Recombinant pichia pastoris for indirect surface display of GnRH fusion protein

Abstract

The invention provides a fusion polypeptide capable of being displayed on the indirect surface of recombinant pichia pastoris, the fusion polypeptide comprises tandem repeat GnRH protein polypeptide, the amino terminal of the fusion polypeptide is fused with a CL7 tag, the carboxyl terminal of the GnRH protein polypeptide is fused with a Flg peptide fragment, and the amino acid sequence of the GnRH protein polypeptide is SEQ ID NO: 1. The Im7 protein can be displayed on the surface of a recombinant pichia pastoris cell with high efficiency and high activity, so that the tandem repeat pentavalent GnRH fusion protein which is recombined and expressed by escherichia coli and fused with CL7 protein and immunologic adjuvant peptide Flg can be indirectly displayed on the surface of the pichia pastoris cell with high efficiency and high activity; therefore, the storage stability of the GnRH fusion protein or the stability of oral immunization on acidic conditions or digestive enzymes in digestive tracts is improved, efficient GnRH antigen specific humoral immune response is induced, and GnRH is neutralized so as to generate an immune sterilization effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and in particular relates to a recombinant Pichia pastoris for indirect surface display of GnRH fusion protein. Background Art

[0002] As a GRAS (Generally Recognized as Safe) microorganism, Pichia pastoris is widely used in life science and biotechnology research, biopharmaceutical R&D and production, food and feed industry. As a eukaryotic microorganism, Pichia pastoris has the functions of mRNA transcription and processing, protein expression and secretion, protein degradation, glycosylation modification, etc.

[0003] The thickness of the yeast cell wall is about 200 to 300 nm, and it consists of two parts, the inner and outer parts. The inner layer of the yeast cell wall is composed of chitin, β-1,3 and β-1,6 glucans. The outer layer of the yeast cell wall is mainly composed of highly glycosylated mannoproteins. Most of these proteins are covalently linked to the inner layer of glucans. The composition and structure of the yeast cell wall structure can efficiently target antigen presenting cells (macrophages, dendritic cells), thereby activating the immune system. The antigens displayed on the surface of yeast cells can induce the body to produce efficient humoral and cellular immune responses.

[0004] Yeast surface display technology is a technology that fuses the exogenous target protein or polypeptide encoding gene with a specific cell wall anchor protein gene sequence, clones it into a yeast secretion expression vector and introduces it into yeast competent cells. The fusion gene is transcribed and translated in the yeast cells, and the fusion protein containing the cell wall anchor protein is positioned on the yeast cell surface with the help of the intracellular protein secretion mechanism, thereby achieving yeast cell surface display of the target protein or polypeptide.

[0005] However, studies have found that many proteins, such as pathogen protective antigens, are difficult to display on the surface of yeast cells with high efficiency or high immune activity. Therefore, it is necessary to develop an efficient Pichia pastoris surface display system to improve the efficiency of antigen display on the surface of Pichia pastoris cells. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant Pichia pastoris for indirect surface display of GnRH fusion protein. The prepared recombinant Pichia pastoris has efficient immune adjuvant activity and can induce efficient GnRH antigen-specific humoral immune response through injection or oral administration, thereby producing an immune sterilization effect.

[0007] The present invention first provides a fusion polypeptide that can be indirectly displayed on the surface of a recombinant Pichia pastoris, comprising a tandemly repeated GnRH protein polypeptide, a CL7 tag that can bind with high affinity to the Im7 protein displayed on the surface of Pichia pastoris is fused to the amino terminus of the GnRH protein polypeptide, and a Flg peptide segment is fused to the carboxyl terminus; the amino acid sequence of the GnRH protein polypeptide is QHWSYGLRPG (SEQ ID NO: 1);

[0008] Furthermore, the amino acid sequence of the CL7 tag is as follows:

[0009] SKSNEPGKATGEGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFESFDDFRETFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKRTSFELNHQKPIEQNGGVYDMDNISVVTPKRNIDIEG(SEQ ID NO:2);

[0010] The nucleotide sequence of its coding gene is as follows:

[0011] agcaaaagcaatgaaccgggtaaagcaaccggtgaaggtaaaccggttaataacaaatggctgaacaa

[0012] tgccggtaaagatctgggtagtccggttccggatcgtattgcaaataaactgcgtgataaagaattcg

[0013] agagcttcgatgattttcgtgaaaccttttgggaagaagttagcaaagatcctgaactgagcaaacag

[0014] tttagccgcaataacaatgatcgtatgaaagttggtaaagcaccgaaaacacgtacccaggatgttag

[0015] cggtaaacgtacctcatttgaactgaatcatcagaaaccgattgaacagaatggtggcgtttatgata

[0016] tggataacattagcgttgttaccccgaaacgcaacattgatattgaaggc (SEQ ID NO: 3).

