B-HEMBP (pyr) -gene recombinant plasmid enzyme cutting identification result (NdeI/XhoI), wherein M is DL5000 DNA Marker; 1 pET21b-HEMBP (pyr) -Nb (small fragment length 1629 bp); pET21b-HEMBP (pyr) -FMDV98 (small fragment length 1881 bp); pET21b-HEMBP (pyr) -PCV2b (small fragment length 1947 bp); pET21b-HEMBP (pyr) -CdIGMP026 (small fragment length 1890 bp); 5 pET21b-HEMBP (pyr) -H5HA10 (small fragment length 1764 bp); pET21b-HEMBP (pyr) -NLSFMD (small fragment length 1914 bp); pET21b-HEMBP (pyr) -Cago60 (small fragment length 2043 bp); pET21b-HEMBP (pyr) -Propacid OAS (small fragment length 2277 bp); pET21b-HEMBP (pyr) -CdIGMP499 (small fragment length 1956 bp); pET21b-HEMBP (pyr) -PCV2VHHC3 (small fragment length 1656 bp); pET21b-HEMBP (pyr) -HAFnt (small fragment length 2193 bp);
b2 is a diagram of pET21B-His6MBP-gene recombinant plasmid enzyme digestion identification result (NdeI/XhoI), wherein M is DL5000 DNA Marker; 1: pET21b-His6MBP-CdIGMP026 (small fragment length 1849 bp); pET21b-His6MBP-Cago60 (small fragment length 2002 bp); pET21b-His6MBP-Nb (small fragment length 1588 bp); pET21b-His6MBP-FMDV98 (small fragment length 1840 bp); pET21b-His6MBP-PCV2b (small fragment length 1906 bp); pET21b-His6MBP-CdIGMP499 (small fragment length 1915 bp); pET21b-His6 MBP-Prop acid OAS (small fragment length 2236 bp); pET21b-His6 MBP-NLSFMD (small fragment length 1873 bp); pET21b-His6MBP-HAFnt (small fragment length is 2152 bp); 10 pET21b-His6MBP-H5HA10 (small fragment length 1723 bp); pET21b-His6MBP-PCV2VHHC3 (small fragment length 1615 bp);
FIG. 5 shows the electrophoresis chart (A) of the constructed pET21B-His6-MBP (pyr) -gene recombinant plasmid and the identification result (B) of the restriction enzyme (NdeI/XhoI); in A picture, M is DL5000 DNA Marker; pET21b-HEMBP (pyr) -Cago60 (target fragment length 1980 bp); pET21b-HEMBP (pyr) -PCV2VHHC3 (target fragment length is 1593 bp); pET21b-HEMBP (pyr) -FMDV98 (the target fragment is 1818 bp in length); 4 pET21b-HEMBP (pyr) -H5HA10 (target fragment length 1701 bp); 5: pET21b-HEMBP (pyr) -NLSFMD (target fragment length 1851 bp) 6: pET21b-HEMBP (pyr) -HAFnt (target fragment length 2130 bp); pET21b-HEMBP (pyr) -Prop acid OAS (target fragment 2214 bp in length); pET21b-HEMBP (pyr) -CdIGMP499 (target fragment length 1893 bp); 9 pET21b-His 6;
in the B picture, M is DL5000 DNA Marker; 1 pET21b-His6-MBP (pyr) -FMDV98 (small fragment length 1839 bp); pET21b-His6-MBP (pyr) -CdIGMP499 (small fragment length of 1914 bp); pET21b-His6-MBP (pyr) -PCV2VHHC3 (small fragment length 1614 bp); pET21b-His6-MBP (pyr) -Prop acid OAS (small fragment length 2235 bp); pET21b-His6-MBP (pyr) -H5HA10 (small fragment length 1722 bp); pET21b-His6-MBP (pyr) -HAFnt (small fragment length is 2151 bp); pET21b-His6-MBP (pyr) -NLSFMD (small fragment length 1872 bp); pET21b-His6-MBP (pyr) -Cago60 (small fragment length 2001 bp);
FIG. 6 shows the difference between the pET21b-HEMBP (pyr) recombinant expression vector and other expression vectors in the expression result of a part of proteins, wherein M is Thermo Scientific PageRuler stabilized Protein Ladder (170 kDa, 130kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15kDa, 10 kDa); 1, not inducing the whole bacteria; 2, inducing the whole strain; 3, supernatant after induction; 4, precipitation after induction;
FIG. 7 is a comparison of the results of expression of a portion of the protein in pET21b-HEMBP (pyr) recombinant expression vector (left) and pET21b-His6MBP expression vector (right), wherein M is Thermo Scientific PageRuler Prestatined protein ladder (170 kDa, 130kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15kDa, 10 kDa); 1, not inducing the whole bacteria; 2, inducing the whole strain; 3, supernatant after induction; 4, precipitation after induction;
