CN113480616B - Heterotrimeric structural domain, heterotrimeric fusion protein, preparation method and application - Google Patents

Abstract

The invention provides a heterotrimeric structural domain, a heterotrimeric fusion protein, a preparation method and application. Experiments show that the heterotrimeric fusion protein can be identified with certain corresponding antibodies and effectively combined with high titer, and can also be used as an antigen to induce the generation of high titer antibodies. Based on the technical platform constructed by the invention, feasible solutions are provided for preparing effective vaccine antigens for preventing epidemic diseases, preparing functional effective cytokines for treating immune system diseases, preparing defensive and therapeutic vaccines aiming at certain cancers, preparing epidemic virus antibody detection preparations or epidemic pathogen monitoring preparations aiming at epidemic disease detection monitoring and the like.

Description

Heterotrimeric structural domain, heterotrimeric fusion protein, preparation method and application

Technical Field

The invention relates to the technical field of bioengineering and virus vaccines, in particular to a heterotrimeric structural domain, a fusion protein containing the heterotrimeric structural domain, a preparation method of the fusion protein, and application of the fusion protein in preparation of vaccines, preparation of epidemic disease virus antibody detection preparations or epidemic disease pathogen monitoring preparations.

Background

In nature, functional oligoproteosomes exist in both complex higher organisms such as mammals and simple lower organisms such as bacteriophage and enveloped viruses, and participate in the functional operation of organisms. An oligomeric proteosome, also called a multimeric protein or a multimeric proteosome, refers to a proteosome composed of two or more subunit proteins. Compared with single subunit protein, the oligomeric protein body has large molecular volume and multi-subunit synergy, and can generate allosteric effect to facilitate cell recognition, signal transduction, function regulation and the like. For example, the human immune system is a highly coordinated system of multiple cells, in which signal transduction pathways involve the mutual recognition and binding of cellular receptors and cellular ligands and the activation of signal transduction pathways, which trigger a series of immune cell responses. In this process, the receptors and corresponding ligands (e.g., cytokines) on the cell membrane often appear as oligomeric protein polypeptides, such as homodimers or trimers. The abnormal changes of the two components can cause the abnormal function of the body, and lead to pathological changes such as rheumatoid arthritis, cancer and the like.

The functional role of the oligoproteosomes is most pronounced in enveloped viruses, especially in the RNA enveloped virus family, such as pneumonia syncytial virus, influenza virus, novel coronaviruses, and the like. When the virus invades the host cell, the virus firstly binds with a corresponding receptor on the host cell membrane by a special glycoprotein on the envelope membrane to cause cell membrane fusion, and then virus genetic materials are injected into the host cell so as to facilitate the mass replication of the virus. This oligosaccharide protein on the viral envelope naturally becomes a major target for stimulating the immune system to produce targeted and effective neutralizing antibodies in vaccine studies. However, the oligosaccharide protein on the envelope of this kind of enveloped viruses, especially RNA viruses, has its specific structure and function. Before and after fusion of the virus and the host cell, the spatial conformation of the virus envelope protein is changed, and the weak stable conformation before the fusion is changed into the strong stable conformation after the fusion, but the form before the fusion of the virus envelope protein contains the antigen epitope which is not existed on the form after the fusion. Taking the currently prevalent new coronavirus as an example, the spike S glycoprotein on the envelope of the virus occurs as a homotrimer, the monomer is composed of both S1 and S2 subunits, the S1 subunit contains the region RBD (Receptor binding Domain) that binds to the host cell ACE2 Receptor. When RBD in the S1 subunit is combined with host cell ACE2, instability change of S protein trimer is triggered, and then the S1 subunit and the S2 subunit are dissociated. The conformation of the S2 subunit becomes a highly stable post-fusion structure, and thus the S protein has both pre-fusion (Prefusion) and post-fusion (Postfusion) conformations. In this variation, the RBD with the immunogenic epitope-bearing portion of the S protein polypeptide at the outermost end of the capsular membrane engrafts underneath and is masked, while the non-antigenic determinant portion is stably exposed at the outer end. This change in spatial conformation presents a challenge to vaccine development because the powerful protective neutralizing antibodies elicited by the immune system need to react with the S protein trimer prior to fusion. Unfortunately, conventional methods for preparing recombinant protein subunits as vaccine antigens only produce fused antigen proteins with strong stable conformations, greatly reducing or even inactivating the immunogenicity of the antigens, resulting in loss of protective efficacy of the vaccine. Because of the important function and conformation variability of the S protein, obtaining a correct S protein trimer structure with stable property and uniform conformation is a key factor for successfully developing vaccines, neutralizing antibodies and serological detection kits so as to stimulate the immune system to generate correct immune response reaction to the maximum extent, and when the natural virus really comes, the neutralizing antibodies lock the S protein of the virus in a form incapable of invading cells, thereby playing a role in protecting human health.

Most oligomeric proteosomes share a common structural feature, i.e., a certain polypeptide of a protein monomer is a functional domain that forms and stabilizes the oligomeric conformation of the proteosome, forming multimeric proteosomes such as homo-or heterodimers, trimers, tetramers, etc. Therefore, the invention provides a heterotrimeric domain and a fusion protein containing the heterotrimeric domain, which provide feasible solutions for preparing effective vaccine antigens for preventing epidemic diseases, preparing functional effective cytokines for treating immune system diseases, preparing defensive and therapeutic vaccines aiming at certain cancers, preparing epidemic virus antibody detection preparations or epidemic pathogen monitoring preparations and the like.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a heterotrimeric domain, a fusion protein containing the heterotrimeric domain, a preparation method of the fusion protein, and application of the fusion protein in preparation of vaccines, preparation of epidemic disease virus antibody detection preparations or epidemic disease pathogen monitoring preparations.

In order to realize the purpose, the invention is realized by the following technical scheme:

the first purpose of the invention is to provide a heterotrimeric domain, which is formed by site-directed mutation of DNA bases of virus envelope glycoprotein domain polypeptide fragments, wherein the N end of each polypeptide in the heterotrimeric domain after the site-directed mutation contains a long a-helical fragment, the C end of each polypeptide in the heterotrimeric domain after the site-directed mutation contains an antiparallel short a-helical fragment, and the two helical fragments are connected by a flexible small loop.

