A polyselenocysteine ​​selenoprotein P organic selenium preparation

Abstract

The present invention provides a polyselenocysteine ​​selenoprotein P organic selenium preparation, belonging to the technical field of feed additive preparation. The polyselenocysteine ​​selenoprotein P organic selenium preparation is prepared by constructing a recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein P, fermenting and culturing the polyselenocysteine ​​selenoprotein P, and spray-drying the fermented bacterial liquid. The selenium-enriched amount of the yeast strain expressing polyselenocysteine ​​selenoprotein P is 1.69 times the selenium-enriched amount of the yeast expressing natural selenoprotein P, and 3.7 times that of wild Pichia pastoris. The polyselenocysteine ​​selenoprotein P organic selenium preparation prepared by fermentation of the recombinant Pichia pastoris has a high organic selenium content, which solves the problem of low selenium conversion efficiency in the preparation of organic selenium by traditional methods.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additive preparation, and in particular relates to a polyselenocysteine ​​selenoprotein P organic selenium preparation. Background Art

[0002] Selenium is an essential trace element for the health of humans and animals. It has important biological functions in anti-oxidation, anti-aging, lowering blood pressure, regulating immunity, preventing cancer and anti-cancer, and anti-atherosclerosis. It is also a component of some enzymes, including glutathione peroxidase, deiodinase iodothyronine and thioredoxin reductase. Selenium can protect cells from the harmful effects of free radicals formed during oxidation. In nature, selenium is mainly divided into two categories: organic selenium and inorganic selenium. Inorganic selenium usually exists in the form of selenium, selenate and selenite, while organic selenium is mainly formed by biotransformation and amino acids. It generally exists in the form of selenoproteins, selenopolysaccharides and selenonucleic acids, and has the characteristics of high bioavailability and low toxicity. Two-thirds of the land in my country is selenium-deficient, and a total of 22 provinces and 72% of counties are in a state of selenium deficiency. Animals cannot synthesize selenium independently and must obtain it from food. Therefore, the development of selenium-enriched products is of great significance to improve the state of selenium deficiency.

[0003] Microorganisms grow fast, are easy to culture, and have the ability to convert and enrich selenium. Using microorganisms to convert inorganic selenium (selenite) into organic selenium is an effective way to prepare animal selenium supplements. However, the selenium conversion ability of wild strains is based on the normal growth of microorganisms, and the selenium enrichment ability is limited. How to develop an efficient selenium conversion method to prepare organic selenium is an urgent problem to be solved in the animal feed industry. Summary of the invention

[0004] The invention aims to provide a polyselenocysteine ​​selenoprotein P organic selenium preparation, belonging to the technical field of feed additive preparation.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Firstly, the present invention provides an organic selenium preparation of polyselenocysteine ​​selenoprotein P, wherein the organic selenium preparation is an organic selenium preparation prepared by expressing polyselenocysteine ​​selenoprotein P by recombinant Pichia pastoris.

[0007] Furthermore, the amino acid sequence of the polyselenocysteine ​​selenoprotein P is SEQ ID NO.1.

[0008] Furthermore, the DNA sequence of the expression gene of the polyselenocysteine ​​selenoprotein P is SEQ ID NO.2.

[0009] Furthermore, the preparation method of the organic selenium preparation is as follows:

[0010] (1) The recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein P was inoculated with YPD culture medium at a volume ratio of 1%, and cultured at 30°C and 250 r / min in a shaking incubator for 24 h as the primary seed liquid;

[0011] (2) According to the fermentation volume, the first-stage seed solution was inoculated with YPD culture solution at a volume ratio of 1% to prepare the second-stage seed solution and multi-stage seed solution;

[0012] (3) The multi-stage seed liquid was inoculated with a YPD culture medium containing 100 mg / L sodium selenite at a volume ratio of 1%, and the fermentation tank parameters were set to a stirring speed of 500-800 r / min, a tank pressure of 9 psi, a temperature of 30° C., a DO value (dissolved oxygen) set at more than 30%, and the fermentation culture was carried out for 24 h;

[0013] (4) The fermentation temperature was adjusted to 25°C and the fermentation was carried out for 96 hours;

[0014] (5) The fermented bacterial liquid is dried into powder by a spray dryer, and then sealed and packaged to prepare a polyselenocysteine ​​selenoprotein P organic selenium preparation.

