CN111440816A

CN111440816A - Surface display type yeast host cell and application thereof in preparation of porcine astrovirus yeast vaccine

Info

Publication number: CN111440816A
Application number: CN202010153131.9A
Authority: CN
Inventors: 张蕾; 李婉情; 张文涛; 赵成雪
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2020-07-24

Abstract

The invention discloses a surface display type yeast host cell and application thereof in preparing a porcine astrovirus yeast vaccine. The vaccine comprises a C-terminal hypervariable region of a Cap protein of a porcine astrovirus, and a GPD promoter, the C-terminal hypervariable region of the Cap protein, a lectin Aga1, a purification tag and the like are connected in series to construct a transcription unit by utilizing a saccharomyces cerevisiae transcription element efficient construction method. The vaccine of the invention can be induced by glucose, has low production cost and is suitable for large-scale production. Experiments show that the saccharomyces cerevisiae strain has use safety and can induce obvious mucosal immune response and humoral immune response.

Description

Surface display type yeast host cell and application thereof in preparation of porcine astrovirus yeast vaccine

Technical Field

The invention belongs to the technical field of biological genetic engineering, and relates to a surface display type yeast host cell and a method for preparing a porcine astrovirus yeast vaccine.

Background

Yeast surface display technology (YSD) refers to the technology of displaying a foreign protein or peptide on the surface of a Yeast cell by fusion with a cell wall protein.1993, Schreuder et al [1] describe the first time the display of a foreign protein on the cell surface of Saccharomyces cerevisiae YSD was originally used for the production of biocatalysts, and with the development of molecular biology and Yeast genetics, YSD has been widely used in various fields.e.g.bioadsorption, bioremediation and biosensor design. in the biomedical field, e.g.the preparation of antibodies and oral vaccines, YSD has been used in the field of basic research to analyze the interaction between protein-protein and protein-ligand. α -lectin system is one of YSD consisting of two parts, the Aga1 subunit (anchoring component) inserted into the cell wall via GPI; the Aga2 subunit (adhesion component) used as anchoring protein for a target peptide or protein.Cys 7 and Cys50 residues of Aga2 are bound to Aga1 via two disulfide bonds.

Biological delivery systems as one of the most effective means for delivering vaccines to mucosal surfaces, there are currently five major delivery vehicles for vaccine antigen production, bacterial, yeast, insect, mammalian and plant expression systems, among which yeast is the dominant host for foreign protein expression due to its unique advantages, (1) saccharomyces cerevisiae is a well-recognized and very safe eukaryotic model organism that does not produce any toxic substances and is not pathogenic after oral administration, (2) saccharomyces cerevisiae has the advantages of eukaryotic organisms, clear genetic background, simple culture conditions, high foreign protein expression, and is an ideal foreign protein expression host, (3) saccharomyces cerevisiae has post-translational modification processes of eukaryotic organisms, glycosylation modified viral proteins have better immunogenicity, (4) saccharomyces cerevisiae has been used as an oral vaccine and drug trail, because the robust cell wall structure of saccharomyces cerevisiae can protect biological macromolecules from digestive breakdown of gastric acid in the presence of metabolites produced by intestinal flora, interacts with small intestinal epithelial cells, stimulates the immune cell wall of the intestinal epithelial cell system of saccharomyces cerevisiae (356) and enhances the immune response of saccharomyces cerevisiae with mannan-specific immune adjuvants.

Astrovirus is a single stranded positive stranded RNA virus without a membrane that was first observed in diarrhea stools in children by Electron Microscopy (EM) by Appleton and Higgins in 1975 [2,3 ]. Since then, reports of astrovirus enteric infections have been made in many countries and regions around the world. Astrovirus can infect a variety of hosts, both mammalian (including human) and avian, and diarrhea is a characteristic clinical manifestation of this viral infection [4,5 ]. Human astrovirus infection is closely associated with diarrhea and gastroenteritis symptoms in elderly and immunocompromised patients and is considered the second leading pathogen to infantile diarrhea [6]. Despite its clinical significance, the virus (diarrheal symptoms) is currently one of the least studied enteroRNA viruses due to, among other reasons, the lack of a virus-stable continuous cell line [7 ].

Astrovirus particles have smooth surfaces and five or six star-like processes (about 10%) on the surface of the particles, the virus particles are in regular icosahedral symmetry, without capsular coating, and have a diameter of 28-41 nm. Risco et al found that the diameter of the virus particles in feces is 28-30nm, whereas the diameter of the virus particles in LL C-MK2 cells is 41nm, with distinct processes but no star-like structures [8 ].

Disclosure of Invention

The invention aims to provide a surface display type yeast host cell based on an Aga1-Aga2 system and a preparation method of a recombinant saccharomyces cerevisiae surface display vaccine strain for expressing a swine astrovirus Cap protein.

The technical scheme of the invention comprises the following steps:

an Aga1-Aga 2-based saccharomyces cerevisiae surface display system, comprising an Aga gene derived from a yeast strain EBY100 and an Aga2 gene derived from a pYD1 plasmid, the Aga gene expressing an Aga subunit (anchor component) inserted into a cell wall via Glycosylphosphatidylinositol (GPI), the Aga2 gene expressing an Aga2 subunit (adhesion component) serving as an anchor protein for a target peptide or protein, and the target peptide or protein being displayed on the surface of saccharomyces cerevisiae by binding the anchor component and the adhesion component.

A vector, which comprises the Aga1 fragment and has a nucleic acid sequence of SEQ ID No. 15.

A saccharomyces cerevisiae host cell comprising the vector.

A surface display yeast host cell comprises an Aga1-Aga2 saccharomyces cerevisiae surface display system.

A method for constructing surface display yeast host cells comprises the steps of taking a laboratory preservation strain JDY52 as an initial strain, cloning a GPD promoter by PCR, replacing a pIU211 lac promoter with the GPD promoter through reverse PCR amplification and seamless connection, transferring linearized plasmids pIU211(GPD) into JDY52 through lithium acetate transformation, screening positive transformants by using an SD-URA selective culture medium and genome PCR, and obtaining a constitutive glucose promoter GPD-regulated and surface efficient display Aga1 yeast host strain ST 1814G.