Further, the site directed mutation introduces 2S-S covalent bond sites on the elongated a-helix fragment.

Further, the site of the targeted mutation is G29C and F30C, respectively, of the polypeptide fragment.

Furthermore, the amino acid sequence of the heterotrimeric domain polypeptide is shown as SEQ ID NO.1, and the DNA sequence is shown as SEQ ID NO. 6.

Still further, the heterotrimeric domain polypeptide amino acid sequence also includes mutants of single or multiple amino acids among the amino acids shown in SEQ ID No.1, chimeras of single or multiple amino acids among the amino acids shown in SEQ ID No. 1.

Further, the envelope glycoprotein is derived from all human influenza viruses and animal influenza viruses.

A second object of the invention is to propose a trimer of fusion proteins, each protein monomer fusion polypeptide of said trimer comprising:

(1) A heterotrimeric domain polypeptide of any one of the above fused at its C-terminus,

(2) A GSGGSG-encoding hinge inserted between its C-terminus and the heterotrimeric domain polypeptide.

A third object of the invention is to propose a heterotrimeric fusion protein composition, comprising three polypeptide chains, wherein each polypeptide chain comprises:

(1) A polypeptide of a heterotrimeric domain portion of any one of the above,

(2) A non-domain fusion protein polypeptide to be trimerized linked to the N-terminus of the polypeptide of the heterotrimeric domain portion as defined in any of the above.

Further, the non-domain fusion protein polypeptide to be trimerized is derived from a homologous secretory protein.

Further, characterized in that the non-domain fusion protein polypeptide to be trimerized is derived from a non-homologous secretory protein.

Further, the non-domain fusion protein polypeptide to be trimerized is a prophylactic vaccine antigenic protein polypeptide molecule.

Still further, the non-domain fusion protein polypeptide to be trimerized is selected from the group consisting of spike S protein polypeptide on the surface of a neocoronavirus membrane, hemagglutinin protein HA polypeptide on the surface of an influenza virus membrane.

It is a fourth object of the present invention to propose a method for the preparation of a heterotrimeric fusion protein composition as defined in any one of the above, comprising the following steps:

(1) Carrying out PCR (polymerase chain reaction) site-specific mutagenesis on the D N A bases of two sites of G29C and F30C of the domain polypeptide to obtain a trimerization domain DNA fragment; recovering a trimerization domain DNA fragment, cloning the DNA fragment to a plasmid vector, and constructing a trimerization domain recombinant plasmid;

(2) Inserting the non-structural domain fusion protein polypeptide gene segment to be trimerized into a trimerization structural domain recombinant plasmid to form a heterotrimeric fusion protein recombinant plasmid;

(3) Cloning the heterotrimeric fusion protein recombinant plasmid to an expression plasmid vector to obtain a recombinant expression plasmid vector of the heterotrimeric fusion protein;

(4) Transfecting host cells with the recombinant expression plasmid vector of the heterotrimeric fusion protein, recovering host cell culture supernatant, and extracting and purifying the heterotrimeric fusion protein composition.

Further, the host cell is selected from a eukaryotic cell or a prokaryotic cell.

Still further, the host cell is selected from any one of an insect cell, a mammalian cell, an escherichia coli cell, a fungal cell.

A fifth object of the invention is to propose a vaccine comprising a heterotrimeric fusion protein composition according to any one of the above.

Further, the vaccine also comprises an adjuvant, and the adjuvant is selected from at least one of an aluminum salt adjuvant, a CpG Toll-Like adjuvant, a water-in-oil-in-water adjuvant, a squalene adjuvant, a vegetable oil adjuvant, a liposome adjuvant, a nanoparticle adjuvant or a Freund's adjuvant.

The sixth purpose of the present invention is to provide an application of any one of the heterotrimeric fusion protein compositions in preparation of a epidemic disease virus antibody detection preparation or an epidemic disease pathogen monitoring preparation, wherein the preparation comprises any one of an ELISA detection kit, a colloidal gold test strip, a chemiluminescent detection kit or a fluorescent luminescent detection kit.

Therefore, compared with the prior art, the invention has the beneficial effects that:

(1) The heterotrimeric domain formed by site-directed mutation of DNA bases of the viral envelope glycoprotein domain polypeptide fragment can be fused with proteins from different sources to form a heterotrimeric fusion protein composition. Experiments have shown that the heterotrimeric fusion protein composition can recognize and effectively bind to high titers of certain corresponding antibodies, and that it can also induce the production of high titers of antibodies as an antigen. Based on the technical platform constructed by the invention, feasible solutions are provided for preparing effective vaccine antigens for preventing epidemic diseases, preparing functional effective cytokines for treating immune system diseases, preparing defensive and therapeutic vaccines aiming at certain cancers, preparing epidemic disease virus antibody detection preparations or epidemic disease pathogen monitoring preparations and the like.

(2) The invention transfects host cells through the recombinant expression plasmid vector of the constructed heterotrimeric fusion protein to obtain cell culture solution, recovers and purifies the cell culture solution to obtain the heterotrimeric fusion protein composition.

Drawings

FIG. 1 is a depiction of the ectodomain of the S protein of the novel coronavirus, the receptor binding domain of the S protein (RBD) and the HA protein trimerization design of avian influenza virus H7N9 in which the native transmembrane and cytoplasmic domains of the protein are replaced by the Trimerization Domain (TD), the parts of the figure not being shown to scale. Wherein: FIG. 1A is a linear representation of the recombinant S protein trimerization design, FIG. 1B is a linear representation of the Receptor Binding Domain (RBD) trimerization design of the recombinant S protein, and FIG. 1C is a linear representation of the recombinant HA protein trimerization design.

FIG. 2 is a PCR amplification electrophoresis chart of the DNA fragment of the recombinant baculovirus target gene in example 2. Wherein: in fig. 2, column a is a 1KB DNA molecule standard, column B is an S protein gene DNA fragment in fig. 2, column C is an RBD protein gene DNA fragment in fig. 2, and column D is an influenza virus H7N9 membrane protein HA gene DNA fragment in fig. 2.