[0015] Secondly, the present invention provides a poly-selenocysteine ​​selenoprotein P, the amino acid sequence of the poly-selenocysteine ​​selenoprotein P is SEQ ID NO.1.

[0016] Thirdly, the present invention provides a gene for expressing polyselenocysteine ​​selenoprotein P, and the DNA sequence of the gene is SEQ ID NO.2.

[0017] The beneficial effects of the present invention are that a recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein P is constructed, polyselenocysteine ​​selenoprotein P is expressed by fermentation culture, and the fermented bacterial liquid is spray-dried to prepare a polyselenocysteine ​​selenoprotein P organic selenium preparation. The selenium-enriched amount of the yeast strain expressing polyselenocysteine ​​selenoprotein P is 1.69 times the selenium-enriched amount of the yeast expressing natural selenoprotein P, and 3.7 times that of wild Pichia pastoris. The yeast strain X33-selenoprotein Pplus is fermented and cultured in a fermenter, and the selenium content in the supernatant of the yeast liquid lysis is 93.69 mg / L, and the selenium conversion rate is 93.69%. The polyselenocysteine ​​selenoprotein P organic selenium preparation prepared by fermentation of recombinant Pichia pastoris has a high organic selenium content, which solves the problem of low selenium conversion efficiency of preparing organic selenium by traditional methods. The method of the present invention has a high selenium conversion rate for preparing the polyselenocysteine ​​selenoprotein P organic selenium preparation, can efficiently convert inorganic selenium into safe, highly bioavailable organic selenium, and is easy to mass-produce, effectively solving the demand for organic selenium in industries such as food and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1: SDS-PAGE detection of selenoprotein P.

[0019] Figure 1 Label numbers: M: Protein ladder, 1: X33 yeast, 2: X33-selenoprotein Pplus yeast, 3: X33-selenoprotein P.

[0020] Figure 2 :Detection chart of selenium content in cultured yeast. DETAILED DESCRIPTION

[0021] Example 1: Design of polyselenocysteine ​​selenoprotein P based on computer-aided design

[0022] The GenBank accession number of the selenoprotein P gene sequence is: NM_005410.4, of which 71 to 1216 bp are the selenoprotein P coding sequence, and the translated selenoprotein P amino acid sequence is NP_005401.3. 1217 to 1974 bp are the selenocysteine ​​UGA translation regulatory region sequence, which regulates the translation of UGA into selenocysteine ​​during protein translation, thereby generating protein selenium; without this regulatory sequence, UGA in eukaryotic cells is a stop codon during protein translation and cannot synthesize amino acids.

[0023] There are 10 selenocysteines on natural selenoprotein P. The amino acid sequence of selenoprotein P NP_005401.3 was analyzed using Discovery Studio software to find areas with less steric hindrance to design selenocysteine ​​entry and exit sites. An additional 8 selenocysteines were designed and inserted to form a polyselenocysteine ​​selenoprotein P, whose amino acid sequence is SEQ ID NO.1.

[0024] Example 2: Construction of recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein P

[0025] 1.1 Gene synthesis

[0026] (1) The coding sequence of polyselenocysteine ​​selenoprotein P was synthesized according to the amino acid sequence of polyselenocysteine ​​selenoprotein P SEQ ID NO.1, followed by a selenocysteine ​​UGA translation regulatory region sequence, and EcoRI and NotI restriction endonuclease sites were added to the upstream and downstream of the synthetic gene, respectively. The DNA sequence of the synthetic gene was SEQ ID NO.2. In the amino acid sequence of SEQ ID NO.1, U represents selenocysteine, and the coding sequence in the corresponding DNA sequence is TGA. The SEQ ID NO.2 gene was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pUC57 plasmid to obtain the pUC57-selenoprotein Pplus plasmid.

[0027] (2) Based on the natural selenoprotein P coding sequence and selenocysteine ​​UGA translation regulatory region sequence in the NM_005410.4 sequence, a 71-1974 bp gene sequence was synthesized, and EcoRI and NotI restriction endonuclease sites were added upstream and downstream of the synthetic gene, respectively. The synthetic gene DNA sequence was SEQ ID NO.3, which was commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis and cloned into the pUC57 plasmid to obtain the pUC57-selenoprotein P plasmid.

