CN111675758A

CN111675758A - Genetic engineering subunit vaccine for resisting sheep echinococcosis infection

Info

Publication number: CN111675758A
Application number: CN202010648762.8A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-07-07
Filing date: 2020-07-07
Publication date: 2020-09-18
Anticipated expiration: 2040-07-07
Also published as: CN111675758B

Abstract

The invention discloses a genetic engineering subunit vaccine for resisting sheep echinococcosis infection, which comprises a coding gene sequence shown as SEQ ID NO: 1. The Eg95 protein is optimized, the genetic engineering subunit vaccine for resisting sheep echinococcosis infection is prepared by using the Eg95 protein, the antigenicity, the immunogenicity and the function of the obtained vaccine are similar to those of natural protein, the expression level is higher, the immunogenicity is strong, a good immune effect can be provided only by a small amount, and no pathogenicity exists for sheep, meanwhile, the optimized Eg95 protein expressed by CHO cells and the like is high in glycosylation level, strong in solubility and high in expression amount (which can reach 2-3g/L), and the vaccine can be prepared by large-scale serum-free suspension culture in a bioreactor, so that the production cost of the vaccine is greatly reduced.

Description

Genetic engineering subunit vaccine for resisting sheep echinococcosis infection

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a genetic engineering subunit vaccine for resisting sheep echinococcosis infection, a preparation method and application thereof, and belongs to the technical field of animal immunity drugs.

Background

Echinococcosis (Hydatidosis, Hydatid Disease), also known as Echinococcosis (Echinococcosis) or Cystic Echinococcosis (CE), is a kind of zoonosis parasitic Disease caused by Echinococcus granulosus (Eg) or Echinococcus multilocularis (e.g. multilocularis) larva infecting sheep and parasitizing in human liver, lung and other organs, and is one of the five major parasitic diseases planned to be prevented and controlled by the ministry of health in china. The disease has wide prevalence and strong pathogenic capability, is difficult to prevent and treat, and causes great economic loss to China every year. Echinococcosis is distributed worldwide, China is one of the countries with the highest incidence rate of echinococcosis, and the Ministry of agriculture ranks echinococcosis as a second class of animal epidemic diseases. Because the effect of treating echinococcosis by using a medicament and a method of cyst removal is not ideal, the disease is mainly prevented at present, and the genetic engineering vaccine is the most ideal method for preventing the disease due to the characteristics of high efficiency and specificity.

Eg belongs to the phylum platyozoa, class Taenia, order Lodales, family Taenia, genus Echinococcus. Current studies have shown that many proteins in Eg are shown to be antigenic during Taenia infection and may serve as vaccine candidate molecules against Hydatidosis. These proteins include: heat-resistant lipoprotein AgB, lipoprotein Ag5, P-29, E96, E914, fatty acid connexin FABP, EgA31 and Eg95 (Zusaming, Lewengui, Echinococcus granulosus molecular biology research progress [ J ] J. China parasite control journal, 2005, 18 (3): 217-220.). Of these, the Eg95 protein is the most effective protective antigen discovered to date. The Eg95 protein is a natural oncosphere antigen, the total length of the coding gene is 715bp, and the relative molecular mass is 24.5kDa (Lightowlers M W, Law repe SB, Gauci CG, et al. Vaccination against hydat doissusising a defined recombinant antigen [ J ]. Parasite Immunol, 1996, 18 (9): 457-462. DOI: 10.1111/j.1365-3024.1996.tb 01029). The Eg95 protein contains fibronectin type III structural domain, has partial homology with immune protein superfamily, cell adhesion molecule, cell surface receptor and carbohydrate binding protein, and plays an important role in the process that Eg oncosphere invades the villus epithelium of small intestine. Research shows that the Eg95 gene is expressed in different growth stages of Echinococcus granulosus and can be used for effective immunoprophylaxis of animal echinococcosis, and the protection rate of the vaccine prepared by the expression product is up to 96-100%.

In recent 20 years, various students have done a lot of work on the study of echinococcus vaccines, including genetic engineering vaccines expressing related recombinant proteins through systems such as escherichia coli, pichia pastoris, insect baculovirus, agrobacterium tumefaciens and the like. For example, CN108066755A provides a method for preparing a genetic engineering subunit vaccine by using an escherichia coli expression system to express and obtain recombinant Eg95 protein, but the folding type of the expressed protein of the escherichia coli expression system is poor, and inclusion bodies are easily formed, so the expression amount is low. For another example, CN102732436A discloses a method for recombinant expression of Eg95 protein by pichia pastoris, which has a certain harm by using methanol as an inducer, and the glycosylation level of the pichia pastoris expression system is relatively low.