FIG. 3 is an SDS-PAGE of purified S-TD, RBD-TD and H7-TD under reducing and non-reducing conditions. Wherein: FIG. 3A is a sample of purified S-TD, FIG. 3B is a sample of purified RBD-TD, and FIG. 3C is a sample of purified H7-TD.

FIG. 4 is a Western Blots electrophoretogram of S-TD, RBD-TD and H7-TD in cell culture supernatants under reducing and non-reducing conditions. Wherein: FIG. 4A is a sample of S-TD cell culture supernatant, FIG. 4B is a sample of RBD-TD cell culture supernatant, and FIG. 4C is a sample of H7-TD cell culture supernatant.

FIG. 5 is a dot blot assay to detect the affinity of trimerization domains for specific mAbs. Points corresponding to A, B, C and D in the figure 5 are a sample RBD-GCN4-6XHis, a sample RBD-TD-6XHis, a sample S-TD and a fresh cell culture medium sample in sequence.

FIG. 6 is a graph showing the hemagglutination titer of H7-TD trimer protein detected by chicken erythrocytes. In the figure, three rows A, B and C correspond to a cell supernatant sample H7-TD #1 of the H7-TD trimer, a control sample H7N9-VLP and a cell supernatant sample H7-TD #2 of the H7-TD trimer, respectively.

FIG. 7 shows the use

The chromatographic system carries out high-resolution analysis on the state of the purified S-TD trimer.

FIG. 8 is a graph showing the results of high titer affinity of the antibodies in S-TD and RBD-TD of example 7 with serum of convalescent patients with new coronary pneumonia, in this example BSA was used as a negative control.

FIG. 9 is a graph showing the results of the measurement of the antibody titer produced by immunizing a mouse with the S protein trimer as an antigen. The dots in the figure represent individual mice, and the horizontal lines represent the Geometric Mean Titer (GMT) of EC50 for each group of mice.

Detailed Description

The following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified, in parallel, from materials, methods, and reaction conditions, all of which are intended to be within the spirit and scope of the present invention. The methods of the following examples are conventional unless otherwise specified.

Example 1: construction of recombinant expression plasmid of S-TD tripolymer protein, RBD-TD tripolymer protein and H7-TD tripolymer protein of avian influenza H7N9

1. PCR (polymerase chain reaction) site-directed mutation trimerization structural domain and construction of trimerization structural domain recombinant plasmid

The heterotrimeric structural domain provided by the invention is formed by site-directed mutation of DNA (deoxyribonucleic acid) bases of a virus envelope glycoprotein structural domain polypeptide segment, and keeps the structural characteristics of the virus envelope glycoprotein structural domain, namely the N end of each polypeptide in the structural domain contains a long a-spiral segment, the C end of each polypeptide contains an antiparallel short a-spiral segment, and the two spiral segments are connected by a flexible small loop. In this example, taking avian influenza virus as an example, PCR site-specific mutagenesis was performed on D na bases at two sites, G29C and F30C, of C-terminal domain polypeptide of H5N1 hemagglutinin protein HA of avian influenza virus to obtain trimerization domain DNA fragment.

A DNA fragment of the C-terminal structural domain of avian influenza virus H5N1 hemagglutinin protein HA is artificially synthesized, and two plasmid-related enzyme cutting sites BamHI and HindIII are added. In order to optimize the function of the polypeptide expressed by the DNA fragment of the part of the structural domain, the technicians of the invention carry out multiple base point mutation on the DNA fragment of the part by adopting the conventional method of PCR (polymerase chain reaction) site-specific mutation. Through screening and comparison, the mutation of two base pairs is finally determined, and two disulfide bonds are directly introduced, so that the trimerization function of the heterologous protein of the structural domain is stronger. This short modified peptide of 72 amino acids in total is called Trimerization Domain TD (Trimerization Domain). The amino acid sequence of the heterotrimeric domain TD polypeptide is shown as SEQ ID NO.1, the two amino acid mutation points are G29C (GGA/TGT) and F30C (TTC/TGC), and the base sequence for coding the heterotrimeric domain TD polypeptide is shown as SEQ ID NO. 6.

The construction and expression of the PCR site-directed mutagenesis and trimerization domain recombinant plasmid are described below:

(1) PCR site-directed mutagenesis domain DNA

Designing and synthesizing oligonucleotide segments with mutation sites (see base sequences SEQ ID NO.9 and SEQ ID NO. 10), and obtaining a Trimerization Domain (TD) DNA segment with two point mutations by using an artificially synthesized domain DNA segment as a template and adopting PCR (polymerase chain reaction) in steps. Purifying and recovering the DNA fragment by gel electrophoresis, inoculating the DNA fragment to bacterial plasmid by T4 ligase, transforming the bacterial plasmid into DH5 alpha competent bacterial cells, selecting positive bacterial spots, culturing and extracting recombinant plasmid with the target DNA fragment.

(2) Construction of trimerization Domain TD recombinant plasmid

The shuttle plasmid pFastBac was digested with BamHI and HindIII, respectively, and purified. And (2) hydrolyzing the recombinant plasmid with the target DNA fragment extracted in the step (1) by the same two enzymes, and purifying to obtain the target Trimerization Domain (TD) DNA fragment. Under the action of T4 link enzyme, connecting the treated trimerization structural domain (TD) DNA fragment to an enzyme-cut pFastBac plasmid vector, selecting a positive bacterial spot to extract a DNA plasmid, and obtaining a recombinant shuttle plasmid pFastBac-TD with the TD fragment.

2. Construction of S protein trimer recombinant plasmid

The DNA of the ectodomain part of the Spike protein (Spike, S protein) on the membrane of SARS-CoV-2 virus (GenBank: MN 908947.3) was codon optimized in combination with antigen analysis and codon optimization to make it more suitable for expression in insect cells Sf9 (Spodoptera frugiperda). The optimized S protein gene DNA fragment is artificially synthesized, and corresponding HindIII restriction enzyme cutting sites are added at two ends. The amino acid sequence of the S protein is shown as SEQ ID NO.2, and the base sequence of the coded S protein is shown as SEQ ID NO. 5.

