CN113322223B - Selenium-enriched yeast genetically engineered bacterium, surface display system thereof and construction method thereof - Google Patents
Selenium-enriched yeast genetically engineered bacterium, surface display system thereof and construction method thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to selenium-enriched yeast genetic engineering bacteria, a surface display system and a construction method thereof. Culturing the yeast genetic engineering bacteria by using a culture medium, and then adding sodium selenite for continuous culture to obtain the selenium-enriched yeast genetic engineering bacteria. Inserting goat gamma-interferon into His tag, and constructing pYD 1-CapIFN-gamma by taking the pYD1 shuttle plasmid as a framework; and then the pYD 1-CapIFN-gamma is introduced into selenium-enriched yeast genetic engineering bacteria to obtain the goat gamma-interferon selenium-enriched yeast display system. The system carries out selenium-rich transformation on the yeast in the system, and widens the application range of a goat gamma-interferon yeast expression system; the surface display of the goat gamma-interferon yeast is realized, and the antiviral activity of the goat gamma-interferon is more favorably exerted; lays a foundation for developing a safer, effective and cheaper novel yeast feed additive with antiviral and organism immunity enhancing functions.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to selenium-enriched yeast genetic engineering bacteria, a surface display system thereof and a construction method thereof.
Background
Interferon (IFN) is a cell secreted nonspecific broad-spectrum antiviral inducer protein, mainly glycoprotein, whose main biological functions include: broad-spectrum antiviral, immunomodulating, immunopotentiating, and antitumor, etc. Among them, the interferon for animals becomes a research hot spot by virtue of unique antiviral activity and immunoregulation, and the research on interferon for human, pig, cow, fowl and dog is relatively more, and the interferon produced by genetic engineering has been widely applied to prevention, control and treatment of animal epidemic diseases of fowl, pig and dog. The natural interferon is only produced under specific induction conditions, the content is low, the purification and the preservation are extremely difficult, the interferon obtained by the genetic engineering technology mainly has the defects of low expression quantity, inclusion bodies, high production cost and the like, and the large-scale popularization and application of the interferon in the livestock and poultry breeding industry are limited. Currently, emerging microbial surface display technologies, mainly including bacterial, phage and yeast surface display systems, have been applied in a number of fields: study of protein interactions and recognition, antibody production, oral vaccine production, whole cell catalyst and cell adsorbent production, and the like. Wherein, only the yeast surface display technology is a eukaryotic expression system, and the other 2 technologies all utilize a prokaryotic expression system, lack of modification after protein translation, and are not suitable for displaying eukaryotic proteins. The genetic engineering technology is used to select a proper system, so that the interferon can be expressed efficiently, which is clearly an effective way for promoting the popularization and application in veterinary clinic.
Selenium exists mainly in two forms of organic selenium and inorganic selenium, has important biological functions for human and animal organisms, and mainly comprises the following components: antioxidant effect, immunity effect, nutrition effect, cancer cell inhibition, animal fertility improvement, and heavy metal poisoning antidote. At present, most areas in China have the phenomenon of selenium deficiency, but the selenium content in natural foods is generally low, so that the selenium supplement is a very important problem. The saccharomyces cerevisiae has good selenium enrichment capability, can absorb and convert extracellular inorganic selenium into organic selenium through biological adsorption and active transportation modes, and is enriched in a large amount in cells to become selenium-enriched yeast, which is the most efficient, safer and balanced selenium-supplementing preparation in China so far. However, the research of using genetically engineered bacteria for selenium enrichment is less at present.