Disclosure of Invention

The invention mainly aims to provide a genetic engineering subunit vaccine for resisting sheep echinococcosis infection, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a recombinant protein, which comprises SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

The embodiment of the invention also provides a coding gene of the recombinant protein, which comprises a sequence shown as SEQ ID NO: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

The embodiment of the invention also provides a recombinant vector containing the coding gene.

The embodiment of the invention also provides a host cell containing the coding gene, mainly a mammalian cell.

Further, the host cell may be obtained from a mammalian cell after transfection with the recombinant vector.

The embodiment of the invention also provides an immune composition, which comprises: the recombinant protein; and a pharmaceutically acceptable carrier.

The embodiment of the invention also provides a method for preparing the recombinant protein, which comprises the following steps:

constructing a recombinant expression vector, wherein the recombinant expression vector comprises a coding gene of the recombinant protein;

introducing the recombinant expression vector into a host cell and culturing the host cell under conditions that allow for expression of the protein, followed by isolation and recovery of the recombinant protein from the cell culture of the host cell.

The embodiment of the invention also provides a preparation method for preparing the recombinant protein, which comprises the following steps:

cloning the encoding gene of the recombinant protein to a eukaryotic expression vector to obtain a recombinant expression vector;

transfecting host cells with the recombinant expression vector, and selecting and screening to obtain the host cells which stably and efficiently express the recombinant protein in a suspended manner;

and (3) fermenting and culturing the host cell which stably and efficiently expresses the recombinant protein in suspension, and then separating and purifying the recombinant protein from a cell culture.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparation of the hydatid ovis detection reagent.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in producing a medicament for inducing an immune response to the hydatid ovis in a test animal.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in the production of a medicament for preventing animals from being infected by the hydatid ovis.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparing a genetic engineering subunit vaccine for resisting sheep echinococcosis infection.

Accordingly, the embodiment of the invention provides a genetic engineering subunit vaccine for resisting sheep echinococcosis infection, which comprises any one of the immune compositions. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of the recombinant vector or the host cell containing the recombinant protein coding gene in producing a reagent for detecting animal infection by the hydatid ovis.

The embodiment of the invention also provides application of the recombinant vector or the host cell containing the recombinant protein coding gene in producing a medicament for inducing an immune response to the hydatid ovis antigen in a test animal.

The embodiment of the invention also provides application of the recombinant vector or the host cell containing the recombinant protein coding gene in producing a medicament for preventing animals from being infected by the hydatid ovis.

Compared with the prior art, the embodiment of the invention optimizes the Eg95 protein by combining the Eg95 protein with tandem repeat epitopes, the Fc fragment of sheep IgG and the like, the obtained recombinant Eg95 protein is stable and can form a dimer, the genetic engineering subunit vaccine for resisting sheep echinococcosis infection prepared by the recombinant protein has antigenicity, immunogenicity and functions similar to those of natural protein, has higher expression level and strong immunogenicity, can provide good immune effect only by a small amount, the goat and other mammals have no pathogenicity, and meanwhile, the embodiment of the invention utilizes CHO cells and other expression optimized Eg95 protein, so that the glycosylation level is high, the solubility is strong, the expression quantity is high (can reach 2-3g/L), and the vaccine can be prepared by a bioreactor in a large-scale serum-free suspension culture way, so that the production cost of the vaccine is greatly reduced.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a gel electrophoresis chart of the PCR amplification product of the gene of Eg95 after codon optimization in example 1, in which the band of interest appeared at the 1.3kbp position.

FIG. 2 is a gel electrophoresis chart of the colony PCR amplification product in example 1, wherein the band of interest appears at the 1.3kbp position.

FIG. 3 is a schematic diagram of the structure of the eukaryotic expression vector pCI-Eg95-GS in example 1.

FIG. 4 is a SDS-PAGE detection profile of the cell culture obtained in example 3.

FIG. 5 is a Western Blot detection pattern of the product after SDS-PAGE in example 4.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

One aspect of the embodiments of the present invention provides a recombinant protein comprising SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

That is, the recombinant Eg95 protein sequence provided by the embodiments of the present invention may be the original sequence, an added or truncated sequence.

Furthermore, in the embodiment of the invention, the optimized Eg95 protein is obtained by adding the tandem repeat epitope to the Eg95 protein and adding the Fc fragment of sheep IgG, and the recombinant protein is stable, can form a dimer and has a good immune effect.