The DNA fragments of the recombinant plasmids pFastBac-TD and S protein are respectively cut by HindIII enzyme, after purification and T4 ligase connection, the DNA fragment of the S protein is inserted into the upstream of the reading frame of the pFastBac-TD plasmid so as to form the secretory fusion protein S-TD. And selecting positive bacterial spots to extract DNA plasmids to obtain recombinant plasmids pFastBac-S-TD. The linear representation of the recombinant S protein trimerization design is shown in figure 1A.

3. Construction of RBD trimer recombinant plasmid

The DNA fragment of Receptor Binding Domain (RBD) of S protein is obtained by PCR amplification using synthesized S protein as template. The amino acid sequence of the RBD protein is shown as SEQ ID NO.3, and the base sequence of the coded RBD protein is shown as SEQ ID NO. 7.

The RBD region of the S protein was amplified by PCR using primers containing the HindIII sites described above. The RBD fragment and the recombinant plasmid pFastBac-TD are respectively cut by HindIII enzyme, purified and linked by T4 enzyme, and the RBD fragment is inserted into the upstream of the reading frame of the pFastBac-TD plasmid to form the secretory fusion protein RBD-TD. And selecting positive bacterial spots to extract DNA plasmids to obtain recombinant plasmids pFastBac-RBD-TD. The linear representation of the Receptor Binding Domain (RBD) trimerization design of recombinant S protein is shown in fig. 1B.

4. Construction of H7 protein trimer recombinant plasmid

The DNA segment of HA (H7) gene of hemagglutinin protein of avian influenza virus H7N9 is also synthesized artificially. The amino acid sequence of the hemagglutinin H7 protein of the avian influenza virus H7N9 is shown as SEQ ID NO.4, and the base sequence of the hemagglutinin H7 protein of the encoded avian influenza virus H7N9 is shown as SEQ ID NO. 8.

The DNA fragments of the recombinant plasmids pFastBac-TD and H7 protein are respectively cut by HindIII enzyme, and after purification and T4 ligase linkage, the DNA fragment of the H7 protein is inserted into the upstream of the reading frame of the pFastBac-TD plasmid so as to form the secretory fusion protein H7-TD. And selecting positive bacterial spots to extract DNA plasmids to obtain a recombinant plasmid pFastBac-H7-TD. The linear representation of the recombinant HA protein trimerization design is shown in figure 1C.

Of course, due to the degeneracy of the amino acid codons, there can be thousands of expression sequences of a given protein amino acid sequence, and thus, the base sequences for expressing the S protein, the trimerization domain TD polypeptide, the RBD protein and the hemagglutinin H7 protein are not unique, and the base coding sequences shown in SEQ ID Nos. 5 to 8 are preferred in this embodiment.

Example 2: preparation, synthesis and detection of S-TD, RBD-TD and H7-TD recombinant baculovirus

(1) Preparation, synthesis and detection of S-TD recombinant baculovirus

Transferring the obtained recombinant shuttle plasmid pFastBac-S-TD into a DH10Bac competent cell, obtaining a positive white spot colony by adopting a colony blue-white spot screening method, and extracting a DNA plasmid of the positive white spot colony to obtain a recombinant baculovirus plasmid Bacmid-S-TD. Insect cells sf-9 were plated in a 6-well cell culture plate, the obtained recombinant baculovirus plasmid Bacmid-S-TD was used for cell transfection, and six days later, cell culture supernatant containing the recombinant baculovirus was harvested.

Adding lysis solution containing SDS into cell supernatant to lyse recombinant baculovirus, extracting with chloroform, and precipitating with ethanol to obtain recombinant baculovirus DNA. The DNA is used as a template, and a PCR primer corresponding to the S protein gene is added to carry out PCR amplification reaction. And running the amplified product on gel electrophoresis to obtain an expected DNA fragment, and proving that the prepared recombinant baculovirus has a target exogenous S protein gene. The results of gel electrophoresis are shown in FIG. 2. The PCR amplification primer is shown as SEQ ID NO. 10-11.

SEQ ID NO.11：Spike-F，ATGTTCGTGTTCTTGGTGCTG

SEQ ID NO.12：Spike-R，TTAGGTGTAGTGCAGCTTGAC

(2) Preparation, synthesis and detection of RBD-TD and H7-TD recombinant baculovirus

The expected DNA fragments were obtained by synthesizing recombinant baculoviruses in which RBD-TD and H7-TD were detected in the same manner as described above. The gel electrophoresis results are shown in FIG. 2. The PCR amplification primer is shown as SEQ ID NO. 13-16.

SEQ ID NO.13：RBD-F，CGTGTGCAGCCCACCGAGTCC

SEQ ID NO.14：RBD-R，GAAGTTCACGCATTTGTTCTTG

SEQ ID NO.15：H7-F，ATGAACACTCAAATCCTGG

SEQ ID NO.16：H7-R，TTATATACAAATAGTGCACC

Example 3: expression, purification and detection of trimerized fusion protein compositions

1. Expression and purification of trimerized fusion protein compositions

(1) Expression and purification of S-TD trimerization fusion protein composition

Laying insect cells sf-9 in a 6-hole cell culture plate, transfecting the recombinant baculovirus plasmid Bacmid-S-TD by adopting a lipid method, and collecting cell supernatant after six days to obtain a first generation of recombinant baculovirus virus seeds which are called P1 generation. Infecting insect cells with P1 generation virus seed for virus seed amplification to obtain second generation recombinant baculovirus virus seed named as P2 generation. Then P2 generation virus seeds are used for infecting insect cells to amplify virus seeds, and P3 generation is obtained to be used as production virus seeds. The good Sf-9 cells are inoculated into a shake flask for suspension culture, and when the cells grow to a logarithmic phase, the cell concentration is diluted to 3.0X 106 cells per milliliter. Inoculating the P3 generation virus seeds into a cell shake flask according to the MOI of 0.1 ratio, harvesting cell supernatant after 3 to 4 days, purifying and detecting the expression synthesis of the S-TD trimerization fusion protein composition.