In recent years, the goat breeding industry in China is rapidly developed, the breeding scale is continuously increased, the epidemic disease types and harm are gradually increased, and the epidemic disease is one of important factors for limiting the development of the goat breeding industry. The harm of conditional pathogenic bacteria and virus diseases is most serious, the harm usually occurs when the goat has low immunity or the immune system is disturbed, once the goat is ill, the goat is often caused to secondarily infect a plurality of epidemic diseases, and the serious disease causes death, so that the goat epidemic disease prevention and control is greatly plagued. At present, the prevention and control of the goat epidemic disease mainly depend on antibiotics and vaccines, but with the arrival of the age of resistance reduction and resistance limitation, the antibiotics which can be used for clinic are very limited. Meanwhile, the department of agriculture has ordered to prohibit the use of antiviral drugs such as amantadine, ribavirin and the like in livestock and poultry farming production. In addition, part of vaccine products have various defects such as quality problems, timeliness problems and the like, even part of epidemic diseases are not available with proper drugs or vaccines, especially the epidemic diseases caused by continuously appearing new strains and variant strains of pathogenic microorganisms, and the prevention and control of goat epidemic diseases are serious, so that the improvement of goat immunity and disease resistance is very important.
Disclosure of Invention
In view of the above, the present invention aims to provide a selenium-enriched yeast genetically engineered bacterium. At present, yeasts used for selenium enrichment mainly comprise conventional strains such as Saccharomyces cerevisiae, russell's yeast, must yeast, phaffia rhodozyma, hansenula, torulopsis and candida, and the research of using genetically engineered bacteria for selenium enrichment is less.
The preparation method of the selenium-enriched yeast genetically engineered bacterium comprises the following steps: and culturing the yeast genetically engineered bacteria by using a culture medium, and adding sodium selenite to continue culturing when the yeast genetically engineered bacteria are cultured to a logarithmic phase.
Preferably, the yeast genetic engineering bacteria comprise Saccharomyces cerevisiae, pichia pastoris or yarrowia lipolytica; the Saccharomyces cerevisiae comprises EBY100, INVSc1 or AH109; the pichia pastoris comprises GS115, KM71, MC100-3, SMD1168, SMD1165 or SMD1163; the yarrowia lipolytica comprises Polf, polg or Polh.
Preferably, the concentration of the sodium selenite is 60 mug/mL.
In some embodiments, the preparation method of the selenium-enriched yeast genetically engineered bacterium comprises the following steps: (1) Selecting Saccharomyces cerevisiae EBY100, streaking and inoculating in YPD solid culture medium, culturing in a constant temperature incubator at 30 ℃, after single colony grows out, selecting single colony, inoculating in 10mL YPD liquid culture, culturing in a constant temperature incubator at 30 ℃, shaking and culturing at 250r/min for 24h, inoculating in 200mL YPD fresh culture medium according to 10% inoculum size, sampling 1mL every 12h, measuring the saccharomycete content, continuously culturing for 72h, and drawing a growth curve according to the thallus content at each time point. The strain EBY100 of Saccharomyces cerevisiae has been found to have a peak cell content of 210×10 when cultured for 36h 6 CFU·mL -1 . (2) According to the characteristics of a growth curve of saccharomyces cerevisiae, sodium selenite with different contents is added during 36h of culture, and the final concentrations are respectively as follows: the total selenium content was determined after incubation for 72h at 0. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL. By measurement, when the optimal addition amount of sodium selenite is 60 mug/mL, the selenium content of saccharomyces cerevisiae unit is 5.61mg/g.
The invention aims to provide a selenium-enriched yeast surface display system. The yeast surface display system comprises an expression vector, engineering bacteria and target proteins.
The engineering bacteria of the selenium-enriched yeast surface display system are the selenium-enriched yeast genetic engineering bacteria, and the selenium-enriched yeast genetic engineering bacteria comprise Saccharomyces cerevisiae EBY100, pichia pastoris GS115, pichia pastoris KM71, pichia pastoris X-33 or yarrowia lipolytica Polg.
The target protein of the selenium-enriched yeast surface display system can be any target protein that can be expressed in the selenium-enriched yeast display system.
