A polyselenocysteine ​​selenoprotein W organic selenium preparation

Abstract

The present invention provides a polyselenocysteine ​​selenoprotein W organic selenium preparation, belonging to the field of biotechnology. The polyselenocysteine ​​selenoprotein W organic selenium preparation is prepared by constructing a recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein W, fermenting and culturing the polyselenocysteine ​​selenoprotein W, and spray-drying the fermented bacterial liquid. The selenium-enriched amount of the yeast strain expressing polyselenocysteine ​​selenoprotein W is 2.98 times the selenium-enriched amount of the yeast expressing natural selenoprotein W, and 3.89 times that of wild Pichia pastoris. The polyselenocysteine ​​selenoprotein W organic selenium preparation prepared by fermentation of recombinant Pichia pastoris has a high organic selenium content, and the organic selenium preparation is easy to absorb when fed, which can effectively increase the selenium content in the blood of white-feathered broilers, reduce the feed-to-meat ratio of white-feathered broilers, and improve the growth performance of white-feathered broilers.

Description

Technical Field

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

[0002] Trace element selenium plays a very important biological function in organisms, such as anti-aging, enhancing body immunity, and promoting growth and development. Selenium deficiency can cause diseases such as reduced body immunity, white muscle disease, diarrhea, edema, liver necrosis, etc., which leads to animal stunting, decreased production level, and decreased fertility. However, the safety limit between the nutritional dose and the toxic dose of selenium is very narrow. In actual production, people have long adopted the addition of inorganic selenium (mainly sodium selenite) to feed to meet the needs of animal growth. Due to the strong toxicity of sodium selenite, it brings many inconveniences to its production and use. Some countries have restricted the use of inorganic salts as a nutritional supplement for selenium, and the development of efficient organic selenium sources has become the focus of selenium nutrition research. In recent years, with the deepening of research, people have found that compared with inorganic selenium, organic selenium has a higher absorption rate and biosafety, and the development of efficient organic selenium sources has become one of the research hotspots of animal nutrition at home and abroad.

[0003] Microorganisms grow fast, are easy to culture, and have the ability to transform and enrich selenium. Using microorganisms to transform inorganic selenium (selenite) into organic selenium is an effective way to prepare animal selenium supplements. However, the selenium transformation ability of wild strains is based on the normal growth of microorganisms, and their selenium enrichment ability is limited. By introducing exogenous selenoproteins into microorganisms with selenium enrichment ability through genetic engineering technology, their selenium enrichment ability can be improved, and then large-scale fermentation can be used to prepare organic selenium preparations, which can effectively solve the problem of shortage of organic selenium resources. Summary of the invention

[0004] The invention aims to provide a polyselenocysteine ​​selenoprotein W organic selenium preparation, belonging to the field of biotechnology.

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

[0006] First, the present invention provides a polyselenocysteine ​​selenoprotein W organic selenium preparation, wherein the organic selenium preparation is an organic selenium preparation prepared by expressing polyselenocysteine ​​selenoprotein W in recombinant Pichia pastoris.

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

[0008] Furthermore, the DNA sequence of the expression gene of the polyselenocysteine ​​selenoprotein W 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 W was inoculated with YPD culture medium at a volume ratio of 1%, and cultured at 30°C and 250 rpm 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 W organic selenium preparation.

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

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

[0017] Fourthly, the present invention provides a gene for expressing selenoprotein W, and the DNA sequence of the gene is SEQ ID NO.3.

[0018] The beneficial effect of the present invention is that two selenocysteine ​​U are additionally introduced into the amino acid sequence of selenoprotein W, and the selenoprotein P gene regulatory sequence, i.e., the selenocysteine ​​UGA translation regulatory region sequence, is used to regulate the translation of UGA on mRNA into selenocysteine ​​during protein translation. A recombinant Pichia pastoris expressing polyselenocysteine ​​selenoprotein W is constructed, and polyselenocysteine ​​selenoprotein W is expressed by fermentation culture, and the fermentation liquid is spray-dried to prepare a polyselenocysteine ​​selenoprotein W organic selenium preparation. The selenium-enriched amount of the yeast strain expressing polyselenocysteine ​​selenoprotein W is 2.98 times the selenium-enriched amount of the yeast expressing natural selenoprotein W, and 3.89 times that of wild Pichia pastoris. The yeast strain X33-selenoprotein Wplus is fermented and cultured in a fermenter, and the selenium content in the supernatant of the yeast liquid lysis is 95.27 mg / L, and the selenium conversion rate is 95.27%. The polyselenocysteine ​​selenoprotein W organic selenium preparation prepared by recombinant Pichia pastoris fermentation has a high organic selenium content, which solves the problem of low selenium conversion efficiency in the preparation of organic selenium by traditional methods. The method of the present invention has a high selenium conversion rate for preparing the polyselenocysteine ​​selenoprotein W organic selenium preparation, can efficiently convert inorganic selenium into safe organic selenium with high bioavailability, is easy to produce on a large scale, and effectively solves the demand for organic selenium in the food, feed and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Chart of detection of selenium content in cultured yeast.