To efficiently purify each trimerized fusion protein composition expressed, the present inventors constructed, screened and prepared monoclonal antibodies directed against the heterotrimeric domain TD. The monoclonal antibody is prepared by adopting a conventional method for preparing the monoclonal antibody by using hybridoma. Briefly, mice were injected three times with the heterotrimeric domain polypeptide, and their spleen cells were taken and fused with tumor cells. The hybridoma cells were cultured in a 96-well cell plate, and positive hybridoma cell lines with high antibody expression were selected and cultured in an enlarged scale. Cell culture supernatant was harvested and monoclonal antibodies were purified by A-protein affinity chromatography.

The monoclonal antibody is coupled with an HP chromatographic column (Cytiva, U.S.A) activated by NHS to obtain a self-made immunoaffinity chromatographic column. Use of

Protein purification system (GE, u.s.a) and home-made affinity chromatography columns, perform affinity chromatography purification on the trimer protein prepared by expression. The specific operation is as follows: the cell supernatant with the S-TD trimerized fusion protein composition after harvesting was centrifuged at 4000 Xg for 10 minutes at 4 ℃ and the centrifuged supernatant was collected and filtered through a filter with a pore size of 0.22. Mu.m. Adding the filtered supernatant to the affinity chromatography column, and performing column hanging and elution. The eluate from the affinity chromatography column was immediately neutralized and buffer exchanged using a ZebaSpin desalting column (ThermoFisher Scientific, u.s.a). NanoDropone (ThermoFisher Scientific, U.S.A.) measures the concentration of the purified trimerized fusion protein composition.

(2) RBD-TD and H7-TD trimerization fusion protein compositions were expressed, prepared and purified in the same manner as described above.

2. Detection of recombinant trimeric proteins

The purified trimerization fusion protein composition is subjected to SDS-PAGE and Western Blots qualitative analysis, and the specific operation is as follows:

(1) SDS-PAGE experiments

Mu.l of the purified sample containing the trimerized fusion protein composition was taken, 4. Mu.l of Laemmli loading solution was added, 1. Mu.l of reducing agent DTT (100 mM) was added and mixed well, heated at 95 ℃ for about 5 minutes, vortexed as needed to ensure complete dissolution, and then rapidly centrifuged in a benchtop microfuge for 1 minute. The samples were spotted into polyacrylamide gel wells prepared by a Protean Minigel instrument (Bio-Rad, U.S.A.). And adding 4 mul of aqueous glycerol sample loading liquid and 1 mul of deionized water into another 15 mul of the same purified sample, uniformly mixing by vortex, heating at 95 ℃ for about 5 minutes, dropping the sample into another hole in the same polyacrylamide gel, and connecting a power supply to run the gel. After completion of the electrophoresis, the gel was rinsed in deionized water, stained with Coomassie brilliant blue G-250 dye solution (Bio-Rad, U.S.A.) and destained in deionized water. FIG. 3 is a SDS-PAGE protein electrophoresis of purified S-TD, RBD-TD and H7-TD under reducing and non-reducing conditions, and FIG. 3 clearly shows that in non-reducing natural state, all three purified proteosome exist in the form of trimer protein, and only under reducing and denaturing conditions, all three trimer proteins are depolymerized into monomer form. It is demonstrated that the heterotrimeric domains of the invention play a decisive functional role in the formation of protein trimers from protein monomers.

(2) Western Blots experiment

By using the same experimental procedure as described above, cell culture supernatant samples containing the trimerized fusion protein composition were separated on SDS-polyacrylamide gels, followed by Western Blots blot membrane transfer. After blocking the transfer film with 5% skim milk solution, the transfer film was diluted 1000x with 5% skim milk and incubated overnight with either rabbit polyclonal antibodies against protein S RBD (against samples S-TD and RBD-TD) (Elabscience, u.s.a) or anti-His-tag antibodies (against His-tagged sample H7-TD) (Applied Biological Materials inc.u.s.a). After 3 washes with PBST, the membrane was incubated with HRP enzyme-bearing goat anti-rabbit secondary antibody diluted 3000x with 5% skim milk powder for 1 hour at room temperature. After washing the membrane again 3 times with PBST, fluorescence signals were generated using Immobilon Crescando Western HRP substrate (MILLPORE, MA, U.S.A.) and Hyperfilm ECL (GE-Amersham, U.S.A.). FIG. 4 is a Western Blots electrophoretogram of S-TD, RBD-TD and H7-TD in cell culture supernatants under reducing and non-reducing conditions. FIG. 4 shows the specific bands indicating the monomeric protein after reductive denaturation and the specific trimeric protein in the non-reduced state. The heterotrimeric domains of the invention are shown to play a decisive functional role in the formation of protein trimers from protein monomers.

Example 4: detection of heterotrimeric Domain TD in trimerization fusion protein compositions by blotting

To accurately and efficiently perform dot blot analysis to identify the heterotrimeric domain in the trimerized fusion protein composition, the heterotrimeric domain TD in the trimerized fusion protein composition was determined using dot blot hybridization experiments using monoclonal antibodies described in example 3 with a targeting heterologous domain. In addition, as a control sample, using public known trimer domain GCN4 sequence construction preparation RBD-GCN4 trimer protein and added with marker 6xHis, to distinguish the identification of the heterotrimeric domain TD specificity, prove the above expressed various trimerization fusion protein composition is due to the heterotrimeric domain function polymerization.

