阅读说明：本技术 西伯利亚鲟球形脂联素蛋白基因原核表达的方法及应用 (Method for prokaryotic expression of acipenser baerii globular adiponectin protein gene and application ) 是由 李志琼 唐妮 徐少奇 李娅 刘晏伶 王美 陈虎 汪斌 田正志 张鑫 周朝伟 陈 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种西伯利亚鲟球形脂联素蛋白基因原核表达的方法及应用,包括：(1)成熟肽基因的克隆；(2)重组质粒的构建；(3)重组质粒诱导表达。本发明的方法使西伯利亚鲟脂联素蛋白能够在原核生物内实现大批量、低成本生产,最后可用于功能验证等注射试验及添加剂。(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.)

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.

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.