Preferably, the target protein is goat gamma-interferon (capfn-gamma), and the nucleic acid Sequence of the goat gamma-interferon comprises a Sequence shown in Sequence No. 1. Based on the selenium-rich characteristic of yeast, the selenium-rich modification is innovatively carried out on a yeast surface display system, a goat gamma-interferon gene sequence containing a His tag is synthesized by people, the selenium-rich yeast is transformed by means of a pYD1 shuttle plasmid through electric shock, and the goat gamma-interferon is displayed on the surface of the selenium-rich yeast in a covalent bond mode through the action of GPI anchoring proteins, so that the goat gamma-interferon recombinant selenium-rich yeast with antiviral and immune activities is obtained, a new idea is provided for yeast surface display, and a foundation is laid for developing a safer, effective and low-cost novel yeast feed additive with antiviral and immunity enhancing functions.
Specifically, the advantages of interferon and selenium-enriched yeast in the aspects of antivirus and immunoregulation are combined, selenium-enriched transformation is carried out on a yeast surface display system, and the recombinant strain obtained after transformation has the biological function of selenium-enriched yeast, so that a new thought is provided for the research and development of novel livestock feed additives for antivirus and immunity improvement, and technical support is provided for the green healthy cultivation of goats and epidemic disease prevention and control. The recombinant yeast is obtained by integrating goat gamma-interferon with a yeast genome by taking a pYD1 shuttle plasmid as a skeleton through an electric shock transformation and homologous recombination mode, and displaying the goat gamma-interferon on the surface of the yeast in a covalent bond mode after induced expression. The biological activity based on gamma-interferon has the property of relying on the receptor on the target cell membrane, and the display of the gamma-interferon on the surface of yeast is more beneficial to the function exertion of the gamma-interferon. Further, a preparation method of a selenium-enriched yeast surface display system with target protein of goat gamma-interferon (CapIFN-gamma) is provided, which comprises the following steps: (1) Inserting the goat gamma-interferon into a His tag, and constructing the recombinant plasmid pYD 1-CapIFN-gamma by taking a pYD1 shuttle plasmid as a framework; (2) And introducing the pYD 1-CapIFN-gamma recombinant plasmid into the selenium-enriched yeast genetically engineered bacterium.
Preferably, in the step (2), the method of introducing the pYD1-CapIFN- γ recombinant plasmid into the yeast expression engineering bacterium is an electrotransformation method.
Preferably, before the pYD 1-CapIFN-gamma recombinant plasmid is introduced into the selenium-enriched yeast genetic engineering bacteria, the selenium-enriched yeast genetic engineering bacteria are prepared into competent cells, and the method for changing yeast expression engineering cells into competent cells is a conventional means in the technical field.
Preferably, the His tag Sequence comprises a Sequence as shown in Sequence No. 2.
In certain embodiments, the method for preparing the selenium-enriched yeast surface display system wherein the target protein is goat gamma-interferon comprises the following steps: (1) Artificially synthesizing a goat gamma-interferon gene sequence according to the codon preference of saccharomyces cerevisiae, inserting a His tag, constructing a recombinant expression plasmid pYD 1-CapIFN-gamma by taking a pYD1 shuttle plasmid as a framework, and carrying out enzyme digestion identification; (2) Based on the selenium-rich characteristic of the saccharomyces cerevisiae, sodium selenite is added in the growth logarithmic phase of the saccharomyces cerevisiae, the culture condition of the saccharomyces cerevisiae EBY100 is optimized, the selenium content is measured by utilizing a spectrophotometry method, the saccharomyces cerevisiae with the selenium-rich function is obtained, and the selenium-rich saccharomyces cerevisiae EBY100 competent cells are prepared for standby; (3) Electrotransformation of the identified correct recombinant plasmid pYD 1-CapIFN-gamma into selenium-enriched yeast EBY100, coating in MD plates, screening surface display type selenium-enriched yeast EBY100/pYD 1-CapIFN-gamma, and primary screening positive recombinant strains by utilizing a PCR method; (4) After the positive recombinant yeast strain with correct molecular identification is induced and expressed by galactose for 72 hours, the positive recombinant yeast strain is centrifuged for 10 minutes at 3000r/min, the supernatant is discarded, and the precipitate is collected.