The detailed procedure of the dot blot experiment is briefly as follows: the Sf-9 cell culture supernatant and S-TD cell culture supernatant which were confirmed to contain a trimer expressing RBD-TD-6XHis protein in example III were diluted with PBS and applied to nitrocellulose membrane (Bio-Rad, U.S.A.) by spotting 15. Mu.l of the dilution with a pipette using a Dot blot Apparatus (Bio-Dot Apparatus, bio-Rad, U.S.A.). As a control, 15. Mu.l of a dilution containing trimeric RBD-GCN4-6XHis was spotted. Negative controls used uninfected Sf-9 cell culture supernatant. Air-drying the spotted nitrocellulose membrane, and sealing the membrane for 1 hour at room temperature by using special skimmed milk powder for sealing the membrane. The self-made monoclonal antibody and the anti-His protein monoclonal antibody are diluted 1000 times by using a PBST solution containing 0.5% skimmed milk powder, and used as a primary antibody to be incubated with the membrane for 1 hour. After 3 washes with PBST, secondary HRP-containing antibodies were diluted 3000-fold with 0.5% nonfat dry milk PBST solution and incubated with the membrane for 1 hour at 37 ℃. Thereafter, the membrane was washed again, and fluorescence was excited using Immobilon Crescando Western HRP substrate (Millipore, MA, U.S.A.) and Hyperfilm ECL (GE-Amersham, U.S.A.) for detection. The results are shown in FIG. 5, and the points corresponding to A, B, C and D in FIG. 5 are sequentially the sample RBD-GCN4-6XHis, the sample RBD-TD-6XHis, the sample S-TD and the fresh cell culture medium sample, and the results show that the expressed oligomeric domain in the RBD-TD-6XHis and the S-TD trimerization fusion protein composition is the expected heterotrimeric domain TD.

Example 5: detecting the hemagglutination titer of the H7-TD trimerization fusion protein

The expression and the existence of the H7-TD trimerization fusion protein in the cell supernatant can be known by a method for detecting the hemagglutination titer. The avian influenza virus hemagglutinin protein HA can be combined with a receptor on the surface of an erythrocyte, and the characteristic can be used as a judgment index for detecting the hemagglutination titer. Therefore, the H7-TD trimer protein in the cell supernatant can be detected by chicken erythrocyte agglutination reaction.

The method comprises the following specific steps: on a micro hemagglutination plate, PBS0.025ml is added from the 1 st hole to the 12 th hole by a pipette, 0.025ml of the harvested cell supernatant is sucked by the pipette, 2-fold dilution is carried out from the 1 st hole to the last hole in sequence, and 0.025ml of liquid in the pipette is discarded. 0.025ml of 1% chicken red blood cell suspension is added to each well, and red blood cell control wells without samples are placed, immediately shaken on a microplate shaker, and incubated at 25 ℃ for 30 minutes. The results were judged when the red blood cells in the control wells were significantly button-shaped. The highest dilution at which erythrocytes were completely agglutinated was used as the determination end point.

The results are shown in FIG. 6, in which three rows A, B and C correspond to the H7-TD trimer cell supernatant sample H7-TD #1, the control H7N9-VLP and the H7-TD trimer cell supernatant sample H7-TD #2, respectively, the blood coagulation titer of the H7-TD #1 is about 9.5, the blood coagulation titer of the H7N9-VLP is about 7.5, and the blood coagulation titer of the H7-TD #2 is about 9.5. Thus, the hemagglutination titers of the two cell supernatant samples 1# and 2# of the H7-TD trimer were 2 power-fold higher than those of the control sample. Indicating that the expressed H7-TD domain fusion protein self-assembles to form the H7-TD trimerization fusion protein and is released into the cell supernatant.

Example 6: size and uniformity analysis of purified S-TD trimerized fusion protein by size exclusion chromatography

In order to effectively determine whether the purified S-TD sample still retains the intact S protein trimer structure, the sample was analyzed by Size Exclusion Chromatography (SEC). The specific method is briefly described as follows:

1. preparation of calibration standards for SEC

The calibrated standard molecular weight markers were 1,350 to 670,000da (Bio-rad, u.s.a). After dissolving the standards with 0.5ml of deionized water, any fine particles were removed by centrifugation. Adjusting the ratio of the prepared standard substance upper column volume and the purification column volume, and taking the OD value detected by the final 280nm as the standard reaching the range of 0.1-0.2.

2. SEC detection of purified samples

Use of

The chromatographic system, superdex 200, 150mm column (Cytiva, U.S. A), performed size exclusion chromatography on the purified S-TD trimer protein. The mobile phase on the column was Phosphate Buffered Saline (PBS) at a flow rate of 0.25 ml/min, and OD280nm was detected. The results of the experiment are shown in fig. 7 and table 1.

TABLE 1

High resolution analysis result of chromatographic system for purified S-TD tripolymer state

FIG. 7 shows the second peak shape, also the peak with the largest peak area, appearing around 5 minutes, with a molecular weight of 558.5kDa, corresponding to the molecular weight of the S protein trimer.

Example 7: high-titer affinity of antibody in serum of new coronary pneumonia rehabilitation patient with S-TD and RBD-TD trimerization fusion protein

In order to identify whether the S-TD and RBD-TD trimerization fusion protein can be combined with the anti-new coronavirus spike protein antibody in an affinity manner, an indirect ELISA method is adopted to detect the S-TD and RBD-TD trimerization fusion protein and Bovine Serum Albumin (BSA).

The three proteins are used as target antigens and coated on a 96-well ELISA plate respectively. Serum of several new coronary pneumonia convalescent patients was mixed, serially diluted by ten-fold dilution, and then added to each coated well. Bovine serum albumin BSA was used as the experimental negative control sample. The affinity titer of the sample is detected according to the conventional indirect ELISA operation steps. As shown in FIG. 8, the S-TD and RBD-TD trimerization fusion proteins can be identified with the antibodies in the serum of the patients and effectively combined with high titer.

Example 8: preparation of vaccine and detection of immune mouse IgG

1. Vaccine preparation

In this example, two kinds of adjuvants (commercial aluminum salt adjuvant and self-made liposome adjuvant similar to AS03 adjuvant) were used to prepare two kinds of vaccines, and whether the purified S-TD trimer protein can be used AS antigen of vaccine to immunize animals for antibody protection was tested. The preparation method of the two vaccines respectively comprises the following steps: (1) Taking the purified S-TD trimerization fusion protein solution and an aluminum salt adjuvant, and mixing the solution with the aluminum salt adjuvant according to a method in an instruction provided by a manufacturer and the ratio of 3:1, mixing uniformly; (2) Taking the purified S-TD trimerization fusion protein solution and a self-made liposome adjuvant according to the weight ratio of 1:1, mixing, stirring and emulsifying to prepare the vaccine for later use.