A method for constructing a surface-displayed yeast host cell, comprising the steps of;

(1) pIU211 construction of recombinant plasmid: the EBY100 yeast genome was extracted, the Aga1 gene was amplified by PCR, and the Aga1 gene was inserted into the integration vector YIpalac 211 by seamless cloning.

(2) pIU211(GPD) recombinant plasmid construction: a pair of reverse amplification primers are designed to amplify pIU211 (the rest parts except lac promoter) by following the principle of seamless cloning technology, then GPD promoter is amplified by using HCKAN-GPD plasmid preserved in a laboratory as a template, and the terminal sequence of the vector is introduced at the 5' end of the GPD forward/reverse amplification primer. The lac promoter on the pIU211 plasmid was replaced by the GPD promoter by seamless cloning.

(3) Preparation of linearized vector: the sequence of the foreign insert gene Aga1 of the recombinant plasmid pIU211(GPD) contains a recognition site of a restriction enzyme BsiWI, and pIU211(GPD) can be digested with BsiWI to obtain a linearized DNA fragment.

(4) Constructing a saccharomyces cerevisiae host strain with the surface efficiently displaying Aga 1: the linearized pIU211(GPD) fragment is transferred into a starting strain JDY52 by a conventional lithium acetate conversion method, and the host strain ST1814G of the Aga1 yeast with the high-efficiency surface display is obtained by SD-URA auxotrophy screening and PCR identification.

The porcine astrovirus fusion protein Aga2-Cap has the nucleic acid sequences of SEQ ID No.16 and SEQ ID No. 17.

The protein gene is obtained by connecting an Aga2 gene derived from a PYD1 plasmid with a C-terminal hypervariable region gene containing the porcine astrovirus in series.

A vector comprising said gene fragment.

A Cap protein surface display Saccharomyces cerevisiae strain, in ST1814G strain, includes the fusion protein.

A Cap protein surface display Saccharomyces cerevisiae strain is prepared by connecting an Aga2 gene derived from a PYD1 plasmid with a C-terminal hypervariable region gene containing porcine astrovirus in series to construct a transcription unit of fusion expression of Aga2-Cap in yeast, realizing the transcription unit and stably integrating the transcription unit into a genome through in vitro enzyme-linked, in vivo yeast transformation and homologous recombination technologies, displaying the transcription unit on the surface of a yeast cell through an Aga1-Aga2 surface display system to obtain a recombinant yeast strain with the surface display Cap protein, and preparing a vaccine by using the obtained strain.

The method for constructing the Cap protein surface display Saccharomyces cerevisiae strain specifically comprises the following steps:

(1) amplification of the C-terminal hypervariable region gene of the Cap protein: a PAStV4 whole genome sequence PAStV4/Tianjin/2018(access number MH425243) is separated from 29 pig diarrhea excrement of Tianjin Silent sea and Ninghe farms through RT-PCR, and a Cap protein C-terminal hypermutation region gene is obtained through PCR amplification, wherein the molecular size is 1272 bp.

(2) The Aga2 gene is connected with a C-terminal hypervariable region gene containing the porcine astrovirus in series: and (3) connecting the Aga2 gene and a Cap protein C-terminal hypervariable region gene in series by using an overlap PCR technology to obtain an Aga2-Cap fusion fragment.

(3) And cloning the C-terminal hypervariable region of the Cap gene to a POT-GPD-TU vector by adopting a seamless cloning method to obtain a recombinant POT/Cap plasmid.

(4) The recombinant expression plasmid is transferred into saccharomyces cerevisiae, HO gene loci integrating exogenous metabolic pathways, namely URR1(SEQ ID No.18), URR2(SEQ ID No.19) and HIS3. BsaI are present on the IV chromosome of a host strain ST1814G, POT/Cap plasmid is digested to release transcription units, BsmBI is digested to PMV-URR1, PMV-URR2 and PMV-L EU2 to release URR1, URR2 and L EU2, the transcription units, URR and a selective marker L EU2 complete the directional assembly of a plurality of gene fragments according to specific joint sequences, thereby forming a long DNA fragment containing the transcription units, the assembled gene fragments can be directionally integrated into HO gene loci [12] of the IV chromosome of ST1814G through yeast transformation, and simultaneously, primers of PAStV4/Tianjin/2018Cap region are used for detection.

An oral vaccine of porcine astrovirus based on a saccharomyces cerevisiae surface display system directly takes a Cap protein surface display saccharomyces cerevisiae strain as an oral vaccine and application.

The invention has the beneficial effects that: the invention is based on an Aga1-Aga2 yeast surface display system, uses saccharomyces cerevisiae JDY52 as an original strain, and constructs a host strain ST1814G which is controlled by a constitutive glucose promoter GPD and has a surface efficiently displayed Aga 1. The Cap protein is displayed on the surface of the saccharomyces cerevisiae to prepare the porcine astrovirus yeast vaccine, and the vaccine is safe and easy to prepare, induces organisms to generate mucosal and humoral immune responses, and provides a tool for immune prevention and control of the porcine astrovirus.

The saccharomyces cerevisiae surface display strain induced by glucose and the porcine astrovirus yeast vaccine constructed by using the saccharomyces cerevisiae surface display strain are first reported at home, and the construction of the surface display strain and the preparation of the vaccine have certain innovativeness and provide ideas and methods for the preparation of the porcine astrovirus and other virus vaccines.