[0028] 1.2 Construction of recombinant expression vector

[0029] (1) Double digestion of pUC57-selenoprotein Pplus and pUC57-selenoprotein P plasmids with restriction endonucleases EcoRI and NotI to recover selenoprotein Pplus and selenoprotein P gene fragments, respectively;

[0030] (2) using restriction endonucleases EcoRI and NotI to double-digest the expression vector pGAPZA and recover the gene fragment;

[0031] (3) The selenoprotein Pplus and pGAPZA nucleic acid fragments were ligated with T4 DNA ligase, and the selenoprotein P and pGAPZA nucleic acid fragments were ligated with T4 DNA ligase, respectively, and transformed into E. coli DH5α competent cells, and the plasmids were extracted and identified by double restriction digestion with EcoRI and NotI to obtain recombinant expression plasmids pGAPZA-selenoprotein Pplus and pGAPZA-selenoprotein P.

[0032] 1.3 Construction of recombinant Pichia pastoris strains expressing selenoprotein P and polyselenocysteine ​​selenoprotein P

[0033] (1) After the recombinant plasmid pGAPZA-selenoprotein Pplus was confirmed to be correct by restriction digestion and sequencing, it was electroporated into Pichia pastoris X33 competent cells. 100 μL of the electroporated cells were spread on a 100 μg / mL bleomycin YPD screening plate and cultured at 25-30°C for 3-5 days. Single colonies on the bleomycin YPD plate were picked and inoculated into a 50 mL Corning mini-bioreactor (EP tube with a breathable membrane hole on the lid). 5 mL of YPD medium was added to each tube and cultured at 30°C, 250 rpm for 48 hours as the seed solution, named X33-selenoprotein Pplus.

[0034] (2) After the recombinant plasmid pGAPZA-selenoprotein P was confirmed to be correct by restriction digestion and sequencing, it was electroporated into Pichia pastoris X33 competent cells. 100 μL of the electroporated cells were spread on a 100 μg / mL bleomycin YPD screening plate and cultured at 25-30°C for 3-5 days. Single colonies on the bleomycin YPD plate were picked and inoculated into a 50 mL Corning mini-bioreactor (EP tube with a breathable membrane hole on the lid). 5 mL of YPD medium was added to each tube and cultured at 30°C, 250 rpm for 48 hours as the seed solution, named X33-selenoprotein P.

[0035] 1.4 Expression of selenoprotein P and polyselenocysteine ​​selenoprotein P

[0036] (1) Take 1 ml of seed liquid of yeast strains X33, X33-selenoprotein Pplus and X33-selenoprotein P, respectively, and inoculate 100 ml of YPD culture medium (containing sodium selenite at a concentration of 100 mg / L) at a volume ratio of 1%, and culture at 30°C, 250 r / min, and shake for 24 h. Each strain was inoculated in three culture bottles.

[0037] (2) Adjust the shaking temperature to 25°C and culture at 250 rpm for 96 h.

[0038] (3) Centrifuge at 6000 rpm for 10 minutes and collect the bacterial precipitate.

[0039] (4) Resuspend the bacterial pellet in 50 ml of sterile PBS, centrifuge at 6000 rpm for 10 min, collect the bacterial pellet, and repeat the washing process three times.

[0040] (5) Resuspend the bacterial pellet in 10 ml of sterile PBS and break up the yeast solution using a high-pressure homogenizer.

[0041] (6) Centrifuge at 12000 rpm for 10 min, collect the supernatant, and detect the expression of selenoprotein P by SDS-PAGE protein gel electrophoresis.

[0042] (7) Take 1 ml of the centrifuged supernatant and add 9 ml of sterile PBS to dilute it to the original fermentation liquid concentration. Determine the selenium content in the supernatant by microwave digestion-atomic fluorescence spectrometry.

[0043] from Figure 1It can be seen that the yeast strain X33-selenoprotein Pplus and X33-selenoprotein P lysate have obvious protein bands expressed between 40 and 55 kD, which is consistent with the size of polyselenocysteine ​​selenoprotein P and selenoprotein P, while the wild-type yeast strain X33 does not have this protein band.

[0044] from Figure 2 It can be seen that the selenium content in the lysis supernatant of the wild-type yeast strain X33 is 24.21 mg / L, and the selenium conversion rate is 24.21%; the selenium content in the lysis supernatant of the yeast strain X33-selenoprotein P is 53.29 mg / L, and the selenium conversion rate is 53.29%; the selenium content in the lysis supernatant of the yeast strain X33-selenoprotein Pplus is 89.86 mg / L, and the selenium conversion rate is 89.86%, which is 1.69 times the selenium-enriched amount of yeast expressing natural selenoprotein P and 3.7 times that of wild Pichia pastoris.