2. Mouse immunization test

A total of 29 BALB/c mice at 6-8 weeks were divided into 5 groups, 6 samples each, and 5 blanks, and the specific grouping schedule is shown in Table 2 below. The mice of the sample immunization group are injected with 10 mu g/0.2 ml/mouse, and the mice of the sample adjuvant control group are injected with 0.2 ml/mouse. On day 21 after the first immunization, second immunization was carried out under the above conditions, and further 14 days after the first booster immunization, 1ml of blood was collected from each eyeball of all mice on day 10. The mouse blood samples were centrifuged and the supernatants were aliquoted and stored at 4 ℃ or-20 ℃ for subsequent testing.

TABLE 2 preparation of vaccines and groups of experimental animals

3. Immune mouse antibody IgG titer detection

The indirect ELISA method is mainly adopted to detect the mouse anti-new crown IgG antibody, and the operation is briefly described as follows: a commercially available standard S protein-coated 96-well ELISA plate (20 ng/100. Mu.l/well) was diluted with the coating solution, and the plate was covered with a preservative film and left in a refrigerator at 4 ℃ overnight. The enzyme label plate is washed in the morning and patted dry. Adding 200 mul/hole of sealing liquid, wrapping with preservative film, and reacting at 37 ℃ for 2h. Washed 3 times with 200. Mu.l/well PBS-T solution and patted dry. The reaction solution was mixed with PBS at a ratio of 1: diluting the collected antibody serum of each experimental mouse and blank control serum by 1000, uniformly mixing, adding 200 mu l of the antibody serum and the blank control serum into the first row of the ELISA plate, then adding 100 mu l of PBS into all the rows of the holes, sucking 100 mu l of diluted serum from the first row, adding the diluted serum into the second row, and performing multiple dilution by analogy. The final row of undiluted serum was used as a background blank for the wells. After addition, the cells were sealed and incubated at 37 ℃ for 1 hour. Pouring out the reaction solution, adding PBS-T200 mu l/hole, washing for 3 times, and patting dry; the reaction solution was washed with PBS 1: a second goat anti-mouse antibody containing Horseradish Peroxidase (HRP) conjugate was diluted to 100. Mu.l/well at 5000 and incubated at 37 ℃ for 45 minutes; pouring out the reaction solution, washing for 3 times by using PBS-T200 ul/hole, and patting dry; adding 100ul of TMB prepared per well, developing for 10-15 min, adding 50 mul of stop solution per well, and measuring OD value of each well by using a microplate reader at a wavelength of 450. The antibody titer of the serum assay results was calculated and judged using the EC 50. The results are shown in FIG. 9, which shows that both S-TD trimer protein vaccines prepared with both adjuvants can induce high titer antibodies in immunized mice.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> xu yan

Nowa Biotech, inc. Canada (Nova Biologiques Inc.)

<120> heterotrimeric structural domain, heterotrimeric fusion protein, preparation method and application

<160> 16

<170> SIPOSequenceListing 1.0

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Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu

1 5 10 15

Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Cys Cys Leu Asp

20 25 30

Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg

35 40 45

Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val

50 55 60

Arg Leu Gln Leu Arg Asp Asn Ala

65 70

<210> 2

<211> 1273

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

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

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

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

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

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

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile

1205 1210 1215

Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile

1220 1225 1230

Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val

1250 1255 1260

Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 3

<211> 223

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 4

<211> 564

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr

1 5 10 15

Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr

20 25 30

Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr

35 40 45

Glu Thr Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly Lys

50 55 60

Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly

65 70 75 80

Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile

85 90 95

Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn

100 105 110

Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys

115 120 125

Glu Pro Met Gly Phe Thr Tyr Asn Gly Ile Arg Thr Asn Gly Val Thr

130 135 140

Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp

145 150 155 160

Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser

165 170 175

Tyr Lys Asn Thr Lys Glu Ser Pro Ala Ile Ile Val Trp Gly Ile His

180 185 190

His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn

195 200 205

Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro

210 215 220

Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp

225 230 235 240

Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe

245 250 255

Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys

260 265 270

Ser Met Gly Ile Gln Ser Arg Val Gln Val Asp Ala Asn Cys Glu Gly

275 280 285

Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln

290 295 300

Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln

305 310 315 320

Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val Pro

325 330 335

Lys Arg Lys Arg Thr Ala Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe

340 345 350

Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg

355 360 365

His Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr

370 375 380

Gln Ser Ala Ile Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Ala

385 390 395 400

Lys Thr Asn Gln Gln Phe Lys Leu Ile Asp Asn Glu Phe Asn Glu Val

405 410 415

Glu Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr

420 425 430

Glu Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln

435 440 445

His Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg

450 455 460

Val Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys

465 470 475 480

Phe Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg

485 490 495

Asn Asn Thr Tyr Asp His Arg Lys Tyr Arg Glu Glu Ala Met Gln Asn

500 505 510

Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val

515 520 525

Ile Leu Trp Phe Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu Ala Ile

530 535 540

Val Met Gly Leu Val Phe Ile Cys Val Lys Asn Gly Asn Met Arg Cys

545 550 555 560

Thr Ile Cys Ile

<210> 5

<211> 3822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgttcgtgt tcttggtgct gctgcccctg gtgtcctctc agtgcgtgaa cctgaccacc 60

aggactcagc tgcctccagc ttacaccaac agcttcaccc gtggtgtcta ctaccccgac 120

aaggtgttcc gttcctccgt gctgcactct acccaggacc tgttcctgcc tttcttctcc 180

aacgtgacct ggttccacgc tatccacgtg tccggtacta acggcaccaa gcgtttcgac 240

aaccccgtgc tgcctttcaa cgacggcgtg tacttcgctt ccaccgagaa gtccaacatc 300

atccgtggct ggatcttcgg tactaccctg gactccaaga ctcagtccct gctgatcgtg 360

aacaacgcta ccaacgtggt catcaaagtg tgcgagttcc agttctgcaa cgaccccttc 420

ctgggcgttt actaccacaa gaacaacaag tcctggatgg aatccgagtt ccgtgtgtac 480

tcctccgcta acaactgcac cttcgagtac gtgtcccagc ctttcctgat ggacctcgag 540

ggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt tcaagaacat cgacggctac 600