Drawings

FIG. 1: a schematic diagram of surface display type saccharomyces cerevisiae construction; firstly, transferring a linearized pIU211(GPD) plasmid into JDY52 yeast cells to obtain a recombinant yeast strain ST1814G, and secondly, realizing transcription units and stably integrating the transcription units into a genome by using in vitro enzyme-linked, in vivo yeast transformation and homologous recombination technologies to obtain a recombinant yeast strain ST 1814G/Cap;

FIG. 2: pIU211 plasmid and pIU211(GPD) plasmid construction; panel A is YIplac211 vector map; panel B shows the pIU211 plasmid insert and linearized vector gel electrophoresis assay, lane 1 is Marker, lanes 2-3 are the Aga1 PCR product, and lanes 4-5 are the YIPALac 211 enzyme cleavage product; panel C shows pIU211(GPD) plasmid insert and linearized vector gel electrophoresis for detection, Marker in

lanes

1 and 5, pIU211 reverse PCR product in lanes 2-4, and GPD PCR product in lane 6;

FIG. 3: SD-URA auxotrophy screening and verification of host bacteria ST 1814G; panel A is a lithium acetate conversion plate of ST 1814G; panel B is a purified plate of ST 1814G; FIG. C is a genomic PCR electrophoretogram of ST 1814G;

FIG. 4 shows the SD-L EU auxotrophy screening result of a Saccharomyces cerevisiae strain displayed on the surface of Cap protein, wherein the A is ST1814G/Cap lithium acetate conversion plate, the B is PCR gel electrophoresis detection of recombinant yeast genome, the Lane 1 is Marker, and the Lane 2-5 is PCR electrophoresis diagram of recombinant yeast ST1814G/Cap genome;

FIG. 5: schematic diagram of host bacterium Aga1 promoter modification;

FIG. 6: assembly of an anchored transcription unit;

FIG. 7: the schematic diagram of the principle of constructing the Cap protein recombinant Saccharomyces cerevisiae expression strain of PAStV 4;

FIG. 8: analyzing the recombinant saccharomyces cerevisiae by western-blot; lane 1 is protein Marker; lane 2 is ST1814G/Cap Whole cell western-blot analysis, 1-2 are different single colonies;

FIG. 9 shows immunofluorescence analysis of recombinant Saccharomyces cerevisiae, left two columns of recombinant yeast after induction by incubation with mouse anti-6 × His monoclonal antibody and fluorescence labeling with FITC-labeled secondary antibody, right two columns of recombinant yeast after induction by incubation with mouse anti-Cap polyclonal antibody and fluorescence labeling with FITC-labeled secondary antibody, upper action negative control ST1814G, lower action experimental group ST1814G/Cap (magnification 40 ×);

FIG. 10: recombining a saccharomyces cerevisiae growth curve;

FIG. 11: detecting a Cap protein specific antibody of an immunized mouse; panel A shows IgA content in mouse feces; panel B shows the IgG content in mouse serum.

Detailed Description

The present invention will be more clearly understood from the following examples. The technical steps of the invention are conventional in the art, and are either commercial or published reagent materials, unless otherwise specified.

Yeast strain: in the following examples, Saccharomyces cerevisiae strain JDY52[12], which is a haploid strain derived from the sporulation of a diploid strain formed after the mating of Saccharomyces cerevisiae BY4727 and Saccharomyces cerevisiae BY4733, is publicly available at the university of Qinghua.

Example 1

pIU211 and pIU211 (GPD):

the saccharomyces cerevisiae surface display system based on the Aga1-Aga2 lectin system requires that both the Aga1 gene and the exogenous gene of the host bacterium are efficiently expressed in order to anchor more exogenous proteins on the surface of a yeast cell. Therefore, we designed PCR amplification primers of the exogenous insertion gene Aga1 according to the method [13,14] established by Boder and Wittrup, following the principle of seamless cloning technology, and the upstream primers are: 5'-GACCATGATTACGCCAAGCTTGATGACATTATCTTTCGCTCATTTTA-3' (SEQ ID No.1), and the downstream primer is: 5'-GACCTGCAGGCATGCAAGCTTTTAACTGAAAATTACATTGCAAGC-3' (SEQ ID No.2), and a linearized vector was prepared by digesting the YIplec 211 plasmid (FIG. 2A) with the restriction enzyme HindIII to construct a recombinant plasmid pIU 211. .

pIU Aga1 promoter in the bacterial strain is lac promoter, and Aga1 gene is over-expressed under the control of galactose. Based on pIU211, we wanted to construct a more stable glucose-induced Aga1 expression vector, so we designed a specific vector pIU211 primer in this study, with the upstream primer: 5'-TTTCACACAGGAAACAGCTATGACCATG-3' (SEQ ID No.3), and the downstream primer is: 5'-GCCTGGGGTGCCTAATGAGTGAGCTAA-3' (SEQ ID No.4), and designing a specific primer of the GPD promoter, wherein the upstream primer is: 5'-TTAGGCACCCCAGGCTCATTATCAATACG-3' (SEQ ID No.5), the downstream primer is: 5'-GTTTCCTGTGTGAAATTTGTTTGTTTATGT-3' (SEQ ID No.6), the lac promoter of vector pIU211 was replaced with the GPD promoter by a seamless cloning technique.

Example 2

Constructing a saccharomyces cerevisiae host strain with the surface efficiently displaying Aga 1:

the linearized pIU211(GPD) fragment is transferred into a starting strain JDY52 by a conventional lithium acetate conversion method, and the host strain ST1814G of the Aga1 yeast with the high-efficiency surface display is obtained by SD-URA auxotrophy screening and PCR identification.

Example 3

Constructing a POT-GPD-Cap-TU plasmid:

using plasmid PYD1 as a template, designing a specific primer, amplifying Aga2 signal peptide, using an upstream primer of 5'-AGCGTGCGTCTCGGATGATGCAGTTACTTCGCTG-3' (SEQ ID No.7) and a downstream primer of 5'-GTGCTGCGTCTCAGCTATCAATGGTGATGGTGA-3' (SEQ ID No.8), detecting the product specificity by 1% agarose gel electrophoresis after PCR reaction is finished, purifying by using a DNA recovery kit, dissolving the purified DNA in 30 mu L ddH₂O。

Performing One-POT reaction at 55 ℃ for 1 hour, adding T4 DNA L igase into the reaction system, directly transforming the mixture into DH5 α competent cells after the reaction is finished, coating the cells on a L B (ampicillin resistance) solid plate, performing inverted culture at 37 ℃ for 12-16 hours, selecting white single colonies for colony PCR verification, and finally, naming the correct plasmid as POT-GPD-TU.