In another aspect of the embodiments of the present invention, there is provided a gene encoding the recombinant protein, which includes a sequence shown in SEQ ID NO: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

In another aspect of the embodiments of the present invention, there is provided a recombinant vector comprising a gene encoding the recombinant protein. The recombinant vector may be a eukaryotic expression vector, and may be selected from, for example, but not limited to, pSV2-GS, pCI-GS, pcDNA4-GS, and preferably pCI-GS.

In another aspect of the embodiments of the present invention, there is provided a host cell comprising a gene encoding the recombinant protein.

Further, the host cell may be formed by transfection with a recombinant vector containing a gene encoding the recombinant protein.

Further, the host cell may be a mammalian cell, such as a CHO cell line, which may be selected from, but is not limited to, DG44, DXB11, CHO-K1, CHO-S cell lines and the like, preferably CHO-S

In another aspect of the embodiments of the present invention, there is also provided an immunization composition comprising: the recombinant protein; and a pharmaceutically acceptable carrier. Further, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of two or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201VG, liquid paraffin, camphor oil, plant cell agglutinin, and the like, preferably MONTANIDE ISA 201 VG.

Yet another aspect of the embodiments of the present invention provides a method of preparing the recombinant protein, which includes:

transfecting host cells by the recombinant expression vector, screening out the host cells which stably and efficiently express the recombinant protein in a suspended manner, fermenting and culturing, and separating and recovering the recombinant protein from a cell culture.

Wherein, the eukaryotic expression vector and the host cell can be as described above.

In the above embodiment of the invention, by adopting the eukaryotic expression system and using the suspension culture of host cells such as CHO cells and the like for expression, the glycosylation level is high, the solubility is strong, the expression level is high (up to 2-3g/L), and the immune effect is good.

In another aspect of the embodiment of the invention, the application of the recombinant protein or the immune composition in preparing the sheep hydatid detection reagent is also provided.

In another aspect of the embodiments of the present invention, there is also provided a use of the recombinant protein or the immunological composition in the manufacture of a medicament for inducing an immune response against a hydatid ovis antigen in a subject animal.

In another aspect of the embodiments of the present invention, there is also provided a use of the recombinant protein or the immune composition in the manufacture of a medicament for preventing infection of an animal by hydatid ovis.

In another aspect of the embodiment of the invention, the application of the recombinant protein or the immune composition in preparing a genetic engineering subunit vaccine for resisting sheep echinococcosis infection is also provided.

Accordingly, another aspect of the embodiments of the present invention provides a genetically engineered subunit vaccine against echinococcosis ovis infection, comprising any one of the immunization compositions described above. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

In another aspect of the embodiment of the invention, the application of the recombinant vector or the host cell containing the recombinant protein coding gene in producing the reagent for detecting the animal infected by the hydatid ovis.

In another aspect of the embodiments of the present invention, there is also provided a use of a recombinant vector or a host cell comprising a gene encoding the recombinant protein in the manufacture of a medicament for inducing an immune response against a hydatid ovis antigen in a test animal.

In another aspect of the embodiment of the invention, the application of the recombinant vector or the host cell containing the recombinant protein coding gene in the production of the medicament for preventing animals from being infected by the hydatid ovis.

Accordingly, another aspect of the embodiments of the present invention also relates to a method for inducing an immune response against Babesia ovis antigen, which comprises administering the genetically engineered subunit vaccine against Babesia ovis infection to a test animal such as sheep.

Accordingly, another aspect of the embodiments of the present invention also relates to a method for protecting a test animal from Babesia ovis infection, which comprises administering the genetically engineered subunit vaccine against Babesia ovis infection to a test animal such as sheep.

Yet another aspect of the embodiments of the present invention provides a vaccine suitable for generating an immune response against hydatid ovis infection in a test animal, the vaccine comprising: recombinant proteins of the invention and adjuvant molecules.

Further, the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

Further, the adjuvant may preferably be related adjuvants produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.

In some embodiments, a method for preparing a genetically engineered subunit vaccine against ovine echinococcosis infection specifically comprises:

1) cloning a eukaryotic expression vector containing an optimized Eg95 protein encoding gene;

2) transfecting CHO cells, and selecting and screening to obtain a CHO cell strain which stably and efficiently expresses Eg95 protein in a suspended manner;

3) fermenting and culturing the cell strain screened in the step 2), and purifying to obtain recombinant Eg95 protein;

4) and fully mixing the recombinant Eg95 protein, and then fully and uniformly mixing the protein with an adjuvant to obtain the recombinant expression subunit vaccine.

The method provided by the embodiment of the invention can harvest the target protein from the cell culture supernatant, and the yield is as high as 2-3g/L, thereby not only shortening the protein purification time and simplifying the vaccine production steps, but also greatly reducing the vaccine production cost.