FIG. 8 shows a comparison of the expression of a portion of the protein in pET21b-HEMBP (Pyr) recombinant expression vector (left) and pET21b-His6MBP (Pyr) expression vector (right), wherein M is Thermo Scientific PageRuler Prestatiinedprotein Ladder (170 kDa, 130kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15kDa, 10 kDa); 1, not inducing the whole bacteria; 2, inducing the whole strain; 3, supernatant after induction; 4, precipitation after induction;
FIG. 9 is a comparison of the expression results of a portion of proteins in pET21b-His6-MBP (pyr) recombinant expression vector (left) and pET21b-His6MBP recombinant expression vector (right), wherein M is Thermo Scientific PageRuler Prestatiinedprotein Ladder (170 kDa, 130kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15kDa, 10 kDa); 1, not inducing the whole bacteria; 2, inducing the whole strain; 3, supernatant after induction; and 4, precipitation after induction.
Detailed Description
The present application is further described with reference to the following examples, which are intended to briefly describe some of the materials involved in the following examples before describing the specific examples.
Biological material:
pUC57 plasmid, pET21b plasmid, available from Novagen;
his6, His6MBP, H5HA10, LSL150-cherry-PCV2b/NLSFMD/HAFnt and other gene sequences are synthesized and provided by Kinsley biotechnology company, and then recombinant plasmids such as pET21b-His6MBP, pET21b-His6, pET21b-His6-cherry-H5HA10, pET21b-LSL150-cherry-PCV2b/NLSFMD/HAFnt and the like are constructed according to the prior art;
the related gene sequencing work in the following examples was also performed by Kinry Biotech;
it should be further explained that the cherry protein is a red fluorescent protein, and after the fusion protein is induced and soluble expressed, the fluorescence of the thalli can be red, which is convenient for observation;
experimental reagent:
BamHI (FD 0054), NdeI (FD 0583), NheI (FD 0973), XhoI (FD 0694), Prestain Protein marker (PageRuler Prestated Protein ladder), etc. as Thermo scientific products;
plasmid DNA small extraction kit, DNA glue recovery kit, products of biological engineering (Shanghai) Limited company;
t4 DNA ligase (# M0202S), New England Biolabs;
ampicillin (Amp), beijing solibao technologies ltd;
DNA molecular mass standard DL5000, available from Bao bioengineering (Dalian) Co., Ltd.;
lysozyme, Actibity >20000 u/mg, Beijing Solaibao Tech Co., Ltd;
30% acrylamide (29:1), 2 xSDS PAGE electrophoresis sample buffer, isopropyl-D-thiogalactoside (IPTG), product of Bijing Dingguo Changsheng biotechnology Limited liability company;
agarose (Agarose), available from sienna bioscience ltd;
LB culture medium: dissolving 10 g of Tryptone, 5 g of Yeast Extract and 10 g of NaCl in 800 mL of ultrapure water, adjusting the pH to 7.0 by using NaOH, adding water to a constant volume of 1L, and sterilizing at high pressure for later use; adding 12 g of agar powder into the solid culture medium;
PBS（pH=7.4）：NaCl 8 g、KCl 0.2 g、Na2HPO4 1.42 g、KH2PO 40.27 g, adding water to dissolve, adjusting pH to 7.4, fixing volume to 1L, autoclaving, and storing at room temperature for use;
50 × TAE electrophoresis buffer: 242g Tris, 37.2g Na were weighed2EDTA·2H2Adding ultrapure water to fully dissolve O, adding 57.1 mL of acetic acid to fully mix, using the ultrapure water to fix the volume of the solution to 1L, and standing at normal temperature for later use; the resulting mixture was prepared as a1 XTAE electrophoresis buffer solution with ultrapure water.