Designing a specific primer, wherein an upstream primer is 5'-GACGATAAGGTACCAGGATCCATGCCAGGTAATGTTGGGCA-3' (SEQ ID No.9), a downstream primer is 5'-GAATTCCACCACACTGGATCCGTATGCCGCATCCCAGGC-3' (SEQ ID No.10), amplifying a PAStV4/Tianjin/2018Cap hypervariable region, detecting the product specificity by using 1% agarose gel electrophoresis after the PCR reaction is finished, purifying by using a DNA recovery kit, and dissolving the purified DNA in 30 mu L ddH₂And O, performing recombination reaction connection by using a seamless cloning kit.

And (3) identifying the bacteria liquid as a positive colony by PCR, inoculating the residual bacteria liquid into L B (ampicillin resistance) liquid culture medium for culturing overnight, extracting plasmids by using a plasmid miniprep kit, and sequencing by Jinzhi Biotech, Suzhou, to obtain the POT-GPD-Cap-TU plasmid.

Example 4

Constructing a saccharomyces cerevisiae host bacterium which is regulated by a constitutive glucose promoter GPD and efficiently displays Aga 1:

a laboratory preserved strain JDY52 is used as an original strain, a linearized plasmid pIU211(GPD) is transferred into JDY52 through lithium acetate transformation, and positive transformants are screened by using an SD-URA selective culture medium and genome PCR to obtain a constitutive glucose promoter GPD-regulated and surface-efficiently displayed Aga1 yeast host strain ST 1814G.

Example 5

Constructing a saccharomyces cerevisiae strain for surface display of the recombinant porcine astrovirus induced by glucose:

(1) construction of Yeast transformation fragments

The plasmid with homology arms (URR1 and URR2) and yeast selection marker (L EU) was digested with BsmB I, mononucleotide sequence L sm (5'-AAGAGACGCAAGACACTGCGGATA-3') (SEQ ID No.11) and Rsm (5'-AAGAGACGCAAGACACTGCGGATAC-3') (SEQ ID No.12) was added, plasmid POT-GPD-Cap-TU with constructed transcription unit was digested with Bsa I90 s according to the procedure, mononucleotide sequence L com was added

(5'-AAGAGACCCAAGACACTGCGGATA-3') (SEQ ID No.13) and Rcom

(5'-AAGAGACCGAGTCACTGCCAACA-3') (SEQ ID No.14), the processed transcription unit described above (GPD-Aga2-CapADH1) was mixed with a plasmid containing homology arms (URR1 and URR2) and yeast selection marker (L EU) at a ratio of 1: 1, and ligated with T4 ligase overnight at 16 ℃ for yeast transformation, by programmed 90 s.

(2) Obtaining of recombinant Saccharomyces cerevisiae strains

Selecting ST 181G single colony to inoculate in 3-5M L YPD liquid culture medium, culturing overnight at 30 ℃ and 220rpm, transferring the overnight cultured bacterial liquid into 5M L new YPD culture medium according to the proportion of 1:50 to ensure that the initial OD is 0.1-0.2, culturing at 30 ℃ and 220rpm until the OD600 is 0.5-0.8.2500 rpm, centrifuging for 5min to collect thallus of 5M L bacterial liquid, washing cells with 1M L sterile water, centrifuging to collect thallus and combine the thallus into a tube, adding 100 mu L0.1M lithium acetate into the centrifuge tube, centrifuging at 12000rpm for 20s to collect thallus, adding 50 mu L.1M lithium acetate into the centrifuge tube, centrifuging at 12000rpm for 20s to collect thallus, sequentially adding 240 mu L% PEG4000, 36 mu L M lithium acetate, 100 mu g of refined DNA and 2 mu g of fragment DNA into the centrifuge tube, violently shaking to completely shake to 30 ℃ and mix uniformly, adding 30 rpm to 30min, inoculating into a YPD culture medium with the centrifuge tube, inoculating the supernatant of 5M L new YPD culture medium, culturing at 30 rpm, centrifuging to collect thallus, inoculating the bacterial strain, inoculating the supernatant, culturing at 30 ℃ and culturing at 30 ℃ for 30 ℃ and 30 rpm to 30 ℃ for 30min, inoculating to 5M, inoculating to a strain, inoculating the bacterial strain for 30 ℃ to 5M, inoculating to 2, centrifuging to 5M YPD culture medium, inoculating to 2, inoculating to a strain, culturing, inoculating to a strain, inoculating the supernatant to a strain, culturing at 30 ℃ to a strain, inoculating to a strain.

Example 6

Displaying the expression characteristics and the immune efficacy analysis of the saccharomyces cerevisiae strain on the surface of the Cap protein of the porcine astrovirus:

(1)Western-blot

collecting ST1814G/Cap bacterial liquid induced for 36h by 1m L, centrifuging at 12000rpm for 2min to collect thalli, resuspending the thalli in 80 mu L sterile water, adding 20 mu L5 × SDS loading buffer solution, centrifuging at 12000rpm and 4 ℃ for 5min in a boiling water bath, carefully sucking the supernatant, and using 20 mu L supernatant for 12% SDS-PAGE electrophoresis.

After SDS-PAGE electrophoresis is finished, transferring the protein separation gel to an NC membrane with the same size as the protein separation gel by a wet transfer method, wherein the membrane transfer condition is 300mA for 100 min; after the membrane transfer is finished, sealing the NC membrane for 1h by using 5% skim milk at room temperature; rinsing the NC membrane for 3 times by using TBST buffer solution; the NC membrane front was completely submerged in murine anti-Cap polyclonal antibody (primary antibody) diluted 1:200 with 2% BSA and incubated overnight at 4 ℃; recovering primary antibody, and rinsing NC membrane with TBST buffer solution for 5min for 3 times; incubated with HRP-labeled goat anti-mouse IgG (secondary antibody) diluted 2% BSA 1:5000 for 1h at room temperature; rinsing the NC membrane with PBST buffer solution for 3 times, 5min each time; and dropping chemiluminescence mixed liquor to the front side of the NC membrane, exposing by using a Bio-Red chemiluminescence imager, and observing the western blot.