The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

An "adjuvant" as described in the present specification means any molecule added to the vaccine described in the present specification to enhance the immunogenicity of the antigen encoded by the encoding nucleic acid sequence described below.

"antibody" as used herein means an antibody of the type IgG, IgM, IgA, IgD or IgE, or a fragment, fragment or derivative thereof, including Fab, F (ab') 2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of an animal, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

By "coding sequence" or "coding nucleic acid" as used herein is meant a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of the subject or animal to which the nucleic acid is administered.

An "immune response" as described herein means the activation of the immune system of a host (e.g., the immune system of an animal) in response to the introduction of an antigen, such as a common antigen from an infection of hydatid ovis. The immune response may be in the form of a cellular response or a humoral response or both.

A "nucleic acid" or "oligonucleotide" or "polynucleotide" as described herein means at least two nucleotides covalently linked together. The description of single strands also defines the sequence of the complementary strand. Thus, nucleic acids also encompass the complementary strand of the single strand described. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also encompass substantially the same nucleic acids and their complements. Single strands provide probes that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also encompass probes that hybridize under stringent hybridization conditions.

In the present specification, a nucleic acid may be single-stranded or double-stranded or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods.

In the present specification, the coding sequence can be optimized for stability and high levels of expression. In some cases, the codons are selected to reduce the formation of RNA secondary structures, such as those due to intramolecular bonds.

In application, the recombinant protein of the invention and a pharmaceutically acceptable carrier can be prepared into a subunit vaccine composition, and an effective amount of the subunit vaccine composition is inoculated to mammals such as sheep. In one embodiment, the pharmaceutically acceptable carrier comprises an adjuvant and/or an immunopotentiator, wherein the adjuvant is not limited in kind, and specific examples thereof may include, but are not limited to, an alumina gel adjuvant, an oily adjuvant (e.g., Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), or any combination thereof.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts.

According to the invention, the eukaryotic expression system and the CHO cell are used for expressing the recombinant protein, the antigenicity, the immunogenicity and the function of the obtained recombinant protein are similar to those of natural protein, the expression level is higher, the immunogenicity is strong, and no pathogenicity is caused to animals such as sheep.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORYMANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and Methodsin Molecular BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 construction of recombinant eukaryotic expression vector pCI-Eg95-GS

Eg95 gene amplification and purification A codon-optimized Eg95 gene (SEQ ID NO: 1) was synthesized by Shanghai Sangni Biotech Co., Ltd and cloned into a pUC-57 vector to obtain a pUC-Eg95 plasmid vector. PCR amplification was carried out using pUC-Eg95 as a template and Eg95-F, Eg95-R as a primer (the gene sequence of Eg95-F, Eg95-R is shown in SEQ ID NO: 3, 4), and the amplification system is shown in Table 1. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45 seconds, renaturation at 60 ℃ for 45 seconds, extension at 72 ℃ for 2 minutes, 30 cycles; extension at 72 ℃ for 10 minutes and storage at 4 ℃.

TABLE 1 Eg95 Gene amplification System

The PCR product was subjected to gel electrophoresis to identify the size of the target gene, and as shown in FIG. 1, a band appeared at a position of about 1.3kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

2. The PCR products of the pCI-GS plasmid and the purified Eg95 gene were digested with Hind III and EcoR I at 37 ℃ for 3 hours, respectively, and the reaction systems are shown in tables 2 and 3. And respectively recovering enzyme digestion products after gel electrophoresis, and purifying by using a gel recovery and purification kit.

TABLE 2 Eg95 Gene restriction system

TABLE 3 pCI-GS plasmid digestion reaction System

3. Ligation the digested pCI-GS plasmid and the digested product of the Eg95 gene were ligated overnight using T4 DNA ligase in a water bath at 16 ℃ in the system shown in Table 4.

TABLE 4 Eg95 Gene and pCI-GS plasmid ligation System

4. Mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l of LB medium without Amp, and incubated at 37 ℃ for 1 hour. 1.0ml of the cell suspension was concentrated by centrifugation to 100. mu.l, applied to LB solid medium containing Amp, and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification single colonies on the picked plates are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial liquid as a template and Eg95-F and Eg95-R as primers. The size of the target gene was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 1.3kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. Obtaining the eukaryotic expression vector pCI-Eg 95-GS. The map of the constructed vector is shown in FIG. 3.