[0020] Figure 2 :Detection chart of selenium content in blood of experimental chickens.

[0021] Figure 3 : Statistical chart of feed-to-meat ratio of experimental chickens. DETAILED DESCRIPTION

[0022] Example 1: Design of polyselenocysteine ​​selenoprotein W based on computer-aided design

[0023] The GenBank accession number of the amino acid sequence of selenoprotein W is AAL00898.1, and the 13th amino acid is selenocysteine ​​U. The amino acid sequence of selenoprotein W was analyzed using Discovery Studio software to find the region with less steric hindrance to design the selenocysteine ​​insertion site, and two additional selenocysteines were designed to insert to form a polyselenocysteine ​​selenoprotein W, whose amino acid sequence is SEQ ID NO.1.

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

[0025] 1.1 Gene synthesis

[0026] (1) The polyselenocysteine ​​selenoprotein W coding sequence was synthesized according to the amino acid sequence of polyselenocysteine ​​selenoprotein W SEQ ID NO.1, followed by a 1217-1974 bp nucleotide sequence of the selenoprotein P translation regulatory region sequence NM_005410.4. This sequence is 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 selenocysteine ​​cannot be synthesized.

[0027] (2) EcoRI and NotI restriction endonuclease sites were added to the upstream and downstream of the synthetic gene, respectively, and the DNA sequence of the synthetic gene was SEQ ID NO. 2. In the amino acid sequence of SEQ ID NO. 1, U is selenocysteine, and the coding sequence in the corresponding DNA sequence is TGA. The SEQ ID NO. 2 gene was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and cloned into the pUC57 plasmid to obtain the pUC57-selenoprotein Wplus plasmid.

[0028] (3) The coding sequence of natural selenoprotein W AAL00898.1 was synthesized based on its amino acid sequence, followed by a 1217-1974 bp nucleotide sequence of the selenoprotein P translation regulatory region sequence NM_005410.4. 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 be synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pUC57 plasmid to obtain the pUC57-selenoprotein W plasmid.

[0029] 1.2 Construction of recombinant expression vector

[0030] (1) Double digesting pUC57-selenoprotein Wplus and pUC57-selenoprotein W plasmids with restriction endonucleases EcoRI and NotI to recover selenoprotein Wplus and selenoprotein W gene fragments, respectively;

[0031] (2) using restriction endonucleases EcoRI and NotI to double-digest the expression vector pGAPZA and recover the gene fragment;

[0032] (3) The selenoprotein Wplus and pGAPZA nucleic acid fragments were ligated with T4 DNA ligase, and the selenoprotein W and pGAPZA nucleic acid fragments were ligated with T4 DNA ligase, and transformed into E. coli DH5α competent cells, respectively. The plasmids were extracted and identified by double digestion with EcoRI and NotI to obtain recombinant expression plasmids pGAPZA-selenoprotein Wplus and pGAPZA-selenoprotein W.

[0033] 1.3 Construction of recombinant Pichia pastoris strains expressing selenoprotein W and polyselenocysteine ​​selenoprotein W

[0034] (1) After the recombinant plasmid pGAPZA-selenoprotein Wplus 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 Wplus.

[0035] (2) After the recombinant plasmid pGAPZA-selenoprotein W 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 W.

[0036] 1.4 Expression of selenoprotein W and polyselenocysteine ​​selenoprotein W

[0037] (1) Take 1 ml of seed liquid of yeast strains X33, X33-selenoprotein Wplus and X33-selenoprotein W, 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.

[0038] (2) Adjust the shaking temperature to 25°C and culture at 250 rpm for 96 h.