(2) Immunofluorescence

Collecting ST1814G/Cap bacterial liquid induced for 36h by 500 mu L, centrifuging at 4000rpm for 5min to collect thalli, taking ST1814G after induction for 36h as a negative control, washing the thalli by 500 mu L PBST for 3 times, 4000rpm for 5min, discarding supernatant, respectively re-suspending the thalli by 6 His-Tag primary antibody and Cap polyclonal antibody diluted by 200 mu L TBST 1:500, rotating and combining for 1h at 4 ℃, recovering primary antibody, washing the thalli by 500 mu L PBST for 3 times, centrifuging at the same time, discarding supernatant, adding FITC-labeled goat anti-light mouse IgG secondary antibody diluted by 200 mu L TBST 1: 1000, incubating for 30min at 37 ℃, recovering secondary antibody, washing the thalli by 500 mu L PBST for 3 times, centrifuging at the same time, discarding supernatant, using 50 mu L PBS, taking 10 mu L bacterial liquid drops on a clean glass slide, covering with a cover glass, coating a cover glass slide for fixing, coating the periphery with the cover glass, and observing under a confocal microscope.

(3) Growth curve

Inducing and culturing recombinant Saccharomyces cerevisiae ST1814G/Cap, and using an original strain JDY52 as a blank control; and measuring OD600 values of 0h, 12h, 24h, 36h, 48h and 60h by using a visible spectrophotometer to determine the growth curve of the recombinant yeast.

(4)ELISA

The experimental group ST1814G/Cap (Yeast-expressing Cap), JDY52 (Yeast-strong), PBS control group (PBS), 3 mice in each group ST1814G/Cap was fed with ST1814G/Cap after inducing for 48h 1m L each time, the unloaded group was fed with equal amount of Y JD 52 thallus, the PBS group was fed with 200 μ L PBS, and fed with mice at 0d, 7d, 14d and 28d for 4 times, and the blood sampling of eyeball 5 days later was performed to detect IgG.

Tanning, namely adding 100 mu L0.2% of tannic acid into a 96-well enzyme label plate, incubating for 1h at 37 ℃, discarding the tannic acid, and washing for 2 times by using sterile water.

Coating, diluting Cap prokaryotic protein with protein coating solution to final concentration of 5 μ g/m L, adding 100 μ L per well, coating overnight at 4 deg.C, discarding coating solution, washing with PBST for 3 times, and air drying.

Blocking, adding 100 mu L5% BSA solution into each well, blocking at 37 ℃ for 1h, removing blocking solution, washing with PBST for 3 times, and air drying.

Primary antibody binding, using post-immune serum and fecal suspension diluted with 2% BSA 1:500 as primary antibody, 100 μ L per well, 3 replicates per sample, simultaneous negative and blank controls, incubation at 37 ℃ for 1h, liquid discard, PBST wash 3 times.

And (3) secondary antibody combination, namely adding 100 mu L1 to 10000 diluted enzyme-labeled secondary antibody (a serogroup is added with a goat anti-mouse IgG (H + L) HRP secondary antibody; a fecal group is added with a goat anti-mouse IgA (H + L) HRP secondary antibody) into each hole, incubating for 1H at 37 ℃, discarding liquid, and washing for 3 times by PBST.

Color development, namely adding 100 mu L TMB single-component color development liquid, and reacting for 15min in a dark place at 37 ℃;

stop, add 50 μ L stop solution and react for 15 min.

Reading: measuring the light absorption value at 450nm within 30 minutes after the reaction is stopped, and calculating the antibody titer according to the formula:

P/N ═ sample absorbance-blank absorbance)/(negative absorbance-blank absorbance)

Results

And detecting the expression condition of Cap protein by using Western blot and immunofluorescence analysis.

As shown in FIG. 8, the Cap prokaryotic protein is approximately 50kDa, and the Western-blot band is larger than 50kDa, presumably because the Cap protein is excessively glycosylated in the yeast cells. Western-blot results prove that the Cap protein is expressed in recombinant saccharomyces cerevisiae cells. Further detection of protein display on the cell surface by immunofluorescence, see FIG. 9, control ST1814G showed no fluorescence (on FIG. 9); whereas ST1814G/Cap of the experimental group showed a clear green fluorescence (FIG. 9), indicating that Cap protein was successfully expressed and localized on the cell surface of recombinant s.cerevisiae.

The growth curve is divided into the influence of the expression of Cap protein on the growth of the recombinant Saccharomyces cerevisiae.

As shown in FIG. 10, there was no significant difference in the growth curves of glucose-induced strains ST1814G/Cap and JDY52 as compared to the control group.

Serum IgG and fecal IgA antibody changes were detected in orally immunized mice by E L ISA.

The result of E L ISA is shown in figure 11, and the titer of serum Cap specific IgG and fecal Cap specific IgA of mice immunized with ST1814G/Cap are both significantly higher than those of the blank strain group and the PBS group.

Although the present invention has been described with reference to the above examples, the scope of the present invention is defined by the appended claims, and any other changes or modifications within the scope of the present invention should be construed as being included in the following equivalents.

References:

[1].Schreuder,M.P.,et al.,Targeting of a heterologous protein to thecell wall of Saccharomyces cerevisiae.Yeast,1993.9(4):p.399-409.

[2].Appleton,H.and P.G.Higgins,VIRUSES AND GASTROENTERITIS ININFANTS.Lancet,1975.305(7919):p.1297.

