CN110724186A - Method for prokaryotic expression of acipenser baerii globular adiponectin protein gene and application - Google Patents
Method for prokaryotic expression of acipenser baerii globular adiponectin protein gene and application Download PDFInfo
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- CN110724186A CN110724186A CN201911035438.2A CN201911035438A CN110724186A CN 110724186 A CN110724186 A CN 110724186A CN 201911035438 A CN201911035438 A CN 201911035438A CN 110724186 A CN110724186 A CN 110724186A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Abstract
The invention discloses a method for prokaryotic expression of a spherical adiponectin protein gene of Acipenser sibirica and application thereof, wherein the method comprises the following steps: (1) cloning mature peptide gene; (2) constructing a recombinant plasmid; (3) the recombinant plasmid induces expression. The method of the invention enables the Siberian sturgeon adiponectin protein to realize mass and low-cost production in prokaryotes, and can be finally used for injection tests and additives such as functional verification and the like.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for prokaryotic expression of a spherical adiponectin protein gene of Acipenser sibirica and application thereof.
Background
Acipenser baeri Brandt belongs to Acipenseridae (Acipenserdae) and has the characteristics of wide food spectrum, easy domestication, fast growth, high caviar quality and the like, and the Acipenser baeri Brandt is popularized to be cultivated in many countries and regions in the world. Meanwhile, China is the biggest sturgeon caviar producing country and export country. It can be seen that the Siberian sturgeon has a high economic value. However, the Siberian sturgeon has long culture period and high requirements on the comprehensive and balance of the nutrition of the feed. Various metabolic-related chronic diseases such as fatty liver are easily generated in the long culture process. Fatty liver caused by fat accumulation will affect growth and development of sturgeons, resulting in decrease of yield and quality of caviar, and affect economic benefit.
Adiponectin (AdipoQ) is an endogenous biologically active polypeptide or protein secreted by adipocytes and plays a role mainly in fat degradation and regulation of glucose levels, as well as in anti-inflammatory and atherogenic, pro-angiogenic and anti-angiogenic factors. Therefore, if adiponectin can be applied to the cultivation of the Siberian sturgeon, the possibility of metabolic diseases of the fish in the cultivation can be reduced, and the cultivation efficiency and the economic benefit can be improved. Therefore, adiponectin has a wide application prospect.
Sturgeon adiponectin includes globular adiponectin (gAd), which is commonly used in functional verification tests of adiponectin proteins. The synthesis of the spherical adiponectin mainly depends on artificial synthesis, has higher cost and lower yield, can only be used for experiments, and cannot meet the requirements in the actual culture production process.
Disclosure of Invention
The invention aims to: aiming at the problems that the artificial synthesis of spherical adiponectin in the prior art has high cost and low yield and can not meet the requirements in the actual culture production process, a method for prokaryotic expression of a acipenser baerii spherical adiponectin protein gene and application thereof are provided.
The technical scheme adopted by the invention is as follows:
a method for prokaryotic expression of a spherical adiponectin protein gene of Acipenser sibirica comprises the following steps:
(1) cloning of mature peptide gene: designing upstream and downstream primers with BamHI enzyme cutting site and Hind III enzyme cutting site respectively according to the amino acid sequence of spherical adiponectin protein, and carrying out PCR amplification to obtain a target nucleotide fragment;
(2) construction of recombinant plasmid: recombining a prokaryotic expression vector and a target nucleotide fragment to form a recombinant plasmid capable of expressing an amino acid sequence;
(3) inducing expression by the recombinant plasmid: introducing the recombinant plasmid into an expression strain, and inducing the expression of the recombinant plasmid in the expression strain by using an inducer.
Further, the expression strain in (3) is E.coli BL21, and the inducer is isopropyl-beta-D-thiogalactopyranoside.
Further, the recombinant plasmid was transformed into an expression strain E.coli BL21, cultured to OD in a liquid LB medium containing ampicillin at 37 ℃ and 200rpm/min600The value reaches 0.4-0.6, and isopropyl-beta-D-thiogalactopyranoside is addedInducing culture to express the recombinant plasmid in the expression strain.
Further, the conditions of the induction culture are as follows: the temperature is 37 ℃, the concentration of the inducer is 1.0-1.2mmol/L, and the induction time is 6-8 h.
The application of the method in protein function test or additive preparation.