Example 2 construction and screening of recombinant CHO cells expressing Eg95 protein

1. Cell transfection

1.1 preparation of cells CHO cells in logarithmic growth phase were sampled and counted at 1 × 10⁶continuously passaging the cells at the cell density of cells/ml, maintaining the seeds, centrifuging the rest cells, centrifuging at 1000rpm for 4 minutes, discarding the supernatant, re-suspending the supernatant by about 20ml of fresh CHO-WM culture medium, centrifuging again, centrifuging at 1000rpm for 4 minutes, discarding the supernatant, re-suspending and counting the supernatant by a small amount of culture medium, and finally adjusting the cell density to 1.43 × 10⁷cells/ml。

1.2 mixing of plasmid and cells 5. mu.g of pCI-Eg95-GS plasmid vector of example 1 was taken, and added to an EP tube, 0.7ml of cells was added thereto, and after mixing uniformly, the mixture was left to stand for 15 minutes.

1.3 electric shock 2 pulses of 280V 20ms, immediately transferring the cells into a shake flask after the electric shock is finished, performing suspension culture, and observing the cells after 48hState, liquid change culture, isocellular density growth to 0.6 × 10⁶For cells/ml, 50. mu.M MSX (L-methionine sulphoxide) was added for pressure screening.

2. Monoclonal screening

2.1 resuspend cells in CHO cell serum-free protein free media CHO-WM cell media + 50. mu.M MSX from Volmer Biotechnology Ltd, Suzhou, and count.

2.2 plating to dilute the cells to 5/mL, add 200. mu.l of the mixed cells to a 96-well plate, stand at 37 ℃ with 5% CO₂And incubating for 4-6h in the cell incubator. Wells of individual cells were recorded.

2.3 when the wells of a single cell in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100. mu.l of 0.25% trypsin-EDTA was digested at room temperature for about 2min, 2mL of CHO-WM medium (containing 10% FBS + 50. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off with a pipette. And transferring the cells to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by Elisa, continuously carrying out expanded culture on the high-efficiency expression positive clone, and freezing and storing.

3. Cell shake flask fermentation

3.1 subculture medium configuration: CHO-WM medium was used to add 50. mu.M MSX as subculture medium and placed in a 37 ℃ water bath to preheat to 37 ℃.

3.2 from CO₂Taking out the shake flask cells by a constant temperature shaking table, and counting.

3.3 dilution of cells to 2.5-3.5 × 10⁵cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO₂Incubate overnight in a constant temperature shaker at 100 rpm/min.

3.4 counting the cell density and the cell activity every 24 hours, measuring the glucose, and adding the glucose to 4g/L when the sugar is lower than 2 g/L; samples were taken at 1mL per day and the supernatant was used to detect protein expression.

Cell lines expressing the proteins shown in Table 5 were also constructed according to the above example:

TABLE 5

Example 3 SDS-PAGE detection

The cell culture supernatant of the Eg95 protein harvested in example 2 and the cell culture supernatant of the Eg95 protein were subjected to endoglycosidase Endo H_fAfter the treatment, SDS-PAGE was performed, and a setup was made such that the supernatant of the cell culture of the Eg95 protein was added to a loading buffer without β -mercaptoethanol for SDS-PAGE, and empty CHO cells were used as a negative control.40. mu.l of the harvested cell culture was taken, and 10. mu.l of 5 × SDS gel loading buffer (1 mol/l Tris-HCI (pH6.8)1.25mL, bromophenol blue 25mg, glycerol 2.5mL, SDS 0.5g was dissolved in ddH)₂And O, diluting to 5mL, subpackaging with 0.5 mL/tube, storing at room temperature, adding 25 μ l of β -mercaptoethanol into each tube before use, uniformly mixing, carrying out 5-minute boiling water bath, centrifuging at 12000r/min for 1 minute, taking supernatant, carrying out SDS-PAGE gel (12% concentration gel) electrophoresis, taking gel after electrophoresis, dyeing and decoloring, and observing target bands.

As shown in FIG. 4, the Eg95 protein showed a target band at a molecular weight of about 45kDa, the Eg95 protein treated with endoglycosidase Endo Hf showed a target band at a molecular weight of about 42kDa, the control group without β -mercaptoethanol showed a target band at a molecular weight of about 90kDa, and the negative control showed no band at the corresponding position. Indicating that the target antigen protein is correctly expressed in the recombinant CHO cell.

Example 4 Western Blot assay

The products of example 3 after SDS-PAGE electrophoresis were transferred to NC (nitrocellulose) membranes, blocked with 5% skim milk for 2 hours, incubated with goat-derived anti-Eg 95 positive serum for 2 hours, rinsed, incubated with HRP-labeled rabbit-anti-goat polyclonal antibody for 2 hours, rinsed, added dropwise with an enhanced chemiluminescent fluorogenic substrate, and photographed using a chemiluminescent imager. The results are shown in FIG. 5, in which the recombinant CHO supernatant sample has a cell band and the negative control has no target band, indicating that the target antigen protein is correctly expressed in the recombinant CHO cells.