The invention further provides a method for improving the expression of the goat gamma-interferon, which is to prepare the goat gamma-interferon into the goat gamma-interferon selenium-enriched yeast surface display system by using the preparation method, so that the expression of the goat gamma-interferon is improved.
The invention further provides a preparation comprising the selenium-enriched yeast surface display system, wherein the preparation is one or more of an antibody, an oral vaccine and a feed additive.
Further, the invention also provides a primer pair for PCR identification of the goat gamma-interferon, wherein the primer pair sequences are shown in Sequence No.3 and Sequence No. 4:
Sequence NO.3(F)：CAGATGTTGCTAAAGGTGGTCC；
Sequence NO.4(R)：AGAAGCTCTTCTACCTCTAAACAA。
the invention has the beneficial effects that:
the yeast display system of the goat gamma-interferon provided by the invention realizes the yeast surface display of the goat gamma-interferon, and is more beneficial to the exertion of the antiviral activity of the goat gamma-interferon.
The goat gamma-interferon yeast display system provided by the invention is subjected to selenium-rich transformation, so that the application range of the goat gamma-interferon yeast expression system is widened.
The yeast display system of goat gamma-interferon lays a foundation for developing a safer, more effective and cheaper novel yeast feed additive with antiviral and organism immunity enhancing functions.
Drawings
FIG. 1 is a schematic diagram of the construction scheme of recombinant plasmid pYD 1-CapIFN-gamma.
FIG. 2 is a graph showing the results of double cleavage assay of recombinant plasmid pYD 1-CapIFN-gamma.
FIG. 3 is a graph of the identification of recombinant selenium-enriched yeast.
FIG. 4 is a diagram showing the result of SDS-PAGE analysis of the CapIFN-gamma recombinant protein.
FIG. 5 is a graph showing the result of Western blot analysis of CapIFN-gamma recombinant proteins.
FIG. 6 is a graph of IFA identification results of recombinant selenium-enriched yeast.
Wherein,,
in FIG. 2, 1 is a double cleavage product, the expected bands occur at about 469bp and 5000bp, respectively, and M is DNA marker DL5000;
in FIG. 3, 1 is a selenium-enriched yeast PCR product transferred into a pYD1 empty vector, 2 is a selenium-enriched yeast PCR product transferred into a pYD 1-CapIFN-gamma recombinant plasmid, and M is a DNA marker DL2000;
in FIG. 4, 1-3 are the expression conditions of the CapIFN-gamma recombinant protein when the selenium-enriched yeast transferred into the recombinant plasmid pYD 1-CapIFN-gamma is induced for 24 hours, 48 hours and 72 hours respectively, a specific protein band appears at the position of about 25kDa, 4 is a selenium-enriched yeast negative control transferred into an empty vector, no target protein band is found at the position of 25kDa, and M is a protein Marker;
in FIG. 5, 1 is a selenium-enriched yeast negative control, no specific band appears, 2 is a selenium-enriched yeast strain transformed with recombinant plasmid pYD 1-CapIFN-gamma, and the expected band appears at about 25 kDa;
in FIG. 6, A is a selenium-enriched yeast strain transformed into a recombinant plasmid pYD 1-CapIFN-gamma, a green fluorescence signal appears, and B is a selenium-enriched yeast negative control transformed into an empty vector pYD1, and a fluorescence signal appears.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1 design and Synthesis of CapIFN-gamma Gene of interest
(1) Design of the Gene of interest
Because pYD1 plasmid is cut by Kpn I/EcoR I double enzyme and still keeps the inherent expression label of V5 epi and His tag, in order to make the target protein expression site and expression quantity better to be inspected later, his tag sequence is added in the primer downstream of CapINF-gamma mature peptide gene sequence, and stop codon TAA is designed to make V5 epi and His tag not expressed, and the target protein can be screened and identified based on His tag. Therefore, the target gene sequence mainly comprises KpnI, capIPFN-gamma, his tag and EcoR I gene sequences, the total length of the gene is 469bp, and the target gene is optimally designed according to the preference of a yeast codon.