[3].Oude Munnink,B.B.,et al.,A Novel Astrovirus-Like RNA VirusDetected in Human Stool.Virus Evolution,2016.2(1):p.vew005.

[4].Benedictis,P.D.,et al.,Astrovirus infections in humans andanimals–Molecular biology,genetic diversity,and interspeciestransmissions.Infection Genetics&Evolution,2011.11(7):p.1529-1544.

[5].Péter,et al.,Detection of a mammalian-like astrovirus in bird,European roller(Coracias garrulus).Infection Genetics&Evolution,2015(34):p.144-121.

[6] Dungjian Jun, enteric astrovirus infection and detection technique Western medicine 2003.1(2): page 165-167.

[7].Meliopoulos,V.and S.Schultz-Cherry,AstrovirusPathogenesis.Astrovirus Research,2012:p.65-77.

[8].Risco,C.,et al.,Ultrastructure of human astrovirus serotype2.Journal of General Virology,1995.76(8):p.2075-2080.

[9].Mendez,E.,E.Salasocampo and C.Arias,Caspases mediate processingof the capsid precursor and cell release of human astroviruses.Journal ofVirology,2004.78(16):p.8601-8608.

[10].Mendez,E.,et al.,Proteolytic Processing of a Serotype 8 HumanAstrovirus ORF2 Polyprotein.Journal of Virology,2002.76(16):p.7996-8002.

[11].Krishna and N.K.,Identification of Structural Domains Involvedin Astrovirus Capsid Biology.Viral Immunology,2005.18(1):p.17-26.

[12].Guo,Y.,et al.,YeastFab:the design and construction of standardbiological parts for metabolic engineering in Saccharomycescerevisiae.Nucleic Acids Research,2015.43(13):p.13.

[13].Boder,E.T.and K.D.Wittrup,Yeast surface display for screeningcombinatorial polypeptide libraries.Nat Biotechnol,1997.15(6):p.553-557.

[14].Boder,E.T.and K.D.Wittrup,Yeast surface display for directedevolution of protein expression,affinity,and stability.Methods Enzymol,2000.328:p.430-444.

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<211>34

<212>DNA

<213> Artificial sequence ()

<400>7

agcgtgcgtc tcggatgatg cagttacttc gctg 34

<210>8

<211>33

<212>DNA

<213> Artificial sequence ()

<400>8

gtgctgcgtc tcagctatca atggtgatgg tga 33

<210>9

<211>41

<212>DNA

<213> Artificial sequence ()

<400>9

gacgataagg taccaggatc catgccaggt aatgttgggc a 41

<210>10

<211>39

<212>DNA

<213> Artificial sequence ()

<400>10

gaattccacc acactggatc cgtatgccgc atcccaggc 39

<210>11

<211>24

<212>DNA

<213> Artificial sequence ()

<400>11

aagagacgca agacactgcg gata 24

<210>12

<211>25

<212>DNA

<213> Artificial sequence ()

<400>12

aagagacgca agacactgcg gatac 25

<210>13

<211>24

<212>DNA

<213> Artificial sequence ()

<400>13

aagagaccca agacactgcg gata 24

<210>14

<211>23

<212>DNA

<213> Artificial sequence ()