Example 5 protein content and agar detection

The content of Eg95 protein in the CHO cell culture supernatant harvested in example 2 was determined using the Elisa method. The operation mode is as follows: goat anti-Eg polyclonal antiserum was diluted with coating buffer to appropriate concentrations, 100 μ l per well, overnight at 4 ℃, washed three times with PBST, and blocked with 1% BSA for 1 h. Adding antigen standard substances (protein obtained by particle exchange chromatography, hydrophobic chromatography and molecular sieve purification) with different concentrations and diluting the sample to be detected in a gradient manner, incubating for 1 hour at 37 ℃, and washing with PBST for three times. Monoclonal antibody for detecting the Eg95 protein was added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. A secondary antibody, i.e., HRP-labeled rabbit anti-sheep IgG, was added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. TMB development for 10 min, 2M H₂SO₄The reaction was terminated. Reading by a microplate reader, and calculating the amount of Eg95 protein in the sample to be detected through a standard curve.

According to example 5, Eg95 protein is prepared on a large scale, and the Elisa test result shows that the average content of the protein in the vaccine stock solution reaches 2.43 g/L.

Detecting the titer of the expressed Eg95 protein by using an agar expansion method, punching plum blossom holes on an agarose gel plate, adding Eg agar expansion detection standard serum in the middle of the plum blossom holes, and adding 2-diluted expression antigens of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9and 10 th power around the plum blossom holes respectively. After incubation in an inverted position for 72h, the line of precipitation was observed. The maximum dilution at which a precipitate line appears is its agar titer. The agar titer detection results are as follows: the Eg95 protein agar titer is 1: 2048.

example 6 vaccine preparation

Diluting Eg95 protein expressed by CHO cell, adding into MONTANIDE ISA 201VG adjuvant (volume ratio is 46: 54) to make final protein concentration be 100 μ g/ml, emulsifying, and storing at 4 deg.C after quality inspection is passed. In the same manner, the control 1 group, the control 2 group and the control 3 group in example 2 were prepared as vaccines, respectively.

Example 7 immunization experiment

Test one:

50 sheep of 4-6 months old are randomly divided into 5 groups, each group comprises 10, 4 groups are immune groups, 1ml of subunit vaccine prepared in example 6 is injected subcutaneously into the neck respectively (the concentration of the vaccine is adjusted to 50 mu g/ml), and the other 1 group is an immunization negative control group, and the first immunization is carried out for four weeks and then the immunization is strengthened once. Jugular vein blood was collected before first immunization, 14 days after first immunization, 28 days, 42 days (14 days after second immunization), and 56 days (28 days after second immunization), and serum was separated and subjected to antibody detection using an antibody ELISA kit available from IDXEE, respectively, and the results are shown in Table 6.

TABLE 6

And (4) judging a result: OD_450nmIs less than or equal to 0.3 +/-0.05, and is judged to be negative; OD_450nmIf the value is more than 0.6 +/-0.05, the product is judged to be positive

And (2) test II:

collecting anticoagulated blood one week after immunization, separating peripheral blood lymphocytes by using lymphocyte separating medium, subpackaging 6 plates, respectively stimulating by using 100 mu l of vaccine, and collecting supernatant for detecting cytokine ELISA after 24 h. The sheep IL-4, IL-10, IFN-gamma and TNF-alpha ELISA kits are respectively adopted for detection, and the specific operation is as follows: the standard sample is added with 50 mul on the enzyme labeling coated plate, the sample diluting solution is added with 40 mul in the sample hole to be detected, and then 10 mul of the sample to be detected is added (the final dilution of the sample is 5 times). And placing the sample at the bottom of the hole of the enzyme-labeled plate, and gently shaking and shaking the sample without touching the hole wall as much as possible. Sealing with sealing plate membrane, incubating at 37 deg.C for 30min, removing liquid, drying, adding washing solution to each well, standing for 30 s, discarding, repeating the above steps for five times, and drying. After removing the blank plate, 50. mu.l of enzyme-labeled reagent was added to each well, and the mixture was incubated and washed as above. Color developing agents A50 μ l and B50 μ l were added to each well, shaken up, and developed in the dark at 37 ℃ for 15 min. The reaction was stopped by adding 50. mu.l of stop solution to each well. The absorbance was measured sequentially at a wavelength of 450nm within 15 min. The specific results are shown in Table 7.