[0017] The amino acid sequence of the Flg peptide segment is as follows: INNNLQRVRELAVQSANSTNSQSDLDS (SEQ ID NO: 4).

[0018] The nucleotide sequence of its coding gene is as follows:

[0019] ATCAACAACAACCTTCAACGGTTTCGGGAATTGGCTGTTCAGTCAGCTAACTCCACGAATTCTCAATCGGACTTGGACAGC (SEQ ID NO: 5).

[0020] As a specific record of the embodiment, the amino acid sequence of the fusion polypeptide is as follows:

[0021] MGSKSNEPGKATGEGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFESFDDFRETFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKRTSFELNHQKPIEQNGGVYDMDNISVVTPKR NIDIEGGGGGSGGGGSKLQHWSYGLRPGKKQHWSYGLRPGGSGQHWSYGLRPGKKQHWSYGLRPGGSGQHWSYGLRPGGGGSASINNNLQRVRELAVQSANSTNSQSDLDSGGGSLEHHHHHH(SEQ ID NO:6).

[0022] The nucleotide sequence of the gene encoding the fusion polypeptide is as follows:

[0023] atgggcagcaaaagcaatgaaccgggtaaagcaaccggtgaaggtaaaccggttaataacaaat

[0024] ggctgaacaatgccggtaaagatctgggtagtccggttccggatcgtattgcaaataaactgcgtgat

[0025] aaagaattcgagagcttcgatgattttcgtgaaaccttttgggaagaagttagcaaagatcctgaact

[0026] gagcaaacagtttagccgcaataacaatgatcgtatgaaagttggtaaagcaccgaaaacacgtaccc

[0027] aggatgttagcggtaaacgtacctcatttgaactgaatcatcagaaaccgattgaacagaatggtggc

[0028] gtttatgatatggataacattagcgttgttaccccgaaacgcaacattgatattgaaggcggaggcgg

[0029] tggatcaggtggaggaggtagtaagcttcagcactggagttacggattgcgtcctggcaaaaagcaac

[0030] actggtcctacggactgagacccggaggcagtggccagcattggtcatacggccttcgtcccggcaag

[0031] aaacagcattggtcttatggcctaaggccaggcggaagtggtcaacattggagttatggacttagacc

[0032] tggaggtggtggttccgctagcatcaacaacaaccttcaacgggttcgggaattggctgttcagtcag

[0033] ctaactccacgaattctcaatcggacttggacagcggaggaggttcgctcgagcaccaccaccacac

[0034] cactga (SEQ ID NO: 7).

[0035] In one aspect, the present invention further provides a recombinant Pichia pastoris, wherein the recombinant Pichia pastoris indirectly displays the above-mentioned fusion polypeptide on its surface.

[0036] Furthermore, the recombinant Pichia pastoris is obtained by fusion-expressing a trivalent tandem Im7 protein from Escherichia coli and a cell wall protein SED1 from Saccharomyces cerevisiae in the original Pichia pastoris GS115 strain, thereby achieving the display of the Im7 protein on the surface of the Pichia pastoris cells.

[0037] The amino acid sequence of the Im7 protein from Escherichia coli is as follows: MELKNSISDYTEAEFVQLLKEIEKENVAATDDVLDVLLEHFVKITEHPDGTDLIYYPSDNRDDSPEGI VKEIKEWRAANGKPGFKQG (SEQ ID NO: 8),

[0038] One of the encoding nucleotide sequences is as follows:

[0039] atggaattgaagaactccatctccgactacactgaggctgagttcgtccagttgttgaaagagatcga

[0040] gaaagagaacgttgctgccaccgatgatgttttggacgttttgttggagcacttcgtcaagatcactg

[0041] aacacccagatggtactgacctgatctactacccatccgacaacagagatgactctccagagggtatc

[0042] gtcaaagaaatcaaagaatggcgtgccgccaacggtaagccaggttttaaacaaggt (SEQ ID NO: 9);

[0043] The amino acid sequence of the cell wall protein SED1 derived from Saccharomyces cerevisiae is as follows:

[0044] QFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPT

[0045] TAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTT

[0046] TTPPYNPSTDYTTDYTVVTEYTTYCPEPTTFTTTNGKTYTVTEPTTLTITDCPCTIEKPTTTSTTEYTV

[0047] VTEYTTYCPEPTTFTTTNGKTYTVTEPTTLTITDCPCTIEKSEAPESSVPVTESKGTTTKETGVTTKQT

[0048] TANPSLVSTVVPVSSSASSHSVVINSNGANVVVPGALGLAGVAMLFL(SEQ ID NO:10);