CapIFN-gamma mature peptide gene sequence information:
the size is 440bp, and KpnI restriction enzyme gene (CGGGTACC) is inserted into 5' of CapIFN-gamma mature peptide gene sequence (shown in sequence 1).
Sequence NO.1：
ATGCAAGGTCCATTCTTTAAAGAAATTGAAAATTTGAAAGAATATTTTAATGCTTCTAATCCAGATGTTGCTAAAGGTGGTCCATTGTTTTCTGAAATTTTGAAAAATTGGAAAGAAGAATCTGATAAAAAGATTATTCAATCTCAAATTGTTTCTTTTTATTTTAAATTGTTTGAAAATTTGAAAGATAATCAAGTTATTCAAAGATCTATGGATATTATTAAGCAAGATATGTTTCAAAAATTTTTGAATGGTTCATCTGAAAAATTAGAAGATTTTAAAAAATTGATTCAAATTCCAGTTGATGATTTGCAAATTCAAAGAAAAGCTATTAATGAATTGATTAAGGTTATGAATGACTTATCTCCAAAATCTAATTTGAGAAAAAGAAAAAGATCTCAAAATTTGTTTAGAGGTAGAAGAGCTTCTATG
His tag sequence information:
1-18bp is His tag gene Sequence, 19-21bp is stop codon gene Sequence, 22-29bp is EcoRI restriction enzyme gene Sequence, sequence No.2: CATCATCACCATCACCATTAAGAATTCCG.
(2) Synthesis of target Gene
The total length of the artificially synthesized gene is 469bp, and the gene sequence is optimized and synthesized by Shanghai Biotechnology Limited company according to the preference of yeast codon.
(3) Construction of pUC 57-CapIFN-gamma recombinant plasmid
The synthesized target gene sequence is directionally inserted into pUC57 cloning plasmid after KpnI/EcoRI double enzyme cutting, and the recombinant plasmid is constructed by Shanghai engineering biological engineering technology Co.
EXAMPLE 2 construction of pYD1-CapIFN-gamma recombinant shuttle plasmid
(1) Extraction and enzyme digestion of plasmids
Referring to FIG. 1, the pYD1 shuttle plasmid and pUC 57-CapIFN-gamma recombinant clone plasmid were extracted with reference to the plasmid miniprep kit instructions, and double digestion was performed using KpnI and EcoRI restriction enzymes, respectively, with a 20. Mu.L digestion system: kpnI/EcoRI 1. Mu.L each, buffer 2. Mu.L, plasmid 12. Mu.L, ddH2O 4. Mu.L, 37℃water bath for 1.5h, after completion of the electrophoresis analysis by 1.0% agarose gel, and the digested pYD1 plasmid and the target fragment were recovered by referring to DNA agarose gel recovery kit instructions, dephosphorylated, and stored at-20℃for use.
(2) Ligation and transformation
The target fragment and the pYD1 plasmid were ligated using T4DNA ligase, with 10 μl ligation system: 1. Mu.L of T4DNA ligase, 5. Mu.L of Buffer, 3. Mu.L of target fragment, 11. Mu.L of pYD and ddH2O, and after mixing, the mixture was ligated overnight at 16℃and the ligation product was transformed into E.coli DH5a by a thermal shock method.
(3) Positive transformant identification
Screening positive transformants by using an ampicillin resistance plate, designing a specific primer gamma F/gamma R (Sequence No.3/Sequence No. 4), and carrying out PCR identification on recombinant strains, wherein a 20 mu L reaction system is as follows: 3. Mu.L of DNA template, 0.5. Mu.L of each primer, 10. Mu.L of 2 XTaq Master mix, and 6. Mu.L of ddH2O, under the following reaction conditions: 95℃for 5min,95℃for 35s,58℃for 30s,72℃for 30s,35 cycles, 72℃for 10min. A single band appeared by 1% agarose gel electrophoresis, and the PCR product was sent to Shanghai Bioworks Co., ltd for sequencing analysis, and the size of the product was 368bp (Sequence No. 5) which was consistent with the expected result. Meanwhile, the recombinant plasmid is extracted by referring to the specification of a plasmid small-extraction medium-quantity kit, double digestion identification is carried out by using KpnI and EcoRI restriction enzymes, expected bands (shown in figure 2) appear, and the identification of the correct recombinant plasmid is named as: pYD 1-CapIFN-gamma, and storing at-20deg.C.