10% Ammonium Persulfate (APS): weighing 1 g of APS, fully dissolving the APS with ultrapure water, fixing the volume to 10 mL, and storing at 4 ℃ for later use;
5 XSDS-PAGE running buffer: weighing 15.15 g of Tris, 5 g of sodium dodecyl benzene sulfonate (SDS) and 72.05 g of glycine, adding ultrapure water, fully dissolving, fixing the volume to 1L, and storing at normal temperature for later use;
coomassie brilliant blue R-250 staining solution: weighing Coomassie brilliant blue R-2501 g, measuring 250 mL of isopropanol, adding 100 mL of glacial acetic acid, adding 650 mL of ultrapure water, fully stirring and dissolving, filtering by using filter paper to remove particles, and storing at room temperature for later use;
coomassie brilliant blue destaining solution: 100 mL of absolute ethyl alcohol and 100 mL of glacial acetic acid, and keeping the volume of ultrapure water to 500 mL at room temperature for later use;
an experimental instrument:
electrophoresis apparatus and electrophoresis tank (JY600C), Beijing Junyi Oriental electrophoresis Equipment Co., Ltd;
amersham Imager 600 gel imaging System, GE Healthcare Life Sciences product;
ultrasonic cell crusher, Ningbo New Ganoderma research institute;
a biopspectrometer uv/vis spectrophotometer, Eppendorf;
example 1
The recombinant expression vector pET21b-HEMBP (pyr) capable of promoting the soluble expression of protein and improving the expression level of protein is constructed by the following steps as shown in a figure 1 (A).
(1) Construction of recombinant plasmid pUC57-HEMBP (pyr) -Nb
The gene sequence of the HEMBP (pyr) -Nb is artificially synthesized, the specific sequence is shown as SEQ ID NO.1, and the gene sequence is connected with a pUC57 plasmid to construct a recombinant plasmid pUC57-HEMBP (pyr) -Nb.
(2) Enzyme digestion and connection, the specific process is as follows:
plasmid pUC57-HEMBP (pyr) -Nb and plasmid pET21b were subjected to double digestion with NdeI and XhoI, respectively, and a 30. mu.L digestion system was designed as follows:
NdeI，1 μL；
XhoI，1 μL；
10 × FastDigest Green Buffer，3 μL；
plasmid pUC57-HEMBP (pyr) -Nb (or plasmid pET21 b), 1. mu.g;
ddH2o is added to 30 mu L;
enzyme digestion is carried out for 1 h at 37 ℃;
carrying out 1% agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product;
the digested products were ligated with T4 DNA ligase and ligated overnight at 16 ℃.
(2) Transformation and screening, and the specific process comprises the following steps:
chemically (CaCl) the ligation product obtained in step (1)2) Transformed into Escherichia coli (E.coli) BL21 (DE 3), then spread in LB solid medium (Amp, 100. mu.g/mL), and cultured overnight by inversion;
positive single colonies were picked, inoculated into resistant LB liquid medium containing ampicillin (100. mu.g/mL), cultured at 37 ℃ for 6 hours at 220 rpm, and then plasmids were extracted according to the instructions of the SanPrep column type plasmid DNA miniprep kit.
The extracted plasmid is identified by double enzyme digestion, and a10 mu L enzyme digestion system is designed as follows:
NdeI，0.25μL；
BamHI，0.25μL；
10 × FastDigest Green Buffer，1μL；
250 ng of the extracted plasmid;
ddH2adding O to 10 mu L;
enzyme digestion is carried out for 1 h at 37 ℃; and carrying out 1% agarose gel electrophoresis separation and identification on the enzyme digestion product.
The results of the identification are shown in FIG. 3. The results in FIG. 3 show that pET21b-HEMBP (pyr) -Nb was successfully constructed. And (4) storing the correctly identified escherichia coli containing the recombinant plasmid for later use.
Example 2
On the basis of example 1, in order to further detect and judge the promotion and improvement effects of the constructed recombinant expression vector pET21b-HEMBP (pyr) on the protein expression quantity and the protein solubility, the inventors performed expression experiments on different foreign proteins by using a prokaryotic expression system, and the related experimental processes are briefly described as follows.