TABLE 7

It is to be understood that the above-described embodiments are part of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

<110> Suzhou Shino Biotechnology Ltd

<120> gene engineering subunit vaccine for resisting sheep echinococcosis infection

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>1269

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>1

aagcttgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggacagga atacaaaggt atgggtgtgg agacccgcac caccgaaacg 120

cccctgcgca aacacttcaa cctgacgcct gtcggttccc agggtatccg cctgtcctgg 180

gaggtgcaac acctgagtga cctgaagggt actgacatct ccctcaaagc cgtgaaccct 240

tccgaccccc tcgtctacaa acgccaaacc gctaagttct ccgacggtca actgaccatt 300

ggtgagctca aaccctccac cctgtataaa atgaccgtgg aggccgtaaa ggccaaaaag 360

accatcctcg gtttcaccgt ggacatcgaa accccccgcg ctggtaaaaa ggagtccacc 420

gtgtccgacg gtcaactgac cattggtgag ctcaaaccct ccaccctgta taaaatgacc 480

gtgtccgacg gtcaactgac cattggtgag ctcaaaccct ccaccctgta taaaatgacc 540

gtgcccggat gcccggaccc atgcaaacat tgccgatgcc caccccctga gctccccgga 600

ggaccgtctg tcttcatctt cccaccgaaa cccaaggaca cccttacaat ctctggaacg 660

cccgaggtca cgtgtgtggt ggtggacgtg ggccaggatg accccgaggt gcagttctcc 720

tggttcgtgg acaacgtgga ggtgcgcacg gccaggacaa agccgagaga ggagcagttc 780

aacagcacct tccgcgtggt cagcgccctg cccatccagc accaagactg gactggagga 840

aaggagttca agtgcaaggt ccacaacgaa gccctcccgg cccccatcgt gaggaccatc 900

tccaggacca aagggcaggc ccgggagccg caggtgtacg tcctggcccc accccaggaa 960

gagctcagca aaagcacgct cagcgtcacc tgcctggtca ccggcttcta cccagactac 1020

atcgccgtgg agtggcagaa aaatgggcag cctgagtcgg aggacaagta cggcacgacc 1080

acatcccagc tggacgccga cggctcctac ttcctgtaca gcaggctcag ggtggacaag 1140

aacagctggc aagaaggaga cacctacgcg tgtgtggtga tgcacgaggc tctgcacaac 1200

cactacacac agaagtcgat ctctaagcct ccgggtaaac atcatcatca tcatcattga 1260

tgagaattc 1269

<210>2

<211>414

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr

20 25 30

Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser

35 40 45

Gln Gly Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys

50 55 60

Gly Thr Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val

65 70 75 80

Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly

85 90 95

Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys

100 105 110

Ala Lys Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg

115 120 125

Ala Gly Lys Lys Glu Ser Thr Val Ser Asp Gly Gln Leu Thr Ile Gly

130 135 140

Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Ser Asp Gly Gln

145 150155 160

Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val

165 170 175

Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu

180 185 190

Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp

195 200 205

Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp

210 215 220

Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn

225 230 235 240

Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn

245 250 255

Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp

260 265 270

Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Ala Leu Pro

275 280 285

Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu

290 295 300

Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser

305 310315 320

Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile

325 330 335

Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr

340 345 350

Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr

355 360 365

Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr

370 375 380

Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

385 390 395 400

Ser Ile Ser Lys Pro Pro Gly Lys His His His His His His

405 410

<210>3

<211>34

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

aagcttgccg ccaccatgga aacagataca ctcc 34

<210>4

<211>49

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

gaattctcat caatgatgat gatgatgatg tttacccgga ggcttagag 49

<210>5

<211>573

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>5

aagcttgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggacagga atacaaaggt atgggtgtgg agacccgcac caccgaaacg 120

cccctgcgca aacacttcaa cctgacgcct gtcggttccc agggtatccg cctgtcctgg 180

gaggtgcaac acctgagtga cctgaagggt actgacatct ccctcaaagc cgtgaaccct 240

tccgaccccc tcgtctacaa acgccaaacc gctaagttct ccgacggtca actgaccatt 300

ggtgagctca aaccctccac cctgtataaa atgaccgtgg aggccgtaaa ggccaaaaag 360

accatcctcg gtttcaccgt ggacatcgaa accccccgcg ctggtaaaaa ggagtccacc 420

gtgtccgacg gtcaactgac cattggtgag ctcaaaccct ccaccctgta taaaatgacc 480

gtgtccgacg gtcaactgac cattggtgag ctcaaaccct ccaccctgta taaaatgacc 540

gtgcatcatc atcatcatca ttgatgagaa ttc 573

<210>6

<211>1149

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>6

aagcttgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggacagga atacaaaggt atgggtgtgg agacccgcac caccgaaacg 120