Sequence NO.3：CAGATGTTGCTAAAGGTGGTCC
Sequence NO.4：AGAAGCTCTTCTACCTCTAAACAA
Sequence NO.5：
CAGATGTTGCTAAAGGTGGTCCATTGTTTTCTGAAATTTTGAAAAATTGGAAAGAAGAATCTGATAAAAAGATTATTCA
ATCTCAAATTGTTTCTTTTTATTTTAAATTGTTTGAAAATTTGAAAGATAATCAAGTTATTCAAAGATCTATGGATATT
ATTAAGCAAGATATGTTTCAAAAATTTTTGAATGGTTCATCTGAAAAATTAGAAGATTTTAAAAAATTGATTCAAATTC
CAGTTGATGATTTGCAAATTCAAAGAAAAGCTATTAATGAATTGATTAAGGTTATGAATGACTTATCTCCAAAATCTAA
TTTGAGAAAAAGAAAAAGATCTCAAAATTTGTTTAGAGGTAGAAGAGCTTCT
EXAMPLE 3 selenium-enriched engineering of Saccharomyces cerevisiae and competent cell preparation
(1) Drawing of expansion culture and growth curve of Saccharomyces cerevisiae
Selecting Saccharomyces cerevisiae EBY100, streaking and inoculating in YPD solid culture medium, culturing in a constant temperature incubator at 30 ℃, after single colony grows out, selecting single colony, inoculating in 10mL YPD liquid culture, culturing in a constant temperature incubator at 30 ℃, shaking and culturing at 250r/min for 24h, inoculating in 200mL YPD fresh culture medium according to 10% inoculum size, sampling 1mL every 12h, measuring the saccharomycete content, continuously culturing for 72h, and drawing a growth curve according to the thallus content at each time point. The strain of Saccharomyces cerevisiae EBY100 was found to have a cell content of 210X 106 CFU.mL-1 at the peak of cell content when cultured for 36 h.
(2) Selenium-rich culture of Saccharomyces cerevisiae
According to the characteristics of a growth curve of saccharomyces cerevisiae, sodium selenite with different contents is added during 36h of culture, and the final concentrations are respectively as follows: the total selenium content was determined after incubation for 72h at 0. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL. By measurement, when the optimal addition amount of sodium selenite is 60 mug/mL, the selenium content of saccharomyces cerevisiae unit is 5.61mg/g.
(3) Preparation of selenium-enriched yeast competent cells
A fresh selenium enriched yeast EBY100 was picked from YPD plates and cultured overnight at 30℃at 250r/min in 10ml YPD liquid medium. The OD600 of the overnight culture was measured to be between 3.0 and 5.0. The 10mLYPD overnight culture was diluted to an OD600 of 0.2-0.4. Culturing in a shaking table at 28-30deg.C for 3-6 hr until OD600 reaches 0.6-1.0. Yeast cells were collected by centrifugation at 1500r/min for 5min at room temperature and the supernatant was discarded. The yeast cells were washed with 10mL of the washing solution, followed by centrifugation at 1500r/min for 5min at room temperature to collect the cells, and the supernatant was discarded. The yeast cells were resuspended with 1mLTE/LiAc and sub-packaged at 100. Mu.L per tube and frozen at-80℃either as-prepared or after preparation.