(one) construction of recombinant expression vector containing target Gene
The target genes include H5HA10, PCV2B, NLSFMD and HAFnt, and referring to the construction process schematic diagram of FIG. 1 (B), the recombinant plasmid expression vectors pET21B-HEMBP (pyr) -H5HA10, pET21B-HEMBP (pyr) -PCV2B, pET21B-HEMBP (pyr) -NLSFMD and pET21B-HEMBP (pyr) -HAFnt containing the target genes are specifically constructed as follows:
BamHI and XhoI double digestion of the recombinant expression vectors prepared in example 1, pET21b-HEMBP (pyr) -Nb and pET21b-His 6-chery-H5 HA10, pET21b-LSL 150-chery-PCV 2b, pET21b-LSL 150-chery-NLSFMD, pET21b-LSL 150-chery-HAFnt plasmids, respectively, with BamHI and XhoI enzymes (digestion system was designed with reference to the 30. mu.L digestion system in example 1);
carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product;
the digested products were ligated with T4 DNA ligase to construct a recombinant plasmid expression vector pET21b-HEMBP (pyr) -H5HA10, pET21b-HEMBP (pyr) -PCV2b, pET21b-HEMBP (pyr) -NLSFMD, pET21b-HEMBP (pyr) -HAFnt;
the ligation product was transformed into E.coli BL21 (DE 3) and screened (see example 1 for screening procedure), and the positive plasmid was identified by double digestion with NdeI and XhoI, and the results are shown in FIG. 4.
The results in FIG. 4 show the success of recombinant plasmid construction.
And (4) storing the strains containing the recombinant plasmids which are correctly identified by enzyme digestion for later use.
The constructed strains containing the correct recombinant plasmid and the strains originally containing pET21b-His6-cherry-H5HA10, pET21b-LSL150-cherry-PCV2b, pET21b-LSL150-cherry-NLSFMD and pET21b-LSL150-cherry-HAFnt plasmids are respectively subjected to induction expression, and the protein expression amount is evaluated by taking thalli after induction. The specific process is as follows:
(1) taking 1 mL of activated bacterium liquid, inoculating the activated bacterium liquid into LB liquid culture medium containing Amp 100 mu g/mL according to the volume ratio of 1:100, and carrying out shaking culture at 37 ℃ and 220 rpm until OD600About = 0.7;
(2) expression was induced overnight at 25 ℃ with the addition of 0.5 mM IPTG.
Respectively preparing an uninduced whole-bacterium sample and an induced expression sample, which are as follows:
1. uninduced whole bacteria sample
Before IPTG induction expression, taking 1 mL of a bacterial liquid sample before induction expression as an uninduced whole bacterial sample, and treating the sample in the following way:
centrifuging the bacterial solution at 9000 rpm for 1 min, discarding the supernatant, and adding (OD)600Value X100) μ L of 1 × loadingbuffer to resuspend the cells; boiling at 99 deg.C for 10min, centrifuging at 12000 g for 2 min, and storing at-20 deg.C for later use.
2. Whole bacteria sample after induction expression
Measuring OD of the bacteria liquid after induction expression by using an ultraviolet spectrophotometer600And then preparing the whole bacteria sample after induction expression by referring to the preparation mode of the whole bacteria sample without induction.
3. Supernatant sample after induced expression and sediment sample after induced expression
Centrifuging 10 mL of the induced bacterial solution, resuspending the cells in (OD 600 value × 100/2). mu.L of PBS (pH = 7.4), adding lysozyme (1 mol/L) and carrying out ice bath for 30 min, and then ultrasonically crushing the cells at low temperature (ultrasonic time 4 s, intermittent 7 s);
crushing to clear, centrifuging at 12000 g at 4 deg.C for 30 min;
sucking 30 mu L of supernatant and adding an equal amount of 2 × loading buffer to prepare a supernatant sample after induction;
after discarding the remaining supernatant, mu.L of PBS was added (OD 600. times. 100/2) to resuspend the pellet, 30. mu.L of resuspension was aspirated and an equal amount of 2 × loading buffer was added to prepare post-induction pellet samples.
The method for detecting the protein content in the sample comprises the following steps:
preparing 12% SDS-PAGE gel, respectively taking 10 mu L of uninduced whole bacteria, induced supernatant and induced precipitate samples for sample loading, carrying out 120V electrophoresis until the samples enter a separation gel interface, adjusting the voltage to 160V, and finishing the electrophoresis when the dye is 0.5 cm away from the bottom of the gel;
stripping the concentrated glue by using a glue unloading plate, dip-dyeing the unloaded separation glue in a dyeing solution for Coomassie brilliant blue dyeing, and shaking and dyeing at room temperature for 2 h;
after the dyeing liquid is recovered, immersing the gel into a decolorizing liquid, and replacing the decolorizing liquid for several times until strips in the gel are clear;
the samples were stored by photography using an Amersham Imager 600 gel imaging system and grey scale analysis was performed.