[0049] The nucleotide sequence of its coding gene is as follows:

[0050] caattttccaacagtacatctgcttcttccaccgatgtcacttcctcctcttccatctccacttcctctggctcagtaactatcacatcttctgaagctccagaatccgacaacggtaccagcacagctgcaccaactgaaacctcaacagaggctccaaccactgctatcccaactaacggtacctctactgaagctccaaccactgctatcccaactaacggtacctctactgaagctccaactgatactactactgaagctccaaccaccgctcttccaactaacggtacttctactgaagctccaactgatactactactgaagctccaaccaccggtcttccaaccaacggtaccacttcagctttcccaccaactacatctttgccaccaagcaacactaccaccactcctccttacaacccatctactgactacaccactgactacactgtagtcactgaatatactacttactgtccagaaccaaccactttcaccacaaacggtaagacttacaccgtcactgaaccaaccacattgactatcactgactgtccatgcaccattgaaaagccaacaaccacatcaaccaccgaatacactgtagtcactgagtacactacttactgtccagaaccaaccactttcaccacaaacggtaagacttacaccgtcactgaaccaaccactttgactatcactgactgtccatgtactattgaaaagagcgaagcccctgagtcttctgtcccagttaccgaatctaagggcactaccaccaaagaaacaggtgttactaccaaacaaaccacagccaacccaagtctaaccgtctccacagtcgtcccagtttcatcctctgcttcttctcattccgttgtcatcaacagtaacggtgctaacgtcgtcgttccaggtgctttaggtttggctggtgttgctatgttattcttataa(SEQ ID NO:11)。

[0051] The recombinant Pichia yeast provided by the present invention can be used to prepare an immune preparation for pet sterilization;

[0052] In one aspect, the present invention also provides an oral immune preparation for pet sterilization, which contains the above-mentioned recombinant Pichia pastoris.

[0053] Compared with the prior art, the present invention has the following advantages:

[0054] Im7 protein can be displayed on the surface of recombinant Pichia pastoris cells with high efficiency and high activity, so that the tandemly repeated 5-valent GnRH fusion protein fused with CL7 protein and immune adjuvant peptide Flg expressed by Escherichia coli can be indirectly displayed on the surface of the above Pichia pastoris cells with high efficiency and high activity;

[0055] Due to the large particle structure and structural stability of Pichia cells, the pentavalent GnRH fusion protein is indirectly displayed on the surface of the above-mentioned recombinant Pichia cells, which can improve the storage stability of the GnRH fusion protein or the stability of oral immunization to acidic conditions or digestive enzymes in the digestive tract;

[0056] Due to the efficient immune adjuvant activity of yeast cells, the pentavalent GnRH fusion protein indirectly displayed on the surface of the above-mentioned recombinant Pichia pastoris cells can induce a highly efficient GnRH antigen-specific humoral immune response through injection or oral immunization, neutralize GnRH and thus produce an immune sterilization effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Agarose gel electrophoresis detection of DNA fragment GS-5×GnRH PCR amplification product;

[0058] Figure 2 Agarose gel electrophoresis detection of DNA fragment pET28a-CL7-Flg PCR amplification product;

[0059] Figure 3 Agarose gel electrophoresis detection of PCR products of pET28a-CL7-5×GnRH-Flg recombinant bacterial solution;

[0060] Figure 4 Agarose gel electrophoresis detection of pET28a-CL7-5×GnRH-Flg plasmid;

[0061] Figure 5 Expression of fusion protein CL7-5×GnRH-Flg in BL21(DE3) and Ni 2+ Affinity purification SDS-PAGE detection image;

[0062] Figure 6SDS-PAGE detection of the supernatant after Pichia pastoris GS115 cells displaying trivalent Im7 protein adsorbed the fusion protein CL7-5×GnRH-Flg. DETAILED DESCRIPTION

[0063] The present invention uses the Pichia pastoris surface display technology to display the immune protein Im7 derived from Escherichia coli on the surface of Pichia pastoris GS115 cells, and through the strong interaction between the Im7 protein and the CL7 protein, the tandem repeat GnRH fusion protein fused with the CL7 protein and the immune adjuvant peptide Flg is indirectly displayed on the surface of the Pichia pastoris cells, thereby obtaining a pet GnRH immune sterilization biological preparation.