[0045] Example 3: Fermentation preparation of polyselenocysteine ​​selenoprotein P

[0046] (1) Take 0.1 ml of seed solution and inoculate 10 ml of YPD culture medium at a volume ratio of 1%, and culture at 30°C and 250 r / min in a shaking incubator for 24 h as the primary seed solution.

[0047] (2) 10 ml of the first-level seed solution was transferred to 1000 ml of YPD culture medium at a volume ratio of 1%, and cultured in a shaking incubator at 30°C and 250 r / min for 24 h as the second-level seed solution.

[0048] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and perform flow calibration of the peristaltic pump.

[0049] (4) Prepare 70 L of YPD medium (containing sodium selenite at a concentration of 100 mg / L), add it to a 100 L fermenter, and sterilize the medium, fermenter, and pipes at 121°C for 30 min.

[0050] (5) When the culture solution in the fermentation tank is cooled to 30°C, 700 ml of the secondary seed solution is added to the fermentation tank and the fermentation tank culture is started. The fermentation tank parameters are set to a stirring speed of 500-800 r / min, a tank pressure of 9 psi, a temperature of 30°C, and a DO value (dissolved oxygen) set above 30%, and the fermentation culture is carried out for 24 hours.

[0051] (6) The fermentation temperature was adjusted to 25° C. and the fermentation was carried out for 96 h. The fermentation broth was taken and the organic selenium content was detected according to the method of Example 2.

[0052] (7) The fermented bacterial liquid is dried into powder by a spray dryer, and then sealed and packaged to prepare a polyselenocysteine ​​selenoprotein P organic selenium preparation.

[0053] The yeast strain X33-selenoprotein Pplus was fermented in a fermenter, and the selenium content in the supernatant of the yeast liquid lysis was 93.69 mg / L, and the selenium conversion rate was 93.69%. The method of the present invention has a high selenium conversion rate for preparing the polyselenocysteine ​​selenoprotein P organic selenium preparation, can efficiently convert inorganic selenium into safe, highly bioavailable organic selenium, and is easy to mass produce, effectively solving the demand for organic selenium in the food, feed and other industries.

Claims

1. A polyselenocysteine ​​selenoprotein P organic selenium preparation, characterized in that: The organic selenium preparation is an organic selenium preparation prepared by expressing polyselenocysteine ​​selenoprotein P by recombinant Pichia pastoris, and the amino acid sequence of the polyselenocysteine ​​selenoprotein P is SEQ ID NO.

1.

2. The organic selenium preparation according to claim 1, characterized in that The DNA sequence of the expression gene of the polyselenocysteine ​​selenoprotein P is SEQ ID NO.

2.

3. The organic selenium preparation according to claim 1, characterized in that The preparation method of the organic selenium preparation is as follows: (1) The recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein P was inoculated with YPD culture medium at a volume ratio of 1%, and cultured at 30°C and 250 r / min in a shaking incubator for 24 h as the primary seed liquid; (2) According to the fermentation volume, the first-stage seed solution was inoculated with YPD culture solution at a volume ratio of 1% to prepare the second-stage seed solution and multi-stage seed solution; (3) The multi-stage seed liquid was inoculated with a YPD culture medium containing 100 mg / L sodium selenite at a volume ratio of 1%, and the fermentation tank parameters were set to a stirring speed of 500-800 r / min, a tank pressure of 9 psi, a temperature of 30° C., a DO value (dissolved oxygen) set at more than 30%, and the fermentation culture was carried out for 24 h; (4) The fermentation temperature was adjusted to 25°C and the fermentation was carried out for 96 hours; (5) The fermented bacterial liquid is dried into powder by a spray dryer, and then sealed and packaged to prepare a polyselenocysteine ​​selenoprotein P organic selenium preparation.

4. A polyselenocysteine ​​selenoprotein P, characterized in that The amino acid sequence of the polyselenocysteine ​​selenoprotein P is SEQ ID NO.

1.

5. A gene for expressing polyselenocysteine ​​selenoprotein P, characterized in that: The DNA sequence of the gene is SEQ ID NO.2.

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