The results of induced expression are shown in FIG. 6. Analysis shows that: the pET21b-HEMBP (pyr) recombinant expression vector can make the non-expressed NLSFMD protein and the HAFnt, PCV2b and H5HA10 protein expressed by inclusion body be expressed in a soluble way. The gray scale analysis result shows that the solubility of the pET21b-HEMBP (pyr) -HAFnt/NLSFMD/PCV2b/H5HA10 recombinant protein is respectively as follows: 75%, 60.7%, 78% and 82.7%; wherein the solubility = amount of recombinant protein in supernatant/(amount of recombinant protein in supernatant + amount of recombinant protein in pellet).
Example 3
Based on the recombinant expression vector constructed in example 1, further, the inventors constructed recombinant expression vectors and performed prokaryotic expression for 10 different target genes, wherein the 10 target genes specifically include: nb, PCV2VHHC3, H5HA10, PCV2b, HAFnt, FMDV98, Cago60, CdIGMP499 (Caulobacter viroids), CdIGMP026 (Phenylobacterium zucineum), and Prop acid OAS, wherein the related operations can be performed by referring to examples 1 and 2 and the prior art, and are not repeated.
The construction flow chart of the recombinant expression plasmid is shown in FIG. 2 (C), and the restriction enzyme digestion identification result of the constructed recombinant expression plasmid is shown in FIG. 4. The results show that the recombinant plasmid was successfully constructed.
In order to further demonstrate the technical effects of the present invention, the 9 target genes were integrated and expressed recombinantly based on the conventional pET21b-His6MBP plasmid, with reference to the procedures and prior art related to examples 1 and 2, and used as comparative examples.
The uninduced whole bacteria, the induced supernatant and the induced precipitate samples of different plasmid expression vectors are subjected to 12 percent SDS-PAGE electrophoresis and then are stained by Coomassie brilliant blue. The electrophoresis results of different recombinant plasmid expression vectors of different genes are shown in FIG. 7, and the grayscale analysis results after gel imaging are shown in Table 1 below.
Table 1 comparison of the differences in soluble expression of two expression vectors promoting protein:
the expression amount (%) is the percentage of the recombinant protein to the total protein in E.coli;
the solubility (%) is the percentage of the amount expressed in the recombinant protein supernatant to (the amount expressed in the supernatant + the amount expressed in the pellet).
Further analysis, in conjunction with fig. 7 and the above statistical results in table 1, can see that:
compared with His6MBP-Nb, the solubility of the HEMBP (pyr) -Nb recombinant protein is improved by 49.2 percent;
the solubility of the recombinant protein HEMBP (pyr) -PCV2b is increased by 11.5 percent compared with that of His6MBP-PCV2 b;
compared with His6MBP-Cago60, the solubility of the recombinant protein HEMBP (pyr) -Cago60 is improved by 28.1 percent;
the solubility of the HEMBP (pyr) -PCV2VHHC3 recombinant protein is improved by 17.4 percent compared with that of pET21b-His6MBP-PCV2VHHC 3;
the solubility of the recombinant protein of the HEMBP (pyr) -CdIGMP499 is improved by 25.9 percent compared with that of the recombinant protein of the His6MBP-CdIGMP 499;
compared with His6 MBP-Prop acid OAS, the solubility of the recombinant protein of the HEMBP (pyr) -Prop acid OAS is improved by 42.7 percent;
compared with His6MBP-FMDV98, the solubility of the recombinant protein HEMBP (pyr) -FMDV98 is improved by 42.2 percent;
compared with His6MBP-H5HA10, the solubility of the recombinant protein HEMBP (pyr) -H5HA10 is improved by 22.4 percent;
no expression is generated after the recombinant protein His6MBP-CdIGMP026 is induced, and the recombinant protein HEMBP (pyr) -CdIGMP026 is expressed and is soluble in 35%.
Combining example 2 and the above statistical results, it can be seen that: aiming at different types of genes, after a new pET21b-HEMBP (pyr) recombinant expression vector is adopted, compared with the original expression vector, the protein solubility and the protein expression quantity are both better improved, and the improvement effect is obvious.