[0064] The CL7 fusion tag protein used in the present invention is a DNA enzyme domain derived from the colicin DNA enzyme CE7, which is about 15 kDa in size. In Escherichia coli cells, the CE7 DNA enzyme active domain with strong cytotoxicity will bind to an inhibitory molecule called Immunity protein 7 (Im7) with a size of about 10 kDa and containing 87 amino acid residues with a strength close to that of a covalent bond. The dissociation constant Kd of the colicin CE7 DNA enzyme domain and its immune protein Im7 reaches 10 -14 ~10 -17 M, has a strong binding ability, thus achieving strict regulation of its DNA enzyme activity. By intercepting the DNA enzyme activity domain of CE7 and modifying it, a protein tag called CL7 was obtained. The CL7 tag lost the original DNA enzyme activity of CE7 but maintained a high affinity with Im7. The CL7 tag contains 131 amino acid residues and has a molecular weight of 14.9kDa.

[0065] The Im7 protein displayed on the surface of Pichia pastoris cells retains the ability to bind to the CL7 tag with high intensity by tandemly repeating the trivalent Im7 protein with a glycine-serine flexible linker peptide and fusing it with the cell wall protein SED1 from Saccharomyces cerevisiae. The exogenously expressed CL7 fusion protein is mixed and incubated with Pichia pastoris displaying the Im7 protein, and the CL7 fusion protein can be efficiently and indirectly displayed on the surface of Pichia pastoris cells.

[0066] The applicant analyzed the amino acid sequence of gonadotropin-releasing hormone (GnRH) of dogs and cats and determined that the amino acid residues of the sequence QHWSYGLRPG can be covalently coupled with a suitable carrier protein or fused with an adjuvant protein in an appropriate structural form to induce the body to produce GnRH antibodies after immunizing animals. High-titer and long-lasting GnRH antibodies can sterilize the immunized animals after binding to GnRH.

[0067] The Flg peptide segment (amino acid residues 85-111) derived from the flagellin FliC of Salmonella typhimurium (GenBank accession no. D13689) is fused with the target antigen and expressed, which has strong immune adjuvant activity and can improve antigen-specific immune response. In addition, the GnRH polypeptide in the present invention improves the immunogenicity of GnRH by tandem fusion expression structure.

[0068] The present invention first constructs a recombinant Pichia pastoris cell that displays a trivalent Escherichia coli Im7 protein on its surface, by fusion-expressing a trivalent tandem Im7 protein derived from Escherichia coli and a cell wall protein SED1 derived from Saccharomyces cerevisiae in the original Pichia pastoris GS115 strain, thereby achieving the display of the Im7 protein on the surface of the Pichia pastoris cell.

[0069] In another aspect, the present invention also provides an E. coli recombinant expression plasmid pET28a-CL7-5×GnRH-Flg of a tandemly repeated 5-valent GnRH fusion protein fused with CL7 protein, GnRH polypeptide fragment, and immune adjuvant peptide Flg.

[0070] The method for constructing a recombinant Pichia pastoris strain displaying a trivalent Im7 protein of the present invention comprises the following steps:

[0071] Using the genome of Saccharomyces cerevisiae strain INVSC 1 as a template, PCR amplified the fragment SED1, and using this fragment as a template, PCR amplified the fragment HA-MCS-SED1 with primers;

[0072] The fragment was cloned into the pPICZαA vector by homologous recombination in E. coli cells to obtain the vector pPICZα-HA-SED1 with a multi-cloning site between the HA tag and SED1;

[0073] Using the Im7 Pichia pastoris secretory expression vector pPICZαA-Im7 as a template, the Im7 DNA fragment was amplified by PCR, and the fragment was cloned into the pPICZαA-HA-SED1 vector by homologous recombination in Escherichia coli cells to obtain the vector pPICZα-HA-Im7-SED1;

[0074] Using vector pPICZαA-HA-Im7-SED1 as a template, PCR amplified the DNA fragment HA-Im7; by restriction endonuclease digestion and DNA ligase ligation, the DNA fragment HA-Im7 was cloned into vector pPICZαA-HA-Im7-SED1 to obtain the bivalent Im7 protein display vector pPICZαA-HA-2×Im7-SED1;

[0075] The same cloning method as above was used to clone the DNA fragment HA-Im7 into the vector pPICZαA-HA-2×Im7-SED1 by endonuclease digestion and DNA ligase ligation to obtain the trivalent Im7 protein Pichia pastoris surface display vector pPICZαA-HA-3×Im7-SED1;

