阅读说明：本技术 一种内切葡聚糖酶表达载体及其构建方法与应用 (endoglucanase expression vector and construction method and application thereof ) 是由 王洪成 沙冲 邵蔚蓝 于 2019-10-24 设计创作，主要内容包括：本发明涉及生物工程领域,具体涉一种内切葡聚糖酶表达载体及其构建方法与应用。本发明提供一种内切葡聚糖酶表达载体,其筛选基因为大肠杆菌glmS基因,原核宿主细胞为glmS基因缺陷型大肠杆菌,转化E.coliΔglmS后生产的蛋白只有大肠杆菌自身蛋白和内切葡聚糖酶蛋白而不含有抗生素等其它外来蛋白,具有相对的安全性；经过发酵和高温灭活处理,既可以消除大肠杆菌的毒性,又可以保证本酶活性的存在；使得来源于热纤梭菌(C.thermocellum ATCC 27405)的CelD基因实现生物安全性高效表达。本发明所提供的载体构建方法简单,安全性好,成本低,具有较高的表达量,可为饲用内切葡聚糖酶的生物发酵提供批量、低价、高质量的原材料。(The invention relates to the field of bioengineering, in particular to endoglucanase expression vectors, a construction method and an application thereof, the invention provides endoglucanase expression vectors, wherein a screening gene of the endoglucanase expression vectors is an escherichia coli glmS gene, a prokaryotic host cell is glmS gene defective escherichia coli, and a protein produced after E.coli delta glmS is converted only contains escherichia coli self protein and endoglucanase protein but does not contain other foreign proteins such as antibiotics and the like, so that the endoglucanase expression vectors have relative safety, can eliminate the toxicity of the escherichia coli and ensure the existence of the activity of the enzyme through fermentation and high-temperature inactivation treatment, and can realize the high-efficiency expression of biological safety of a CelD gene from clostridium thermocellum ATCC 27405.)

1, endoglucanase expression vector, characterized in that, its nucleotide sequence is shown in SEQ ID No. 9.

2. The endoglucanase expression vector of claim 1, wherein the selectable gene of the expression vector is the glmS gene of E.coli, and the prokaryotic host cell is a glmS gene-deficient E.coli; and inserting the endoglucanase celD gene into a pHsh-GlmS plasmid vector after the screening gene is connected with the pHsH-Amp vector fragment, and transforming the plasmid vector into a prokaryotic host cell to obtain the endoglucanase expression vector.

3. The method for constructing an endoglucanase expression vector of claim 1, comprising the steps of:

(1) amplifying a glmS gene cDNA fragment by taking escherichia coli as a template, amplifying by taking pHsH-Amp as a template to obtain a pHsh vector fragment, connecting the phosphorylated glmS gene cDNA fragment with the flat end of the pHsh vector fragment, converting into gfa deletion type escherichia coli, and screening to obtain a pHsh-GlmS plasmid;

(2) and (2) amplifying a celD gene fragment by using the C.thermocellum genome as a template, inserting the celD gene fragment into the pHsh-GlmS plasmid obtained in the step (1), connecting the blunt ends, transforming into Escherichia coli E, coli delta glmS, selecting a transformant, and sequencing to obtain an expression vector pHsh-GcellD.

4. The construction method according to claim 3, wherein in the step (1), the nucleotide sequences of the primers for amplifying the glmS gene cDNA fragment are shown as SEQ ID Nos. 1 and 2, and the nucleotide sequences of the primers for amplifying the pHsh vector fragment are shown as SEQ ID Nos. 3 and 4.

5. The construction method according to claim 3, wherein in the step (1), the molar ratio of the glmS gene cDNA fragment to the pHsh vector fragment during blunt end ligation is 3: 1.

6. the construction method according to claim 3, wherein in the step (2), the nucleotide sequences of the primers for amplifying the celD gene fragment are shown as SEQ ID Nos. 5 and 6, and the nucleotide sequences of the primers for amplifying the pHsh-GlmS vector fragment are shown as SEQ ID Nos. 7 and 8.

7. The method for constructing the recombinant plasmid of claim 3, wherein in the step (2), the celD gene fragment is inserted into the Hsh promoter expression frame of the pHsh-GlmS plasmid.

8, recombinant bacteria comprising the endoglucanase expression vector of claim 1.

9. Use of an endoglucanase expression vector of claim 1 in the fermentative preparation of a feed and/or feed additive.

Technical Field

The invention relates to the field of bioengineering, in particular to endoglucanase expression vectors and a construction method and application thereof.

Background

Endoglucanase (CelD) and cellobiase (CelS) are used as components of endo-cellulase and exo-cellulase with highest enzymatic activity in a cellulosome of Clostridium thermocellum, and are often used as feed enzymes, at present, the industrial production of the feed enzymes mainly adopts a eukaryotic cell fermentation method, but the cost of the eukaryotic fermentation production is usually not low without the production of prokaryotic cells, and the industrial production of the feed enzymes by using the prokaryotic cells is not realized because of the problems of self-toxin of prokaryotic organisms such as Escherichia coli and drug resistance genes contained in expression vectors.