The upstream and downstream primers used in the above method are specifically:
an upstream primer: 5'-AAAGGATCCGCCCTCCTCTACCGC-3', respectively;
a downstream primer: 5'-GGAAGCTTGCGATCTCCGTTTACTGT-3' are provided.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the invention synthesizes Siberian sturgeon globular adiponectin protein by genetic engineering technology, selects a prokaryotic vector which can be used for the in vitro expression of Siberian sturgeon adiponectin protein, obtains recombinant plasmid which can be used for quantitative production, and leads Siberian sturgeon adiponectin to realize mass and low-cost production in prokaryotes and be prepared in a common molecular laboratory, thus the invention has the outstanding advantages of high yield, low cost and simple method;
2. the invention optimizes the inducing conditions, selects the most suitable temperature and time and the concentration of the inducer, and has good inducing expression effect;
3. the protein produced by the invention can be used for protein function tests or used as an additive to be applied to actual culture production, and the economic benefit is effectively improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is an electropherogram of a PCR product of the gAd gene; wherein M is DL2000 Marker; 1-4 are PCR products of gAd gene;
FIG. 2 is a graph of the results of IPTG-induced concentration optimization of gAd/pET32a expression; wherein, M: protein marker; 1: induction of pET-32 alpha (+); 2: pET-32a-gAd uninduced sample; 3-8: the IPTG concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mmol/L respectively induce pET-32a-gAd expression;
FIG. 3 is a graph of the results of IPTG induction time optimization of gAd/pET32a expression; wherein, M: protein marker; 1, inducing pET-32 alpha (+); 2: pET-32a-gAd uninduced sample; 3-8: pET-32a-gAd transformants induced samples for 1.5, 3, 4.5, 6, 7.5 and 9h, respectively;
FIG. 4 is a graph showing the results of the solubility assay of recombinant mature protein; wherein, Marker, pET32a are unloaded (1), solution (5) after the super-break of gAd-32a, sediment (6) after the super-break of gAd-32a and supernatant (7) after the super-break of gAd-32 a;
FIG. 5 is a diagram of the purification of recombinant proteins.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Examples
The method for prokaryotic expression of the spherical adiponectin protein gene of the Acipenser sibirica provided by the preferred embodiment of the invention comprises the following specific steps:
(1) cloning of mature peptide genes
Total brain RNA of the Siberian sturgeon is extracted by using the kit, and is subjected to reverse transcription to synthesize first strand cDNA. According to the sequence of Siberian sturgeon adiponectin obtained by cloning, the gene sequence of the mature peptide of spherical adiponectin (gAd) is obtained by comparison and analysis with other fish adiponectin genes, and then an upstream primer gAd-F (P1) is designed: 5'-AAAGGATCCGCCCTCCTCTACCGC-3' and downstream primer gAd-R (P2): 5'-GGAAGCTTGCGATCTCCGTTTACTGT-3' are provided. BamHI site (GGATCC) and protective base are added to the 5 'end of P1, and HindIII site (AAGCTT) and protective base are added to the 5' end of P2. The primers are adopted to carry out PCR amplification, and the PCR amplification conditions are as follows: denaturation at 94 ℃ for 5min, 35 cycles (94 ℃, 30 s; 67 ℃, 30 s; 72 ℃, 50s), extension at 72 ℃ for 10 min. And cutting the target fragment into gel, recovering, connecting the gel to a pMD19-T vector, and selecting a positive clone for sequencing verification.
The gel electrophoresis pattern after the PCR reaction is shown in FIG. 1.
(2) Construction of recombinant plasmid
Extracting recombinant gAd clone plasmid and expression vector pET-32a, double enzyme digestion with BamHI and Hind III respectively, cutting glue and recovering, connecting with T4 ligase to obtain expression recombinant plasmid gAd/pET32a, transforming into DH5 alpha competent cell, after bacterial liquid culture, carrying out PCR detection and double enzyme digestion verification, and sequencing correct recombinant plasmid.
(3) Expression of recombinant plasmids in expression strains
The expression recombinant plasmid with correct sequencing is transformed into an expression strain E.coli BL21(DE3) and cultured in a liquid LB culture medium (containing ampicillin) to OD600The value reaches 0.4-0.6 (37 ℃, 200rpm/min), isopropyl-beta-D-thiogalactoside (IPTG, 1mmol/L) is added for induction culture, and the recombinant plasmid is expressed in an expression strain.
The size of pET32a-gAd recombinant expression plasmid is estimated to be 36.807 kD.
(4) Optimization of Induction conditions
Culturing the bacterial liquid at 37 deg.C to OD600When the value is 0.4-0.6, adding IPTG to the final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mmol/L respectively, culturing for 6 hours by shaking, and researching the induction effect of different IPTG concentrations on recombinant protein expression.
The results are shown in FIG. 2: when the IPTG induction concentration is 1.2mM, the expression amount of pET32a-gAd recombinant protein is higher than that of IPTG treatment groups with other concentrations.
Method for optimizing reference concentration, and culturing bacterial liquid at 37 deg.C to OD600When the value is 0.4-0.6, IPTG is added to the final concentration of 1.0mmol/L, and time gradients are set for 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 hours. SDS-PAGE was then performed to investigate the induction of recombinant protein expression at different induction times.
The results are shown in FIG. 3: the induction time of pET32a-gAd protein can be 1.5h-9 h.