[0076] The recombinant plasmid pPICZα-HA-3×Im7-SED1 was linearized with restriction endonuclease Pme I and then electroporated into Pichia pastoris GS115 competent cells to obtain the recombinant Pichia pastoris strain GS115 / pPICZα-HA-3×Im7-SED1;

[0077] A single colony of strain GS115 / pPICZα-3×Im7-SED1 was selected for colony PCR to identify positive recombinants containing the target gene;

[0078] The GS115 / pPICZα-3×Im7-SED1 recombinant single colony with positive colony PCR was picked for enrichment culture and methanol induction culture, and the HA-3×Im7-SED1 fusion protein displayed on the surface of the recombinant Pichia pastoris cells was detected with anti-HA tag mouse antibody, PE-Cy7-coupled goat anti-mouse antibody, and fusion protein CL7-eGFP to obtain the GS115 / pPICZα-HA-3×Im7-SED1 recombinant Pichia pastoris strain;

[0079] The recombinant Pichia pastoris inducing expression method of the present invention comprises the following steps:

[0080] The recombinant Pichia yeast seed solution was inoculated into BMGY medium and cultured until OD 600 2 to 6, collect the cells by centrifugation and transfer them to BMMY medium, add 0.5% to 1% methanol for induction; add 0.5% or 1% methanol for induction every 12 hours or 24 hours, collect the cells after 72 to 84 hours of induction, and store them at 4°C or in a freezer;

[0081] The present invention discloses the above-mentioned methanol-induced recombinant Pichia pastoris inactivation method, which comprises the following steps:

[0082] The Pichia pastoris cells in the above step were resuspended in 1×PBS buffer, treated at 56-60°C for 1-2 hours, and the Pichia pastoris cells were collected and stored at 4°C or frozen;

[0083] The present invention discloses a construction of an expression vector of a tandemly repeated 5-valent GnRH fusion protein CL7-5×GnRH-Flg fused with CL7 protein and immune adjuvant peptide Flg and a protein expression method, the method comprising the following steps:

[0084] The amino acid residue sequence of 5-valent GnRH was fused using the connecting peptides GSG and KK. The DNA coding sequence and primers of the fusion peptide were designed according to the codon preference of E. coli using DNAworks. The primers were synthesized by Shanghai Bioengineering, and the GS-5×GnRH DNA fragment was synthesized using overlapping PCR.

[0085] Using pET28a-CL7-CL7-PCV2bCap-Flg as a template, PCR amplified the vector pET28a-CL7-Flg, and then cloned the above-mentioned GS-5×GnRH fragment into the above-mentioned vector by non-restricted cloning mediated by homologous recombination to obtain the expression vector pET28a-CL7-5×GnRH-Flg.

[0086] Chemically competent E. coli BL21(DE3) cells were transformed with pET28a-CL7-5×GnRH-Flg plasmid, and single colonies were picked for protein expression;

[0087] The bacterial cells are collected and broken by ultrasonic or high-pressure sterilizer, the broken bacterial solution is centrifuged or filtered at high speed, and the obtained supernatant is purified by nickel bead affinity chromatography;

[0088] The present invention discloses a method for indirectly displaying CL7-5×GnRH-Flg fusion protein by the inactivated recombinant Pichia pastoris, the method comprising the following steps:

[0089] Take the above-mentioned inactivated recombinant Pichia pastoris GS115 / pPICZα-3×Im7-SED1 cells, add an appropriate amount of the above-mentioned CL7-5×GnRH-Flg protein solution and fully resuspend the above-mentioned yeast cells, and shake in a constant temperature shaker at 4°C or 18°C ​​at a low speed for 1 to 1.5 hours or longer;

[0090] Collect Pichia cells by centrifugation and wash them 3-5 times with 1× PBS, and store them at 4°C or in a freezer;

[0091] The present invention discloses a method for immunizing animals with the recombinant Pichia pastoris that indirectly displays the CL7-5×GnRH-Flg fusion protein, and the method comprises the following steps:

[0092] The inactivated recombinant Pichia pastoris cells indirectly displaying the CL7-5×GnRH-Flg fusion protein were resuspended in 1×PBS buffer;

[0093] Take a certain amount and dilute it according to the proportion, and measure the OD 600 The original Pichia yeast culture OD 600 Numeric value;

[0094] The above-mentioned Pichia pastoris cell suspension indirectly displaying CL7-5×GnRH-Flg fusion protein was diluted to OD 600 It is 20 or 40 or other concentrations;

[0095] Take the above mixed Pichia pastoris cell suspension indirectly displaying CL7-5×GnRH-Flg fusion protein and perform intramuscular, subcutaneous, intraperitoneal or oral immunization at 100 μL, 200 μL or other doses.