<400>14

aagagaccga gtcactgcca aca 23

<210>15

<211>2202

<212>DNA

<213>Saccharomyces cerevisiae

<400>15

atgaccatga ttacgccaag cttgatgaca ttatctttcg ctcattttac ctacctgttc 60

acaatattgt tgggattaac taatattgcc ttggcatctg atccagaaac gattctagtg 120

acgataacca agacaaacga tgcaaatggg gttgttacaa ctacagtttc acccgcgcta 180

gtctccacat ccactatcgt tcaagctggc actacgacat tgtatacgac ttggtgtcca 240

ttgacggtat ccacttcatc tgctgccgaa ataagtcctt caatatcgta cgctactacc 300

ctatccagat ttagtacttt gacattatct acagaagtct gctcccatga ggcatgtcct 360

tcgtcatcga cgttgccaac caccacctta tctgtgactt ccaagttcac ttcatatatt 420

tgccctactt gtcacacaac cgctatcagc tcattatccg aagtaggaac tacaaccgtg 480

gtatcatcca gcgccattga accatcaagt gcctctataa tctcacctgt cacctctaca 540

ctttcgagta caacatcgtc caatccaact actacctccc taagttcgac atctacatct 600

ccaagctcta catctacatc tccaagctct acatctacct catcaagttc gacatctacc 660

tcatcaagtt cgacatctac ctcatcaagt tcgacatcta catctccaag ttcgacatcc 720

acatcttcaa gtttgacatc cacatcttca agttctacat ctacatccca aagttctaca 780

tctacctcat caagttcgac atctacatct ccaagctcta catctacctc atcaagttca 840

acatctacat ctccaagttc taaatctact tctgcaagct ccacttccac ttcttcatat 900

tcaacatcta catccccaag tttgacttct tcatctccaa ctttggcttc cacttctcca 960

agttcaacat ctattagctc tacttttact gattcaactt catcccttgg ctcctctata 1020

gcatcttcat caacgtctgt gtcattatac agcccatcca cacctgttta ctccgtccct 1080

tcgacttcgt caaatgttgc aactccttct atgacttctt caactgttga aacaactgtt 1140

agttcacaaa gttcgtctga atatatcacc aaatcctcaa tttctactac tatcccatca 1200

ttttccatgt ctacatattt caccactgtt agtggagtca ctacaatgta tacgacatgg 1260

tgtccttata gctctgaatc tgagactagc acattaacca gtatgcatga aacggttaca 1320

acagacgcta cagtctgcac tcacgagtct tgcatgccct cgcagacaac aagtttgatt 1380

acatcttcta taaaaatgtc cactaaaaac gtcgcaactt ctgtaagcac ctcaacggtt 1440

gaatcctcat atgcatgctc cacatgtgct gaaacgtcac actcgtattc ttccgtgcaa 1500

acagcttcat caagttctgt aacacagcag accacatcca caaagagttg ggtaagttca 1560

atgacaactt cggatgaaga tttcaataag cacgctaccg gtaagtatca tgtaacatct 1620

tcaggtacct caaccatttc gactagtgta agtgaagcca cgagtacatc aagcattgac 1680

tcagaatctc aagaacaatc atcacactta ttatcgacat cggtcctttc atcctcctcc 1740

ttgtctgcta cattatcctc tgacagtact attttgctat tcagttctgt atcatcacta 1800

agtgtcgaac agtcaccagt taccacactt caaatttctt caacatcaga gattttacaa 1860

cccacttctt ccacagctat tgctacaata tctgcctcta catcatcact ttccgcaaca 1920

tctatctcta caccatctac ctctgtggaa tcgactattg aatcttcatc attgactccg 1980

acggtatctt ctattttcct ctcatcatca tctgctccct cttctctaca aacatctgtt 2040

accactacag aagtttccac tacttcaatc tccatacaat accaaacttc atcaatggta 2100

acaattagcc aatatatggg cagtggatcg caaacgcgtt tgccattagg aaagttggtc 2160

ttcgccatca tggcagttgc ttgcaatgta attttcagtt aa 2202

<210>16

<211>531

<212>DNA

<213> Artificial sequence ()

<400>16

atgcagttac ttcgctgttt ttcaatattt tctgttattg cttcagtttt agcacaggaa 60

ctgacaacta tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttg 120

tcaacgacta ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaa 180

tcagtaacgt ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagcccc 240

ataaacacac agtatgtttt taagcttctg caggctagtg gtggtggtgg ttctggtggt 300

ggtggttctg gtggtggtgg ttctgctagc atgactggtg gacagcaaat gggtcgggat 360

ctgtacgacg atgacgataa ggtaccagga tccagtgtgg tggaattctg cagatatcca 420

gcacagtggc ggccgctcga gtctagaggg cccttcgaag gtaagcctat ccctaaccct 480

ctcctcggcc tcgattctac gcgtaccggt catcatcacc atcaccattg a 531

<210>17

<211>1272

<212>DNA

<213>Porcine astroviruses

<400>17

atgccaggta atgttgggca acaacaggaa tcaatagcag ggttacgaag gacagtagaa 60

ccaacactgg atcccatcac aattatttca tcttctctac ttggacaggc accactctac 120

gggtcacttc acccaactga acctttgaat gctataacga tgcagggcct aggtggttgg 180

gcacagaaga tacatacctt ctccattcat gaattgaata agccagtctt tttgcagggt 240

gataatgaag ttgatccaca gagtttgcag ataaatacct acccaatcta caaaaagacc 300

tcatctacat atgatgtaat agggaaagtt catgctgcat catacgcccg tactgggcgt 360

caacccctaa actggaccac atgcctttgg agagcaacac aaactacaac atttactatc 420

cagggcacta gttctaacac ccagttcctt tttgttaggc caaacatcca acctaatcca 480

gtaaatctac ctaacttcac ctatgtggtg caggcaacta atgcaaagaa tatagcacag 540

acacaaatta gcattcagga aggcaactgg tacctttctg tgtttgcatc ttggggaggc 600

gccaaaaagt atgatatcta tggggtggaa ttttgggctg gagatcaaac tgttaaccca 660

agtcaaactt atgaggttga cccaacattt gatgcgtata aggtaggtat tattttggga 720

acagcgcaac ctttgcagct tcaacttcct gtccaaccaa cagcgctaac caccagtgag 780

attctggccc tcagagacat tgttagtggc cagtaccacc cctttccaac tcttccacca 840

ccaccaccat cagaatacga caatctggaa gtgccacctt tggaggatga ggaagaagaa 900

gaacaaggtg ccactggagg acatgccccc ttggaaagag ccagtcaaca gagagttaga 960

ggaccaaatg cagttgggga gatgtctctt gagaaaaccc ttgacaccaa aaactgggtt 1020

gaatttggcc atataaaaag accaccaaca cctttttcac ctatagaaga ggaggatgag 1080

gaagatgagg attctgattt agatgatgat gattatgctg aacctccctc tgccattaaa 1140

aacttactta ctcctgaagc taaggatctt tatggtcatc tgagacagaa gggtctcagt 1200

catgagcagg caaccaatgc tgcccaagcc gcgttccctc attttgctct tgaagcctgg 1260

gatgcggcat ac 1272

<210>18

<211>500

<212>DNA

<213> Artificial sequence ()

<400>18

gtcatctaag cacagtcgcg cgtccgaacc tagctctact ttagaggccc cggattctgg 60

tgctcgtaga ccgcagaacc gattgggggg atgtacaaca atatttgtta gtcacctttg 120

ggtcacgatc tcccacctta ctggaattta gtccctgcta taatttgcct tgcatataag 180

ttgcgttact tcagcgtcct aaccgcaccc ttagcacgaa gacagatttg ttcattccca 240

tactccggcg ttggcagggg gttcgcatgt cccacgtgaa acgttgctaa accctcaggt 300

ttctgagcga caaaagcttt aaacgggagt tcgcgctcat aacttggtcc gaatgcgggt 360

tcttgcatcg ttcgactgag tttgtttcat gtagaacggg cgcaaagtat acttagttca 420

atcttcaata cctcgtatca ttgtacacct gccggtcacc acccaacgat gtggggacgg 480

cgttgcaact tcgaggacct 500

<210>19

<211>500

<212>DNA

<213> Artificial sequence ()