cccctgcgca aacacttcaa cctgacgcct gtcggttccc agggtatccg cctgtcctgg 180

gaggtgcaac acctgagtga cctgaagggt actgacatct ccctcaaagc cgtgaaccct 240

tccgaccccc tcgtctacaa acgccaaacc gctaagttct ccgacggtca actgaccatt 300

ggtgagctca aaccctccac cctgtataaa atgaccgtgg aggccgtaaa ggccaaaaag 360

accatcctcg gtttcaccgt ggacatcgaa accccccgcg ctggtaaaaa ggagtccacc 420

gtgcccggat gcccggaccc atgcaaacat tgccgatgcc caccccctga gctccccgga 480

ggaccgtctg tcttcatctt cccaccgaaa cccaaggaca cccttacaat ctctggaacg 540

cccgaggtca cgtgtgtggt ggtggacgtg ggccaggatg accccgaggt gcagttctcc 600

tggttcgtgg acaacgtgga ggtgcgcacg gccaggacaa agccgagaga ggagcagttc 660

aacagcacct tccgcgtggt cagcgccctg cccatccagc accaagactg gactggagga 720

aaggagttca agtgcaaggt ccacaacgaa gccctcccgg cccccatcgt gaggaccatc 780

tccaggacca aagggcaggc ccgggagccg caggtgtacg tcctggcccc accccaggaa 840

gagctcagca aaagcacgct cagcgtcacc tgcctggtca ccggcttcta cccagactac 900

atcgccgtgg agtggcagaa aaatgggcag cctgagtcgg aggacaagta cggcacgacc 960

acatcccagc tggacgccga cggctcctac ttcctgtaca gcaggctcag ggtggacaag 1020

aacagctggc aagaaggaga cacctacgcg tgtgtggtga tgcacgaggc tctgcacaac 1080

cactacacac agaagtcgat ctctaagcct ccgggtaaac atcatcatca tcatcattga 1140

tgagaattc 1149

<210>7

<211>453

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>7

aagcttgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggacagga atacaaaggt atgggtgtgg agacccgcac caccgaaacg 120

cccctgcgca aacacttcaa cctgacgcct gtcggttccc agggtatccg cctgtcctgg 180

gaggtgcaac acctgagtga cctgaagggt actgacatct ccctcaaagc cgtgaaccct 240

tccgaccccc tcgtctacaa acgccaaacc gctaagttct ccgacggtca actgaccatt 300

ggtgagctca aaccctccac cctgtataaa atgaccgtgg aggccgtaaa ggccaaaaag 360

accatcctcg gtttcaccgt ggacatcgaa accccccgcg ctggtaaaaa ggagtccacc 420

gtgcatcatc atcatcatca ttgatgagaa ttc 453

Claims

1. A recombinant protein comprising SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

2. A gene encoding the recombinant protein according to claim 1; preferably, the coding gene comprises a sequence shown in SEQ ID NO: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

3. A recombinant vector comprising the encoding gene of claim 2; preferably, the recombinant vector comprises pSV2-GS, pCI-GS or pcDNA4-GS, particularly preferably pCI-GS.

4. A host cell comprising the gene encoding claim 2; preferably, the host cell comprises a CHO cell line, preferably selected from the group consisting of DG44, DXB11, CHO-K1 or CHO-S cell lines, particularly preferably CHO-S cells.

5. An immunological composition characterized by comprising: the recombinant protein of claim 1; and a pharmaceutically acceptable carrier.

6. The immunogenic composition of claim 5, wherein: the pharmaceutically acceptable carrier comprises one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201VG, liquid paraffin, camphor oil and plant cell agglutinin, preferably MONTANIDE ISA 201 VG.

7. A method for producing a recombinant protein, comprising:

cloning a coding gene of the recombinant protein of claim 1 onto a eukaryotic expression vector to obtain a recombinant expression vector;

8. The method of claim 7, wherein: the eukaryotic expression vector comprises pSV2-GS, pCI-GS or pcDNA4-GS, preferably pCI-GS; and/or, the host cell comprises a CHO cell line, preferably selected from the group consisting of DG44, DXB11, CHO-K1 or CHO-S cell lines, particularly preferably CHO-S cells.

9. Use of a recombinant protein according to claim 1 or an immunogenic composition according to claim 5 or 6 in the preparation of a fasciola caprinae detection reagent, in the manufacture of a medicament for inducing an immune response against a fasciola caprinae antigen in a subject animal, or in the manufacture of a medicament for preventing infection of an animal by fasciola caprinae.

10. Use of the recombinant protein according to claim 1 or the immunogenic composition according to claim 5 or 6 for the preparation of a genetically engineered subunit vaccine against echinococcosis ovis infection.