阅读说明：本技术 利用外源金属硫蛋白构建酵母发酵产高含硒蛋白的方法 (Method for producing high-selenium-content protein by constructing yeast fermentation by using exogenous metallothionein ) 是由 马云峰 王松廷 李华 于 2019-06-27 设计创作，主要内容包括：本发明公开了利用外源金属硫蛋白构建酵母发酵产高含硒蛋白的方法,以酵母菌作为原始菌株,设计引物对YES2-MT2和引物对MT2-yes2-F,经遗传转化得到基因工程菌,基因工程菌再经过发酵生产高含硒蛋白；本发明以酿酒酵母(Saccharomyces cerevisiae)作为原始菌株,经过外源基因遗传转化得到基因工程菌,基因工程菌再经过发酵生产以及分离纯化得到高含硒蛋白,无机硒转化为硒蛋白的总效率高达22％,可实现工业化生产。(The invention discloses a method for producing high-selenium protein by constructing yeast fermentation by utilizing exogenous metallothionein, which comprises the steps of taking yeast as an original strain, designing a primer pair YES2-MT2 and a primer pair MT2-YES2-F, obtaining genetic engineering bacteria through genetic transformation, and fermenting the genetic engineering bacteria to produce the high-selenium protein; the invention takes Saccharomyces cerevisiae as an original strain, obtains genetically engineered bacteria through exogenous gene genetic transformation, obtains high selenium-containing protein through fermentation production and separation and purification of the genetically engineered bacteria, has the total efficiency of converting inorganic selenium into the selenium protein as high as 22 percent, and can realize industrial production.)

1. A method for producing high-selenium protein by yeast fermentation through constructing exogenous metallothionein is characterized in that yeast is used as an original strain, a primer pair YES2-MT2 and a primer pair MT2-YES2-F are designed, genetic engineering bacteria are obtained through genetic transformation, and the high-selenium protein is produced through fermentation of the genetic engineering bacteria;

designing a primer pair YES2-MT2 and a primer pair MT2-YES2-F, wherein the sequences are as follows:

YES2-MT2-R：AGCAGTTGGGGTCCATGGATCCGAGCTCGGTACCAAGCTTA

YES2-MT2-F：GCTGTGCCGACGACGACAAGatGGGTAAGGGAGAAGAACTT

MT2-yes2-F:GGTACCGAGCTCGGATCCATGGACCCCAACTGCTCCTGTG

MT2-Yes2-R:TTACCCATCTTGTCGTCGTCGGCACAGCAGCTGCACTTGT

wherein the primer pair YES2-MT2 is used for reversely amplifying the vector sequence with MT2 homologous arm, and the primer pair MT2-YES2-F is used for amplifying the MT2 segment.

2. The method for producing selenoprotein by fermentation of yeast constructed from exogenous metallothionein as claimed in claim 1, wherein said yeast is Saccharomyces cerevisiae.

3. The method for producing the high-selenium protein by yeast fermentation through exogenous metallothionein construction as claimed in claim 1, wherein the genetically engineered bacteria are subjected to active culture, inoculated into a seed culture medium according to the inoculation amount of 1-10%, and aerobically cultured for 4-8 h at 28-30 ℃; and then inoculating the cultured seeds into a liquid fermentation culture medium according to the inoculation amount of 2-5%. Wherein the seed culture medium is a potato liquid culture medium, and the pH of the culture medium is natural.

4. The method for producing high selenium protein by fermentation of yeast constructed by exogenous metallothionein as claimed in claim 3, wherein the seed culture is aerobically cultured at 28-30 deg.C for 4-8 h, sodium selenite is added into the culture medium, and the concentration is 0.1-10 mmol/L; the method is carried out in a shake flask, and the rotation speed of a shaking table is 160-230 r/min; the fermentation culture is carried out for 4-8 h at 28-30 ℃ in an aerobic way, the ventilation quantity of a fermentation tank is 0.5-1 vvm, and the stirring speed is 100-600 r/min.