[0096] The present invention is described in more detail below with reference to specific embodiments.

[0097] Example 1: Preparation of pet sterilization immune preparation and animal immunization using Pichia pastoris indirect surface display of tandem repeat pentavalent GnRH

[0098] 1. Preparation and inactivation of Pichia pastoris cells displaying tandem trivalent Im7 proteins on their surface

[0099] 1.1. Preparation of Pichia pastoris cells displaying tandem trivalent Im7 proteins on their surface

[0100] Inoculate a single colony of the GS115 / pPICZαA-HA-3×Im7-SED1 strain into a 500 mL Erlenmeyer flask containing 100 mL of BMGY medium and culture at 28°C and 200 rpm until the yeast cell OD 600 2 to 6;

[0101] The cells were collected by centrifugation at 5000 g for 5 minutes, and the BMGY medium was completely discarded;

[0102] Add 100 mL of BMMY medium containing 1% methanol (v / v) to resuspend the bacteria and transfer the bacterial solution to a sterile 500 mL Erlenmeyer flask, and continue culturing in a constant temperature shaker at 28°C and 200 rpm;

[0103] Methanol was added every 24 hours to a final concentration of 1% (v / v);

[0104] After 72 h of methanol induction, the cells were collected by centrifugation at 5000 g for 5 min and stored at 4°C or frozen;

[0105] 1.2. Inactivation of Pichia pastoris cells displaying tandem trivalent Im7 proteins

[0106] Take 50 mL of methanol-induced Pichia pastoris GS115 / pPICZαA-HA-3×Im7-SED1 bacterial solution in a 50 mL centrifuge tube and centrifuge at 8000 g for 5 minutes to collect the bacteria;

[0107] Resuspend the cells in 50 mL of 1× PBS buffer and collect them by centrifugation at 8000 g for 5 min;

[0108] Resuspend the cells in 50 mL of 1× PBS buffer and place in a 56-60°C constant temperature water bath for 1-2 hours;

[0109] The cells were collected by centrifugation at 8000 g for 5 min and stored at -80 °C for future use;

[0110] 2. Construction of E. coli expression vector of fusion protein CL7-5×GnRH-Flg and protein expression and purification

[0111] 2.1. Construction of expression vector pET28a-CL7-5×GnRH-Flg

[0112] Table 1: pET28a-CL7-Flg PCR amplification primer sequence information

[0113] F GGTGGTTCCgctagcATCAACAACAACCTTCAACGGG CL7R gccttcaatatcaatgttgcgtttcg

[0114] Table 2: GS-5×GnRH gene synthesis primers

[0115] CL7GSF gaaacgcaacattgatattgaaggcGGAGGCGGTGGATCAGGTGGAGGAGGTAGT GnRH1 CAGGACGCAATCCGTAACTCCAGTGCTGAAGCTTCTACTACCTCCTCCACCTGATCC GnRH2 CAGGACGCAATCCGTAACTCCAGTGCTGAAGCTTACTACCTCCTCCACCTGATCC GnRH3 AGTTACGGATTGCGTCCTGGCAAAAAGCAACACTGGTCC GnRH4 GGCCACTGCCTCCGGGTCTCAGTCCGTAGGACCAGTGTTGCTTTTTGC GnRH5 CCGGAGGCAGTGGCCAGCATTGGTCATACGGCCTTCGTCCCGGCAAGAAACAGCA GnRH6 GCCTTAGGCCATAAGACCAATGCTGTTCTTGCCGGG GnRH7 TTGGTCTTATGGCCTAAGGCCAGGCGGAAGTGGTCAACAT GnRH8 CAGGTCTAAGTCCATAACTCCAATGTTGACCACTTCCGCC GnRH9 TGGAGTTATGGACTTAGACCTGGAGGTGGTGGTTCCCGCT FlgGnRH10 AAGGTTTGTTGTTGATgctagcGGAACCACCACCTCCAGG

[0116] Table 3: PCR amplification system

[0117] PCR components Volume (μL) 2×Phanta Max Master Mix 25 Forward primer F (10 μM) 2 Reverse primer R (10 μM) 2 template - <![CDATA[ddH2O]]> Add to 50 μL

[0118] Referring to the system in Table 3, pET28a-CL7-PCV2bCap-Flg was used as a template, and the primers in Table 1 were used to PCR amplify the pET28a-CL7-Flg vector. The PCR product was electrophoresed with 0.8% agarose gel and then recovered with a Sigma DNA agarose gel recovery kit;