Example 4
Based on the above examples, to further demonstrate the technical effects of the tag sequence carried by the recombinant expression vector provided in the present invention, the inventors used NheI and XhoI to double-cleave the recombinant plasmid, which is used in example 3 and is based on pET21b-His6 vector plasmid, with reference to the flow chart of fig. 2 (D) for constructing the recombinant plasmid expression vector, with NheI and XhoI, and which incorporates the target genes (PCV 2VHHC3, H5HA10, NLSFMD, HAFnt, FMDV98, Cago60, cdimpg 499, and Prop acid OAS) with NheI and XhoI, while double-cleaving NheI and XhoI from pET21b-His6 vector plasmid; the enzyme products are connected, transformed, screened and identified by enzyme digestion, and the recombinant expression plasmid pET21b-His6-MBP (pyr) -gene (gene represents the target gene PCV2VHHC3, H5HA10, NLSFMD, HAFnt, FMDV98, Cago60, CdIGMP499 or Prop acid OAS) is constructed.
The results of restriction enzyme identification of the recombinant plasmid are shown in FIG. 5. The result shows that the recombinant plasmid is successfully constructed.
For the related operations, reference is made to embodiments 1, 2 and 3 and the prior art, which are not described again.
And performing induced expression on the constructed recombinant plasmid expression vector, performing 12% SDS-PAGE electrophoresis on uninduced whole bacteria, induced supernatant and induced precipitate samples respectively, performing Coomassie brilliant blue staining, and determining and analyzing the protein expression amount.
The results of the differences in protein expression levels and soluble expression between the HE-MBP (pyr) tag and the His6-MBP (pyr) tag in some of the target genes are shown in FIG. 8. As can be seen from the figure:
the expression level of HE-MBP (pyr) -PCV2VHHC3/CdIGMP499/H5HA10 recombinant protein is significantly higher than that of His6-MBP (pyr) -PCV2VHHC3/CdIGMP499/H5HA10 recombinant protein, and the solubility is higher than that of the latter;
the expression amount of HE-MBP (pyr) -Cago60/FMDV98/NLSFMD/Prop acid OAS recombinant protein is slightly higher than that of His6-MBP (pyr) -Cago60/FMDV98/NLSFMD/Prop acid OAS recombinant protein, and the solubility is respectively improved by 8.9%, 19.5%, 4.8% and 15.2%.
The difference in the effect of His6-MBP (pyr) tag and His6MBP tag on the expression level and soluble expression of recombinant proteins in some target genes is shown in FIG. 9. The results in FIG. 9 show that:
the expression amount of the His6-MBP-HAFnt recombinant protein is higher than that of the HE-MBP (pyr) -HAFnt recombinant protein, but the expression amount is only 59.4 percent of the solubility, and the HE-MBP (pyr) -HAFnt recombinant protein is all expressed in a soluble way;
the expression level of the His6-MBP-Prop acid OAS/H5HA10 recombinant protein is also higher than that of the HE-MBP (pyr) -Propacid OAS/H5HA10 recombinant protein, but the solubility is respectively 32% and 57.1%, and is lower than that of the HE-MBP (pyr) -Propacid OAS/H5HA10 recombinant protein, respectively 59.5% and 71.7%;
the expression level of HE-MBP (pyr) -FMDV98/Cago60 recombinant protein is not obviously different from that of His 6-MBP-FMDV 98/Cago60, but the solubility is higher than that of the His 6-MBP-FMDV 98/Cago 60.
The results of the gray scale analysis after protein expression are shown in tables 2 and 3 below.
Table 2: comparison of the differences in protein-soluble expression promoted by pET21b-HE-MBP (pyr) and pET21b-His6-MBP (pyr) expression vectors:
the expression amount (%) is the percentage of the recombinant protein to the total protein in E.coli;
the solubility (%) is the percentage of the amount expressed in the recombinant protein supernatant to (the amount expressed in the supernatant + the amount expressed in the pellet).
Table 3: pET21b-His6-MBP (pyr) and pET21b-His6MBP expression vectors facilitate comparison of the differences in protein-soluble expression:
the expression amount (%) is the percentage of the recombinant protein to the total protein in E.coli;
the solubility (%) is the percentage of the amount expressed in the recombinant protein supernatant to (the amount expressed in the supernatant + the amount expressed in the pellet).