5. The method for producing high selenium protein by yeast fermentation using exogenous metallothionein as claimed in claim 1, wherein after fermentation is finished, the fermentation broth is centrifuged, the precipitate is retained, buffer solution is added, and cell wall is broken; centrifuging to obtain supernatant, hydrolyzing with enterokinase, and centrifuging to obtain supernatant.

Technical Field

The invention relates to a method for producing high selenium-containing protein by constructing yeast through exogenous metallothionein, in particular to a method for producing high selenium-containing protein by fermenting Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is used as an original strain, obtaining genetic engineering bacteria through exogenous gene genetic transformation, and producing the high selenium-containing protein through the fermentation of the genetic engineering bacteria.

Background

Selenium is a trace element essential to organisms, and has the functions of resisting oxidation, resisting aging, improving immunity, preventing cardiovascular diseases, cancers and the like. However, the selenium nutrition of people in many areas of China is insufficient, selenium-containing food needs to be supplemented, and researches show that the organic selenium compound can be absorbed and utilized by human bodies better. The microbial fermentation synthesis of organic selenium has the characteristics of rapidness, stability and high efficiency. However, the following disadvantages exist in the existing studies: if the separation and analysis of the organic selenium compounds in the thalli are not sufficient; insufficient research on the limiting factors of the synthesis of the organic selenium and the like.

The biological organic of selenium is mainly characterized in that inorganic selenium is synthesized into organic selenium through animals, plants and microorganisms, compared with the organic selenium synthesized by plants and animals, the microbial fermentation synthesis has the characteristics of short production period, industrial amplification and stable quality and yield, is more suitable for the industrial development of selenium-rich products, and achieves certain progress at home and abroad. Metallothionein (MT) is metal-binding protein which is widely present in organisms, has low molecular weight, is rich in cysteine, can be induced by metal, has wide biological functions, is mainly involved in storage, transportation and metabolism of trace elements, antagonizes ionizing radiation, eliminates hydroxyl free radicals, detoxifies heavy metals and the like. MT can protect cells from being damaged by heavy metals, is a high-efficiency scavenger of free radicals, can protect yeast from oxidative stress, is mainly involved in stress-resistant secondary metabolism from the function of metallothionein, is not or rarely involved in the basic metabolism of organisms, is more importantly rich in cysteine, can replace sulfur in the metallothionein to form selenomethionin, has small influence on the basic metabolism of the organisms, and enables organic selenium to be efficiently enriched in the cells.

The Zaojia utilizes the overlap extension PCR technology to split the mouse MT2 type gene into two target fragments, adopts the mode of two times of PCR to successfully synthesize the mouse MT2 type coding gene, and successfully clones the coding gene into a pMD18-T vector. The young cymbidium utilizes heavy metals to induce the production of metallothionein by Saccharomyces cerevisiae BD101 and Hansenula anomala BD 102. The problems of difficult later purification and the like can be caused by heavy metal induction, and the problems of difficult transformant detection and the like can be faced when the MT gene is directly constructed into a vector in a host. Therefore, a method for simply and rapidly detecting a successful transformant by transferring a foreign gene into a host is required. At present, no report of expressing and producing high selenium-containing protein in yeast by using exogenous metallothionein gene exists.

Disclosure of Invention

The invention aims to solve the technical problem that the method for efficiently enriching organic selenium in cells in the prior art is to overcome the defects that the problem that the later purification is difficult and the like due to heavy metal induction or direct expression of MT gene construction vectors in hosts and the problem that the transformant is difficult to detect due to direct expression of MT gene construction vectors in hosts and the like, and provides a method for producing high-selenium-containing protein by constructing yeast through exogenous metallothionein and fermenting.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention discloses a method for producing high selenium-containing protein by fermenting yeast genetic engineering bacteria transformed by a vector constructed by exogenous metallothionein and fusion protein genes, wherein a bacterial strain is Saccharomyces cerevisiae. Specifically, yeast is used as an original strain, a primer pair YES2-MT2 and a primer pair MT2-YES2-F are designed, genetic engineering bacteria are obtained through genetic transformation, and the genetic engineering bacteria are fermented to produce the high-selenium protein;

designing a primer pair YES2-MT2 and a primer pair MT2-YES2-F, wherein the sequences are as follows:

YES2-MT2-R：AGCAGTTGGGGTCCATGGATCCGAGCTCGGTACCAAGCTTA

YES2-MT2-F：GCTGTGCCGACGACGACAAGatGGGTAAGGGAGAAGAACTT

MT2-yes2-F:GGTACCGAGCTCGGATCCATGGACCCCAACTGCTCCTGTG

MT2-Yes2-R:TTACCCATCTTGTCGTCGTCGGCACAGCAGCTGCACTTGT

wherein the primer pair YES2-MT2 is used for reversely amplifying the vector sequence with MT2 homologous arm, and the primer pair MT2-YES2-F is used for amplifying the MT2 segment. The primer pair YES2-MT2 and the existing EGFP-pGK plasmid are used as template amplification vector frameworks, and the second primer pair and the previously constructed plasmid are used as templates to amplify the MT2 fragment. And processing the amplification product and then carrying out seamless cloning. Then, competent cells are transformed, LB plates containing Amp are coated, the culture is carried out at 37 ℃, the grown monoclone is subjected to colony PCR identification, and the identified positive clone is the recombinant clone. Extracting the plasmid for later use. Adding the plasmid into yeast competent cells, performing electrotransformation, screening positive recombinants, inoculating identified positive monoclonals into an MD culture medium, culturing at 30 ℃ for 3-5 days, respectively sampling, detecting protein expression condition, performing fluorescence detection, directly placing culture supernatant under an ultraviolet lamp, and observing the generation of fluorescence; WesternBlotting detection, and the generation of a target band is successful, so that the genetic engineering strain MT22 is obtained.

Inoculating the strain MT22 into a culture medium containing a carbon source, a nitrogen source, inorganic salt and water for culture and enzyme production, centrifugally collecting MT22 cells, and breaking the cells to separate and purify the high-selenium protein.

Performing active culture on the genetically engineered bacterium MT22, inoculating the genetically engineered bacterium MT22 into a seed culture medium according to the inoculation amount of 1-10%, and performing aerobic culture at 28-30 ℃ for 4-8 h; and then inoculating the cultured seeds into a liquid fermentation culture medium according to the inoculation amount of 2-5%. Wherein the seed culture medium is a potato liquid culture medium, and the pH of the culture medium is natural.

Further, carrying out aerobic culture on the seeds at 28-30 ℃ for 4-8 h, and adding sodium selenite into a culture medium, wherein the concentration is 0.1-10 mmol/L; the method is carried out in a shake flask, and the rotation speed of a shaking table is 160-230 r/min; the fermentation culture is carried out for 4-8 h at 28-30 ℃ in an aerobic way, the ventilation quantity of a fermentation tank is 0.5-1 vvm, and the stirring speed is 100-600 r/min. Preferably, the nitrogen source of the liquid fermentation medium is: one or more of yeast powder, beef extract, peptone and corn steep liquor.

Further, after fermentation is finished, centrifuging the fermentation liquor, keeping precipitate, adding a buffer solution, and breaking cell walls; centrifuging to obtain supernatant, hydrolyzing with enterokinase, and centrifuging to obtain supernatant. And then, performing SDS-PAGE separation determination to collect bands according to a protein Marker and a standard substance, and calculating the content of selenium in the target protein. The determination method comprises digesting the selenoprotein strip, carrying out derivatization reaction with 2, 3-diaminobenzene under acidic condition, and determining the content of selenium by high performance liquid chromatography. Preferably, the hydrolysis is carried out by incubation with enterokinase at 37 ℃ for 30 minutes.

Selenoprotein conversion rate definition method: (selenium amount in high-selenium protein/amount of selenium put in) x 100%

The invention has the following beneficial effects: the invention takes Saccharomyces cerevisiae as an original strain, obtains genetically engineered bacteria through exogenous gene genetic transformation, obtains high selenium-containing protein through fermentation production and separation and purification of the genetically engineered bacteria, has the efficiency of converting inorganic selenium into the selenium protein as high as 22 percent, and can realize industrial production.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.