[0039] (3) Centrifuge at 6000 rpm for 10 minutes and collect the bacterial precipitate.

[0040] (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.

[0041] (5) Resuspend the bacterial pellet in 10 ml of sterile PBS and break up the yeast solution using a high-pressure homogenizer.

[0042] (6) Centrifuge at 12000 rpm for 10 minutes and collect the supernatant.

[0043] (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.

[0044] from Figure 1 It can be seen that the selenium content in the lysis supernatant of the wild-type yeast strain X33 is 23.99 mg / L, and the selenium conversion rate is 23.99%; the selenium content in the lysis supernatant of the yeast strain X33-selenoprotein W is 31.26 mg / L, and the selenium conversion rate is 31.26%; the selenium content in the lysis supernatant of the yeast strain X33-selenoprotein Wplus is 93.25 mg / L, and the selenium conversion rate is 93.25%, which is 2.98 times the selenium-enriched amount of yeast expressing natural selenoprotein P and 3.89 times that of wild Pichia pastoris.

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

[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 W organic selenium preparation.

[0053] The yeast strain X33-selenoprotein Wplus was fermented in a fermenter, and the selenium content in the supernatant of the yeast lysis liquid was 95.27 mg / L, and the selenium conversion rate was 95.27%. The selenium content in the freeze-dried polyselenocysteine ​​selenoprotein W organic selenium preparation was 3658 mg / kg.

[0054] Example 4: Effects of polyselenocysteine ​​selenoprotein W on broilers

[0055] (1) One hundred 7-day-old healthy white-feathered broilers were randomly divided into two groups, 50 in each group, and kept in isolation. The first group was the control group, which was fed with a basic feed; the second group was the experimental group, which was fed with 10 g / kg of polyselenocysteine ​​selenoprotein W organic selenium preparation in the basic feed.

[0056] (2) The chickens were fed once at 8:00 and 16:00 every day, and were allowed to eat and drink freely. The experimental period was 21 days. During the feeding period, the chickens were immunized and managed according to the conventional immunization procedures for white-feathered broilers. The growth of the experimental chickens was observed every day. Seven days before the start of the experiment, 10 experimental chickens were randomly selected from each group every day, and anticoagulated blood was collected. The selenium content in the blood was determined by microwave digestion-atomic fluorescence spectrometry, and the geometric mean was calculated. At the end of the experiment, the feed-to-meat ratio of each group was calculated.

[0057] from Figure 2 It can be seen that in the first 5 days of feeding the organic selenium preparation, the selenium content in the blood of the white-feathered broilers fed with 10g / kg of polyselenocysteine ​​selenoprotein W organic selenium preparation in the basic feed increased day by day, from 0.4mg / L to 1.32mg / L, and then remained stable. In the control group of chickens fed only the basic feed, the selenium content in the blood only increased from 0.4mg / L to 0.58mg / L within the 8 days of the test, and remained at a low level. Feeding organic selenium preparations is easy to absorb and can effectively increase the selenium content in the blood of white-feathered broilers.

[0058] from Figure 3It can be seen that the feed-to-meat ratio of the control group chickens fed only with basic feed was 2.08:1 after 21 days of feeding, and the feed-to-meat ratio of the white-feathered broilers fed with 10g / kg of polyselenocysteine ​​selenoprotein W organic selenium preparation added to the basic feed was 1.78:1. Feeding organic selenium preparations can reduce the feed-to-meat ratio of white-feathered broilers and improve the growth performance of white-feathered broilers.

[0059] The method of the present invention has a high selenium conversion rate for preparing the polyselenocysteine ​​selenoprotein W organic selenium preparation, can efficiently convert inorganic selenium into safe organic selenium with high bioavailability, is easy to produce on a large scale, and effectively solves the demand for organic selenium in the food, feed and other industries.

Claims

1. A polyselenocysteine ​​selenoprotein W organic selenium preparation, characterized in that: The organic selenium preparation is an organic selenium preparation prepared by expressing polyselenocysteine ​​selenoprotein W by recombinant Pichia pastoris, and the amino acid sequence of the polyselenocysteine ​​selenoprotein W 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 W 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 W 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 YPD culture medium containing sodium selenite at a concentration of 90 mg / L 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 120 h; (4) The fermented bacterial liquid is dried into powder by a spray dryer, and then sealed and packaged to prepare a polyselenocysteine ​​selenoprotein W organic selenium preparation.

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

1.

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

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