SEQUENCE LISTING
<110> Henan university of agriculture
<120> a recombinant expression vector capable of promoting soluble expression of proteins and increasing expression level
<130>none
<160>1
<170>PatentIn version 3.5
<210>1
<211>1242
<212>DNA
<213> Artificial design
<400>1
catatgaaat attataaaca cgagcacgag catgagcacg aacacgagca cgaacacgag 60
ggtgctagcg gcatgaagat cgaggaaggt aaggttgtta tttggcacgc gatgcagccg 120
aacgaactgg aagtgtttca aagcctggcg gaggaataca tggcgctgtg cccggaagtg 180
gagatcgttt tcgagcagaa gccgaacctg gaagacgcgc tgaaagcggc gattccgacc 240
ggtcagggtc cggacctgtt tatctgggcg cacgattgga ttggtaaatt cgcggaggcg 300
ggtctgctgg aaccgatcga cgagtacgtt accgaagatc tgctgaacga atttgcgccg 360
atggcgcagg atgcgatgca atacaagggt cactactatg cgctgccgtt cgcggcggag 420
accgtggcga tcatttataa caaggagatg gttagcgaac cgccgaaaac ctttgacgag 480
atgaaggcga ttatggaaaa atactatgat ccggcgaacg aaaaatacgg catcgcgtgg 540
ccgattaacg cgtatttcat cagcgcgatt gcgcaggcgt ttggtggcta ctatttcgac 600
gataagaccg agcaaccggg tctggacaaa ccggaaacca tcgagggctt taagttcttt 660
ttcaccgaaa tttggccgta catggcgccg accggtgatt ataacaccca gcaaagcatc 720
ttcctggagg gtcgtgcgcc gatgatggtg aacggcccgt ggagcatcaa cgacgttaag 780
aaagcgggta ttaacttcgg cgtggttccg ctgccgccga tcattaagga tggtaaagaa 840
tactggccgc gtccgtatgg tggcgtgaaa ctgatctact ttgcggcggg cattaagaac 900
aaagacgcgg cgtggaagtt cgcgaaatgg ctgaccacca gcgaggaaag catcaagacc 960
ctggcgctgg agctgggtta tattccggtg ctgaccaaag ttctggacga tccggaaatc 1020
aagaacgacc cggtgattta cggttttggc caggcggttc aacacgcgta tctgatgccg 1080
aagagcccga aaatgagcgc ggtgtggggt ggcgttgatg gtgcgatcaa cgagattctg 1140
caggacccgc aaaacgcgga tatcgagggc attctgaaga aataccagca agaaatcctg 1200
aacaacatgc aggaggaaaa cctgtacttc caatccggat cc 1242
Claims (2)
1. A recombinant expression vector capable of promoting protein soluble expression and improving expression quantity is characterized in that the recombinant expression vector pET21b-HEMBP (pyr) is a prokaryotic expression vector and is used for a prokaryotic expression system of escherichia coli, and the recombinant expression vector is obtained by the following steps:
(1) construction of recombinant plasmid pUC57-HEMBP (pyr) -Nb
Artificially synthesizing a gene sequence of the HEMBP (pyr) -Nb, wherein the base sequence is shown as SEQ ID NO.1, and connecting the gene sequence with a plasmid pUC57 to construct a recombinant plasmid pUC57-HEMBP (pyr) -Nb;
(2) enzyme digestion and connection, the specific process is as follows:
carrying out double enzyme digestion on the recombinant plasmid pUC57-HEMBP (pyr) -Nb in the step (1) and the plasmid pET21b by NdeI and XhoI respectively; recovering the enzyme digestion product; connecting the enzyme digestion products;
(3) transformation and screening, and the specific process comprises the following steps:
transforming the ligation product in the step (2) into escherichia coli BL21 competent cells for resistance screening; selecting a positive single colony, extracting plasmids, and performing NdeI and BamHI double enzyme digestion identification; the correctly identified vector is the recombinant expression vector pET21b-HEMBP (pyr).
2. The use of the recombinant expression vector pET21b-HEMBP (pyr) capable of promoting protein solubility expression and increasing expression level in claim 1 for protein expression, which is used for expressing monoclonal antibodies Nb, PCV2VHHC3, antigens H5HA10, PCV2b cap, NLSFMD, HAFnt, FMDV98, polyprotein Cago60, CdIGMP499 (Caulobacter viroids), CdIP 026 (Phenylobacterium zucinum) or Propacid OAS in a prokaryotic expression system of Escherichia coli.
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