The development of molecular biology technology makes it possible to express and produce recombinant protein in large scale by using host cells, and the safety of prokaryotic cells of feed enzymes is expressed by constructing an expression vector by using a gene expression method. However, when a target protein is expressed using a prokaryotic cell, the target protein is produced mainly using an antibiotic or an auxotrophic selection marker, which has a problem of resistance to the antibiotic and is susceptible to reversion.

Glutamine-6-phosphofructosylaminotransferase (Gfa), also known as 6-phosphoglucamine synthetase (Glm), is intracellular enzymes that catalyze the formation of 6-phosphoglucosamine and glutamate from the substrates glutamine and fructose-6-phosphate, i.e., the th reaction in the hexosamine anabolic pathway Gfa is present in almost every species and tissue and is essential for life activities.

Disclosure of Invention

The invention provides a endoglucanase expression vector, a construction method and application thereof, wherein the endoglucanase from clostridium thermocellum is subjected to prokaryotic expression, the expression vector takes a biologically safe glmS gene without antibiotic markers as a screening gene, and is transformed to a glmS deletion strain E.coli delta glmS to produce the endoglucanase.

In order to realize the purpose, the invention adopts the technical scheme that:

in embodiments, the invention provides endoglucanase expression vectors pHsh-GcelD, the nucleotide sequence of which is shown in SEQ ID No.9, wherein the screening gene of the expression vectors pHsh-GcelD is Escherichia coli glmS gene, the prokaryotic host cell is glmS gene-deficient Escherichia coli, the Escherichia coli glmS gene is connected with pHsH-Amp vector segment to obtain pHsh-GlmS plasmid vectors, and the endoglucanase celD gene is inserted into the pHsh-GlmS plasmid vectors, transformed into glmS gene-deficient Escherichia coli, and extracted.

In embodiments, the invention provides a method for constructing the endoglucanase expression vector pHsh-GcelD, which comprises the following steps:

(1) amplifying a glmS gene cDNA fragment by taking escherichia coli as a template, amplifying by taking pHsH-Amp as a template to obtain a pHsh vector fragment, connecting the phosphorylated glmS gene cDNA fragment with the flat end of the pHsh vector fragment, converting into gfa deletion type escherichia coli, and screening to obtain a pHsh-GlmS plasmid;

(2) and (2) amplifying a celD gene fragment by using the C.thermocellum genome as a template, inserting the celD gene fragment into the pHsh-GlmS plasmid obtained in the step (1), connecting the blunt ends, transforming into Escherichia coli E, coli delta glmS, selecting a transformant, and sequencing to obtain an expression vector pHsh-GcellD.

In the step (1), the nucleotide sequences of the primers for amplifying the glmS gene cDNA fragment are shown as SEQ ID No.1 and 2, and the nucleotide sequences of the primers for amplifying the pHsh vector fragment are shown as SEQ ID No.3 and 4;

in the step (1), the molar ratio of the glmS gene cDNA fragment to the pHsh vector fragment in the blunt end connection is 3: 1.

in the step (2), the nucleotide sequences of the primers for amplifying the celD gene fragment are shown as SEQ ID Nos. 5 and 6, and the nucleotide sequences of the primers for amplifying the pHsh-GlmS vector fragment are shown as SEQ ID Nos. 7 and 8.

In the step (2), the insertion of the celD gene fragment into the pHsh-GlmS plasmid obtained in the step (1) is to insert the celD gene fragment into an Hsh promoter expression frame of the pHsh-GlmS plasmid.

In embodiments, the invention also provides recombinant escherichia coli, wherein the recombinant escherichia coli is obtained by transforming the pHsh-GcelD plasmid into an escherichia coli E.coli delta gfa strain.

In embodiments, the invention provides the use of the endoglucanase expression vector pHsh-GcelD in the field of fermentation, in particular in the preparation of feed and/or feed additives by fermentation.

The invention has the beneficial effects that:

the invention provides endoglucanase expression vectors, wherein a screening gene of the endoglucanase expression vectors is an escherichia coli glmS gene, a prokaryotic host cell is glmS gene defective escherichia coli, a protein produced after E.coli delta glmS transformation is only escherichia coli self protein and endoglucanase protein but does not contain other foreign proteins such as antibiotics and the like, and the endoglucanase expression vectors have relative safety, can eliminate toxicity of escherichia coli and ensure activity of endoglucanase through fermentation and high-temperature inactivation treatment, so that the CelD gene from clostridium thermocellum (C. thermocellum ATCC 27405) realizes high-efficiency expression of biological safety.

Drawings

FIG. 1 is a schematic diagram of a constructed endoglucanase expression vector pHsh-GcelD;

FIG. 2 is an SDS-PAGE image of intracellular proteins following transformation of E.coli with the pHsh-GcelD plasmid; in the figure, M is a DNA molecular weight standard, lane 1 represents no-load transformation of Escherichia coli, lane 2 represents pHsh-GcelD transformed Escherichia coli intracellular protein expression.

Detailed Description

The present invention is further defined by the following examples , from which the basic features of the invention can be ascertained by those skilled in the art and from the above descriptions and examples, and various modifications and changes can be made to the invention to adapt it to various usages and conditions without departing from the spirit and scope of the invention.