[0119] Referring to the system shown in Table 3, the primers in Table 2 were used as templates, and the GS-5×GnRH DNA fragment was synthesized through two rounds of PCR using overlapping PCR;

[0120] The GS-5×GnRH fragment and the pET28a-CL7-Flg vector were transformed into Escherichia coli DH5α by the conventional transformation method of Escherichia coli chemical competence at a molar ratio of 3:1, and coated on LB solid medium (kalamycin, 100 μg / mL) plates;

[0121] Pick a single colony from the above plate, inoculate it into an Eppendorf tube containing 0.5 mL LB liquid medium (kalamycin, 100 μg / mL), and culture it in a 37°C constant temperature shaker at 200 rpm for 8 to 12 hours;

[0122] The PCR reaction system was prepared according to Table 3 using primers CL7GSF and FlgGnRH10, the above bacterial solution was used as a template, the GS-5×GnRH fragment was used as a positive control template, and the above LB liquid culture medium was used as a negative control template. The E. coli containing the target plasmid was identified by bacterial solution PCR and sequenced to obtain the E. coli expression vector pET28a-CL7-5×GnRH-Flg and the plasmid was extracted for protein expression;

[0123] 2.2. Expression and purification of fusion protein CL7-5×GnRH-Flg in E. coli

[0124] Take an appropriate amount of pET28a-CL7-5×GnRH-Flg plasmid in a sterile Eppendorf tube, transform Escherichia coli BL21 (DE3) chemically competent cells according to the conventional transformation method of Escherichia coli chemically competent cells, apply LB (kalamycin, 100 μg / mL) solid culture medium, and culture inverted overnight in a 37°C constant temperature incubator; pick single colonies from the above-mentioned overnight cultured BL21 (DE3) plates, inoculate them in LB liquid culture medium (kalamycin, 100 μg / mL), and culture them in a 37°C constant temperature shaker at 200 rpm until OD 600 When the p-value was about 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the culture was continued at 37°C and 200 rpm for 4-6 hours, and then the cells were collected by centrifugation at 8000 g and 4°C for 5 minutes;

[0125] E. coli cells were washed once with 1× PBS (pH 7.4) buffer containing 10 mM imidazole, centrifuged and the remaining PBS buffer was aspirated, then quickly frozen with liquid nitrogen and stored at -80°C;

[0126] To the frozen E. coli cells, 1×PBS (pH 7.4) buffer containing 10 mM imidazole and PMSF with a final concentration of 1 mM were added, and the cells were disrupted by ultrasonication on an ice-water mixture;

[0127] After the ultrasonic disruption of cells, PMSF was added again with a final concentration of 0.1 mM;

[0128] Centrifuge at 10,000 g and 4 °C for 30 min, and filter the supernatant with a 0.22 μm disposable filter;

[0129] The Ni was equilibrated with 3 to 5 column volumes of 1× PBS (pH 7.4) buffer containing 10 mM imidazole. 2+ Affinity purification column, and then the supernatant solution of the E. coli ultrasonic disruption containing the fusion protein CL7-5×GnRH-Flg is respectively loaded onto the column for binding;

[0130] After loading onto the column, wash the impure proteins with 5 to 10 column volumes of 1× PBS (pH 7.4) buffer containing 10 mM imidazole;

[0131] After washing, the target protein was eluted with 3 to 5 column volumes of 1×PBS (pH 7.4) buffer containing 1 M imidazole, and the target protein was frozen and stored at 4°C, -20°C or -80°C for future use;

[0132] 3. Preparation of Pichia pastoris indirectly displaying CL7-5×GnRH-Flg fusion protein

[0133] Take two tubes (50 mL centrifuge tubes) of the inactivated recombinant Pichia pastoris GS115 / pPICZα-3×Im7-SED1 cells and add 5 mg of the Ni 2+ The column-purified CL7-5×GnRH-Flg protein solution was used to fully resuspend the yeast cells;

[0134] Incubate in a constant temperature shaker at 4°C or 18°C, 60 rpm for 1 to 1.5 hours or longer;

[0135] Take 0.1 mL of the above yeast cells to a 1.5 mL Eppendorf tube, add 0.4 mL of 1× PBS (pH 7.4), then add 0.5 mL of CL7-eGFP fusion protein and mix well, and shake in a constant temperature shaker at 4°C or 18°C, 120 rpm for 1 hour;

[0136] The yeast cells were collected by centrifugation at 8000 g for 1 min and washed 2-3 times with 1× PBS (pH 7.4);

[0137] The Pichia pastoris cells without CL7-eGFP fusion protein were used as a control, and the green fluorescence of the Pichia pastoris cells was observed under a blue light instrument;