EXAMPLE 4 construction and identification of recombinant selenium-enriched Yeast
(1) Shock conversion
Taking 100 mu L of prepared selenium-enriched yeast EBY100 competent cells, adding 5 mu L of plasmids, blowing and sucking uniformly by a pipetting gun, transferring to a 4mm electrorotating cup precooled at 4 ℃, and standing for 5min in an ice bath. Wiping the electric rotating cup and electric shock: the Bio-rad Gene Pulser Xcell electroporator selects the preset program Fungal 2, voltage 2.5kV, one shock. After electric shock, 1mL of a pre-chilled 1M sorbitol solution at 4℃was added to the electric beaker, and the mixture was blown uniformly with a pipette and placed in an ice bath. Transfer to a 1.5mL EP tube and stand in a water bath at 30℃for 1h. After 1h, 100-150. Mu.L of the plate was removed and incubated in leucine MD solid medium at 30℃until colonies grew to a size of 2-3mm in diameter (approximately 2-3 d). Meanwhile, the electrotransfer pYD1 plasmid is used as a negative control.
(2) PCR identification of recombinant selenium-enriched yeast
Single colonies were picked from MD plates and inoculated into 5mLYPD liquid medium at 30℃for overnight incubation at 250 r/min. Recombinant yeast DNA was extracted with reference to the yeast genome extraction kit instructions and identified by PCR using the primers γf/γr designed in example 2, and PCR products were analyzed by 1% agarose gel electrophoresis, with expected bands appearing, and a blank control without bands (as shown in fig. 3), and the identified correct recombinant strain was designated EBY100/pYD1-CapIFN- γ.
(3) Inducible expression of recombinant selenium-enriched yeast
The recombinant selenium-enriched yeast with correct identification is inoculated with YNB-CAA liquid culture medium containing 2% galactose, cultured at 30 ℃ and 250r/min, sampled 1 time every 24 hours, centrifuged at 3000r/min for 10min after 72 hours of induced expression, and the supernatant is discarded, and the precipitate is collected.
(4) SDS-PAGE of recombinant yeasts
100. Mu.L of 5 XSDS loading buffer was added to the pellet, after mixing, the pellet was discarded after 10min in a 100℃water bath, after the completion of 12,000r/min, centrifuged for 5min, the supernatant was carefully aspirated. The supernatant was loaded at 15. Mu.L, analyzed by electrophoresis on 10% SDS-PAGE, and EBY100/pYD1 samples were processed as negative controls. After the electrophoresis was completed, staining and decoloring treatment were performed, and it was found by analysis that a specific protein band appeared at about 25kd position, and that the negative control had no corresponding band, consistent with the expected result. CapIFN-gamma has appeared as a band of interest 24h after induction of expression (as shown in FIG. 4).
(5) Western blot analysis
After induction expression of the positive recombinant yeast strain for 72 hours, the samples were treated according to method 4. After electrophoresis, the electrophoresis product is transferred to a PVDE transfer film by a transfer film instrument. At room temperature, the membrane was washed 3 times with TBST solution for 10min each with a blocking solution (5% nonfat dry milk) for 2 h. The PVDE transfer film is placed in a clean plate, then rabbit anti-His tag polyclonal antibody is added, primary antibody incubation is carried out, after treatment for 1h at 4 ℃, the film washing operation is repeated. After washing, goat anti-rabbit IgG (H+L) antibody (alkaline phosphatase-labeled) diluted 30000 times with TBST was added thereto, and the membrane was incubated at 4℃for 1 hour, and the above-described washing operation was repeated. The nitrocellulose membrane was placed in BCIP/NBT color development solution to develop color in the dark, and after the bands appeared, the reaction was stopped by repeated rinsing with deionized water. As a result, the expected band appeared at the approximately 25kDa position, and the control wells were free of bands (as shown in FIG. 5).