<400>19

cgctagtaac atcagctaac gaaagagtta gaggctcgct aaatcgcact gtcggggtcc 60

cttgggtatt ttacactagc gtcaggacga ctagcatgtg tctttccttc caggggtatg 120

cgggtgcgtg gacaaatgag cagcatacgt atttactcgg cgtgcctgct ctctcgtatt 180

tctcctggag atcaaggaaa tgtttcatgt ccaagcgaaa agccgctcta cggaatggat 240

ctacgttact gcctgcataa ggaaaccggt gtagccaagg acgaaagcga ccctaggttc 300

taaccatcga ctttggcgga aaggtttcac tcaggaagca gacactgatt gacacggttt 360

agcagaacgt ttgaggacta ggtcaaattg agtggtttaa tatcggcatg tctggcttta 420

aaattcagta tagtgcgctg atcggaaacg aattaaaaac acgagttccc aaaaccaggc 480

gggctcgcca cgctaatcgg 500

Claims

1. The surface display system based on the Aga1-Aga2 saccharomyces cerevisiae is characterized by comprising an Aga gene derived from a yeast strain EBY100 and an Aga2 gene derived from a PYD1 plasmid, wherein the Aga gene expresses an Aga subunit as an anchoring component and is inserted into a cell wall through glycosyl phosphatidyl inositol, the Aga2 gene expresses an Aga2 subunit as an adhesion component and is used as an anchoring protein of a target peptide or protein, and the target peptide or protein is displayed on the surface of the saccharomyces cerevisiae through the combination of the anchoring component and the adhesion component.

2. A vector comprising the Aga1 fragment of claim 1 having the base sequence SEQ ID No. 15.

3. A surface-displayed yeast host cell comprising the Aga1-Aga2 Saccharomyces cerevisiae surface display system of claim 1.

4. A method for constructing surface display type yeast host cells is characterized in that a laboratory preservation strain JDY52 is used as an initial strain, a GPD promoter is cloned by PCR, a PIU211 lac promoter is replaced by the GPD promoter through reverse PCR amplification and seamless connection, a linearized plasmid PIU211(GPD) is transferred into JDY52 through lithium acetate conversion, a positive transformant is screened by an SD-URA selective culture medium and genome PCR, and a constitutive glucose promoter GPD-regulated and surface efficient display Aga1 yeast host strain ST1814G is obtained.

5. The method for constructing a surface-displayed yeast host cell according to claim 4, which comprises the steps of;

(1) construction of the PIU211 recombinant plasmid: extracting an EBY100 yeast genome, amplifying an Aga1 gene by PCR, and inserting the Aga1 gene into an integrated vector YIpalac 211 by seamless cloning;

(2) construction of the PIU211(GPD) recombinant plasmid: following the seamless cloning technology principle, a pair of reverse amplification primers is designed to amplify PIU211 (the rest parts except lac promoter), then GPD promoter is amplified by using HCKAN-GPD plasmid preserved in a laboratory as a template, and the terminal sequence of the vector is introduced at the 5' terminal of the GPD forward/reverse amplification primers. Replacing the lac promoter on the PIU211 plasmid with the GPD promoter by seamless cloning;

(3) preparation of linearized vector: the sequence of the exogenous insertion gene Aga1 of the recombinant plasmid PIU211(GPD) contains a recognition site of a restriction enzyme BsiWI, and a linearized DNA fragment can be obtained by digesting the PIU211 or the PIU211(GPD) with the BsiWI;

(4) constructing a saccharomyces cerevisiae host strain with the surface efficiently displaying Aga 1: the linearized PIU211 or PIU211(GPD) fragment is transferred into a starting strain JDY52 by a conventional lithium acetate conversion method, and the Aga1 yeast host strain ST1814G with high-efficiency surface display is obtained by SD-URA auxotrophy screening and PCR identification.

6. The porcine astrovirus fusion protein Aga2-Cap has the nucleic acid sequences of SEQ ID No.12 and SEQ ID No. 17.

7. A strain of Cap protein surface-displayed s.cerevisiae comprising the fusion protein of claim 6 in strain ST 1814G.

8. A Cap protein surface display Saccharomyces cerevisiae strain is characterized in that an Aga2 gene derived from a PYD1 plasmid is connected in series with a C-terminal hypervariable region gene containing a porcine astrovirus to construct a transcription unit of fusion expression of Aga2-Cap in a yeast, the transcription unit is realized and stably integrated into a genome through in vitro enzyme-linked, in vivo yeast transformation and homologous recombination technologies, and the transcription unit is displayed on the surface of a yeast cell through an Aga1-Aga2 surface display system to obtain a recombinant yeast strain with the surface display Cap protein.

9. The method for constructing a Cap protein surface display Saccharomyces cerevisiae strain according to claim 8, comprising the following steps:

(1) amplification of the C-terminal hypervariable region gene of the Cap protein: a PAStV4 whole genome sequence PAStV4/Tianjin/2018(access number MH425243) is separated from 29 pig diarrhea feces of Tianjin Silent sea and Ninghe farm by RT-PCR, and a Cap protein C-terminal hypervariable region gene is obtained by PCR amplification;

(2) the Aga2 gene and a C-terminal hypervariable region gene containing the porcine astrovirus: serially connecting the Aga2 gene and a Cap protein C-terminal hypervariable region gene by using an overlap PCR technology to obtain an Aga2-Cap fusion fragment;

(3) cloning Cap genes to a POT-GPD-TU vector by adopting a seamless cloning method to obtain a recombinant plasmid POT/Cap plasmid;

(4) the recombinant expression plasmid is used for transforming Saccharomyces cerevisiae, HO gene loci integrating exogenous metabolic pathways exist on chromosome IV of a host strain ST1814G, URR1(SEQ ID No.18), URR2(SEQ ID No.19) and HIS3, BsaI digests POT/Cap plasmid to release transcription units, BsmBI digests PMV-URR1, PMV-URR2 and PMV-L EU2 to release URR1, URR2 and L EU2, the transcription units, the URR and a selective marker L EU2 complete the directional assembly of a plurality of gene segments according to specific joint sequences so as to form long DNA segments containing the transcription units, and the assembled gene segments can be directionally integrated to HO gene loci of chromosome IV of ST1814G through yeast transformation.

10. The porcine astrovirus oral vaccine based on the saccharomyces cerevisiae surface display system is characterized in that a Cap protein surface display saccharomyces cerevisiae strain is directly used as the oral vaccine.