[0138] If there is no fluorescence, it means that the Im7 protein on the surface of the Pichia pastoris cells is completely bound by the CL7-5×GnRH-Flg fusion protein. The Pichia pastoris cells and supernatant are collected by centrifugation and washed 3 to 5 times with 1×PBS (pH 7.4) and stored at 4°C or frozen;

[0139] If there is fluorescence, it means that there is still Im7 protein on the surface of the yeast cells that has not been bound by the CL7-5×GnRH-Flg fusion protein, and it is necessary to add the fusion protein and continue binding;

[0140] 4. Animal Immunization with Recombinant Pichia Pastoris Cells Indirectly Displaying 5×GnRH Fusion Protein

[0141] The above-mentioned Pichia cells indirectly displaying 5×GnRH protein were resuspended in 1×PBS (pH 7.4) buffer;

[0142] Take 0.1-0.5 mL and dilute it with 1× PBS (pH 7.4) buffer at a ratio of 1:2. Select the appropriate dilution of the bacterial solution to measure the OD 600 The original Pichia yeast culture OD 600 Numeric value;

[0143] The above Pichia pastoris cell suspension indirectly displaying 5×GnRH protein was diluted with 1×PBS (pH 7.4) buffer to an OD 600 is 20;

[0144] The mixed Pichia cell suspension was taken and 100 μL / mouse was used for intramuscular immunization. The second immunization was performed 14 days after the first immunization, and the third immunization was performed 14 days after the second immunization.

[0145] Blood was collected 1 day before immunization 1, 1 day before immunization 2, 1 day before immunization 3, 14 days after immunization 3, and 14 days after immunization 3, and antibody titer was tested using GnRH fusion protein. The results are shown in Table 4;

[0146] Table 4: GnRH antibody titer data table

[0147] Immune serum 1. Antibody titer 2. Antibody titer 3. Antibody titer GS115 / 3×Im7 400 1600 160 GS115 / 3×Im7 / CL7-5×GnRH-Flg 1600 64000 256000

[0148] 3. A mating experiment was conducted 14 days after immunization, and the litter size of the immunized mice and the control group mice was counted to calculate the sterilization efficiency. The results are shown in Table 5.

[0149] Table 5: Immunization sterilization efficiency data table

[0150]

[0151] The experimental results show that, through injection or oral immunization, the GnRH fusion protein indirectly displayed on the surface of the recombinant Pichia pastoris cells of the present invention can induce a highly efficient GnRH antigen-specific humoral immune response, neutralize GnRH, and thus produce an immune sterilization effect.

Claims

1. A fusion polypeptide capable of indirect surface display on a recombinant Pichia pastoris, characterized in that: The fusion polypeptide comprises a tandemly repeated GnRH protein polypeptide, and a CL7 tag capable of binding with high affinity to the Im7 protein displayed on the surface of Pichia pastoris is fused to the amino terminus of the GnRH protein polypeptide, and a Flg peptide segment is fused to the carboxyl terminus of the GnRH protein polypeptide, wherein the amino acid sequence of the GnRH protein polypeptide is SEQ ID NO:

1.

2. The fusion polypeptide according to claim 1, characterized in that The amino acid sequence of the CL7 tag is SEQ ID NO:

2.

3. The fusion polypeptide according to claim 1, characterized in that The amino acid sequence of the Flg peptide segment is SEQ ID NO:

4.

4. The fusion polypeptide according to claim 1, characterized in that The amino acid sequence of the fusion polypeptide is SEQ ID NO:

6.

5. The fusion polypeptide according to claim 4, characterized in that The nucleotide sequence of the gene encoding the fusion polypeptide is SEQ ID NO:

7.

6. A recombinant Pichia pastoris, characterized in that: The recombinant Pichia pastoris indirectly displays the fusion polypeptide of claim 1 on its surface.

7. The recombinant Pichia pastoris according to claim 6, characterized in that The recombinant Pichia pastoris is obtained by fusion expression of a trivalent tandem Im7 protein derived from Escherichia coli and a cell wall protein SED1 derived from Saccharomyces cerevisiae in the original Pichia pastoris GS115 strain.

8. The recombinant Pichia pastoris according to claim 7, characterized in that The amino acid sequence of the Im7 protein is SEQ ID NO:8, and the amino acid sequence of the cell wall protein SED1 is SEQ ID NO:

10.

9. Use of the recombinant Pichia pastoris according to claim 6 in preparing an immune preparation for pet sterilization.

10. An oral immune preparation for pet sterilization, characterized in that: The immune preparation contains the recombinant Pichia pastoris according to claim 6.

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