(6) IFA analysis
Taking EBY100/pYD 1-CapIFN-gamma bacterial liquid 12 000r/min after galactose induction for 72h of 1mL, centrifuging for 5min at 4 ℃, discarding the supernatant, retaining the precipitate, and taking EBY100/pYD1 after galactose induction for 72h as a negative control. PBS was washed 3 times, 2min each time, 6000rpm each time, 4℃and centrifuged for 5min, and the supernatant was discarded. The meat rabbits are immunized by using the CapIFN-gamma prokaryotic expression product, the CapIFN-gamma polyclonal antibody is prepared, the rabbit anti-CapFN-gamma polyclonal antibody (primary antibody) is diluted by sterilized PBS according to the proportion of 1:200, the primary antibody diluted by 500 mu L is incubated overnight at 4 ℃, and after PBS is washed 3 times, 6000rpm,4 ℃ is centrifuged for 5min. mu.L of FITC-labeled goat anti-rabbit IgG diluted 1:5000 with sterile PBS was added and incubated for 1h at 37 ℃. After 3 washes with PBS, the mixture was centrifuged at 6000rpm and 4℃for 5min. After 50 μl of sterilized PBS was resuspended, 10 μl of bacterial droplets were taken on a clean slide, covered with a cover slip, and the edges were covered with a clear nail polish fixing cover slip. Under a fluorescence microscope, the recombinant selenium-enriched yeast EBY100/pYD 1-CapIFN-gamma excites a green fluorescence signal, and the control group has no fluorescence signal (shown in figure 6).
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Sequence listing
<110> Chongqing City academy of livestock sciences
<120> selenium-enriched yeast genetically engineered bacterium, surface display system and construction method thereof
<130> 2021-5-25
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tctgataaaa agattattca atctcaaatt gtttcttttt attttaaatt gtttgaaaat 180
ttgaaagata atcaagttat tcaaagatct atggatatta ttaagcaaga tatgtttcaa 240
aaatttttga atggttcatc tgaaaaatta gaagatttta aaaaattgat tcaaattcca 300
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Claims (4)
1. The selenium-enriched yeast surface display system is characterized in that engineering bacteria of the selenium-enriched yeast surface display system are selenium-enriched yeast genetic engineering bacteria, and the selenium-enriched yeast genetic engineering bacteria are Saccharomyces cerevisiae @Saccharomyces cerevisiae) An EBY100; the target protein of the selenium-enriched yeast surface display system is goat gamma-interferon CapIFN-gamma, and the nucleic acid sequence of the goat gamma-interferon is shown as SEQ ID NO. 1; the preparation method of the selenium-enriched yeast genetically engineered bacterium comprises the following steps: culturing yeast genetically engineered bacteria by using a culture medium, and adding sodium selenite with the concentration of 60 mug/mL to continue culturing when the yeast genetically engineered bacteria are cultured to a logarithmic phase;
the preparation method of the selenium-enriched yeast surface display system comprises the following steps:
(1) Inserting the goat gamma-interferon into a His tag, and constructing a recombinant plasmid pYD 1-CapIFN-gamma by taking a pYD1 shuttle plasmid as a framework; (2) Introducing the recombinant plasmid pYD 1-CapIFN-gamma into the selenium-enriched yeast genetic engineering bacteria by using an electrotransformation method, screening surface display type selenium-enriched yeast EBY100/pYD 1-CapIFN-gamma, and primarily screening positive recombinant strains by using a PCR method; and (3) carrying out induced expression on the positive recombinant yeast strain with correct molecular identification by utilizing galactose for 72 hours, centrifuging for 10 minutes at 3000r/min, discarding the supernatant, and collecting the precipitate to obtain the selenium-enriched yeast surface display system.
2. The selenium-enriched yeast surface display system of claim 1, wherein in the preparation method of claim 1, the selenium-enriched yeast genetically engineered bacterium is prepared into competent cells prior to introducing the pYD1-CapIFN- γ recombinant plasmid into the selenium-enriched yeast genetically engineered bacterium.
3. Use of the selenium enriched yeast surface display system of claim 1 in the preparation of a formulation for enhancing immunity and disease resistance in goats.
4. A formulation comprising the selenium enriched yeast surface display system of claim 1, wherein the formulation is one or more of an antibody, an oral vaccine, a feed additive.
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