(5) Solubility assays for recombinant mature proteins
Culturing the bacterial liquid at 37 deg.C to OD600When the value is 0.4-0.6, 1.2mM IPTG is added for induction for 6 h. Centrifugation was carried out at 10000g for 5min in a centrifuge, and the precipitate was collected and washed once with 30mL of PBS. Adding 30mL of PBS for resuspending the precipitate, and crushing in ultrasonic waves under the conditions of ice water bath, 30% amplitude, 5s ultrasonic waves and 5s stop time, and repeatedly circulating for 10 min. After the ultrasonic operation is finished, the ultrasonic wave is transmitted to the ultrasonic generator,centrifuging at 10000g for 5min in a centrifuge, collecting supernatant and precipitate, performing SDS-PAGE electrophoresis, and observing whether the expression product of the target gene mainly exists in the supernatant or the precipitate, thereby judging whether the expression product is soluble protein or inclusion body protein. As shown in fig. 4.
(5) Western-blotting validation of recombinant proteins
Expression cells induced with IPTG (1.2mmol/L) for 6h were collected, subjected to SDS-PAGE, and then proteins were transferred to PVDF membrane by wet transfer method, and washed and then blocked with blocking solution (Solarbio). 6His monoclonal antibody (TransGen) was used as a primary antibody, and incubated at 4 ℃. HRP-labeled goat anti-mouse IgG (TransGen) was used as a secondary antibody and incubated at room temperature for 1 h. After developing with ECL kit (Solambio) for 2 minutes, photographs were taken by gel electrophoresis imaging system (Bio-rad).
(6) Purification and renaturation of recombinant proteins
Taking recombinant bacteria for IPTG (1.2mmol/L) induced expression for 6h at 37 ℃, centrifuging (10000g/min,5min), collecting precipitate, washing with PBS, resuspending the bacteria with 1/10-volume ultrasonic crushing liquid, ultrasonically crushing the resuspended bacteria liquid in ice bath, collecting precipitate, dissolving the precipitate in 6M guanidine hydrochloride solution, filtering with 0.45 mu M microporous filter membrane, and using Ni to filter2+-NTA affinity chromatography column (GE) for separation and purification of the fusion protein, and SDS-PAGE electrophoresis for detection of protein purity. As shown in FIG. 5, the purified protein eluate was subjected to gradient renaturation by dialysis bag, the concentration of the purified recombinant protein was measured by BCA protein quantification kit, and then filtered through a 0.22 μm microporous membrane for sterilization and stored in an ultra-low temperature freezer at-80 ℃ for further use.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
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Claims (6)
1. A method for prokaryotic expression of a spherical adiponectin protein gene of Acipenser sibiricum is characterized by comprising the following steps:
(1) cloning of mature peptide gene: designing upstream and downstream primers with BamHI enzyme cutting site and Hind III enzyme cutting site respectively according to the amino acid sequence of spherical adiponectin protein, and carrying out PCR amplification to obtain a target nucleotide fragment;
(2) construction of recombinant plasmid: recombining a prokaryotic expression vector and a target nucleotide fragment to form a recombinant plasmid capable of expressing an amino acid sequence;
(3) inducing expression by the recombinant plasmid: introducing the recombinant plasmid into an expression strain, and inducing the expression of the recombinant plasmid in the expression strain by using an inducer.
2. The method for prokaryotic expression of the globular adiponectin protein gene of Acipenser sibirica according to claim 1, wherein: the expression strain in the step (3) is E.coli BL21, and the inducer is isopropyl-beta-D-thiogalactopyranoside.
3. The method for prokaryotic expression of the globular adiponectin protein gene of Acipenser sibirica according to claim 2, wherein: the recombinant plasmid is transformed into an expression strain E.coli BL21, and cultured to OD in a liquid LB culture medium containing ampicillin at 37 ℃ and 200rpm/min600And adding isopropyl-beta-D-thiogalactopyranoside for induction culture to enable the recombinant plasmid to be expressed in an expression strain, wherein the value reaches 0.4-0.6.
4. The method for prokaryotic expression of the globular adiponectin protein gene of Acipenser sibirica according to claim 3, wherein: the conditions of the induction culture are as follows: the temperature is 37 ℃, the concentration of the inducer is 1.0-1.2mmol/L, and the induction time is 6-8 h.
5. Use of the method according to any one of claims 1 to 4 for protein function tests or for the preparation of additives.
6. Upstream and downstream primers for use in the method according to any one of claims 1 to 4, in particular:
an upstream primer: 5'-AAAGGATCCGCCCTCCTCTACCGC-3', respectively;
a downstream primer: 5'-GGAAGCTTGCGATCTCCGTTTACTGT-3' are provided.
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| CN102181437A (en) * | 2011-02-28 | 2011-09-14 | 东莞市畜牧科学研究所 | gAd gene of porcine globular adiponectin and protein encoded by gAd gene and application |
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