ttcaagatct actccaagca cacccctatc aacctcgtgc gtgacctgcc tcagggcttc 660

tctgctctgg aacctctggt ggacctgcca atcggtatca acatcacccg tttccagact 720

ctgctggctc tgcaccgttc ctacttgacc cctggcgact cctcttctgg atggactgct 780

ggcgctgctg cttactacgt gggttacctg cagcctcgta ccttcctgct gaagtacaac 840

gagaacggaa ccatcaccga cgctgtggac tgcgctctgg accctttgtc cgagactaag 900

tgcaccctga agtccttcac cgtcgagaag ggcatctacc agacctccaa cttccgtgtg 960

cagcccaccg agtccatcgt gcgtttccct aacatcacca acttgtgccc cttcggcgag 1020

gtgttcaacg ctactcgttt cgcttccgtg tacgcttgga accgcaagcg catctctaac 1080

tgcgtggccg actactccgt cctgtacaac tccgcttcct tcagcacctt caagtgctac 1140

ggtgtctccc ctaccaagct gaacgacctg tgcttcacca acgtctacgc tgactccttc 1200

gtgatccgtg gcgacgaagt gcgtcagatc gctcctggtc aaaccggcaa gatcgctgac 1260

tacaactaca agctgcccga cgacttcacc ggttgcgtga tcgcctggaa ctccaacaac 1320

ctggactcta aagtcggcgg caactacaat tacctgtacc gtctgttccg caagtccaac 1380

ctgaagcctt tcgagcgtga catctctacc gagatctacc aggctggttc taccccttgc 1440

aacggtgtcg agggtttcaa ctgctacttc ccactgcagt cctacggttt ccagcctacc 1500

aacggcgtcg gttaccagcc ttaccgtgtg gtggtgctgt ccttcgaact gctgcacgct 1560

cctgctactg tgtgcggtcc caagaaatcc accaacctgg tcaagaacaa atgcgtgaac 1620

ttcaacttca acggcctgac cggcaccggt gtcctgaccg agtctaacaa gaagttcctg 1680

ccattccagc aattcggccg tgatatcgct gacaccactg acgctgtgcg cgaccctcag 1740

actctggaaa tcctggacat caccccatgc agcttcggtg gtgtctccgt gatcacccct 1800

ggtactaaca cctccaacca ggtggccgtg ctgtaccagg acgtgaactg cactgaagtg 1860

cccgtggcta ttcacgctga ccagctgact ccaacctggc gcgtgtactc caccggttcc 1920

aacgtgttcc agacacgtgc tggttgcctg atcggtgctg agcacgtcaa caactcctac 1980

gagtgcgaca tccccatcgg cgctggtatc tgcgcttcct accagactca gaccaactct 2040

ccccgtcgtg ctcgttccgt ggcttcccag tccatcattg cttacactat gtccctgggt 2100

gctgagaact ccgtcgctta ctctaacaac tctatcgcta tccccaccaa cttcaccatc 2160

tccgtcacca ccgagatcct gcctgtgtcc atgaccaaga cctccgtgga ctgcaccatg 2220

tacatctgcg gcgactccac cgagtgctct aacctgctgc tgcagtacgg ttccttctgc 2280

acccagctga accgtgctct gaccggtatc gctgtcgagc aggacaagaa cacccaagag 2340

gtgttcgctc aagtgaagca gatctacaag acccctccta tcaaggactt cggcggattc 2400

aacttctccc agatcttgcc cgatccttct aagccctcca agcgctcctt catcgaggac 2460

ctgctgttca acaaagtgac cctggctgac gctggtttca tcaagcagta cggcgactgc 2520

ctgggcgaca ttgctgctcg tgatctgatc tgcgctcaga agttcaacgg attgaccgtc 2580

ctgcctcctc tgctgaccga cgagatgatc gctcagtaca cctccgctct gctcgctggc 2640

actatcacct ccggatggac tttcggagct ggtgctgccc tgcagatccc cttcgctatg 2700

cagatggctt accgtttcaa cggtatcggt gtcacccaga acgtgctcta cgagaaccag 2760

aagctgatcg ccaaccagtt caactccgcc atcggaaaga tccaggattc cctgtcctcc 2820

accgcttccg ctctgggaaa gctgcaagac gtggtcaacc agaacgctca ggctctgaac 2880

accctcgtga agcagctgtc ctctaacttc ggtgctatct cctctgtcct gaacgacatc 2940

ctgtctcgtc tggacaaggt cgaggccgag gtgcaaatcg accgtctgat cactggtcgt 3000

ctgcagtctc tgcagaccta cgtgacccag cagttgatcc gcgctgctga gatccgtgct 3060

tccgctaact tggctgctac caagatgtcc gagtgcgtgc tgggacagtc caagcgtgtt 3120

gacttctgcg gcaagggtta ccacctgatg agcttccctc agtccgctcc tcacggtgtc 3180

gtgttccttc acgtcaccta cgtgcccgct caagagaaga acttcactac cgctccagct 3240

atctgccacg acggcaaggc tcatttccca cgtgaaggcg tgttcgtgtc caacggcacc 3300

cattggttcg tcacccagcg caacttctac gagccccaga tcatcactac cgacaacacc 3360

ttcgtgtccg gcaactgcga cgtcgtgatc ggtatcgtta acaatactgt gtacgaccct 3420

ctgcagcccg agctggactc cttcaaagag gaactggaca agtacttcaa aaaccacact 3480

agccccgacg tggacctggg agacatctct ggtatcaacg cttccgtcgt gaacatccag 3540

aaagagatcg accgcctgaa cgaggtggcc aagaacctca acgagtccct gatcgacctg 3600

caagagctgg gcaaatacga gcagtacatc aagtggccct ggtacatctg gctgggtttc 3660

attgctggcc tgatcgctat cgtgatggtc actatcatgc tgtgctgtat gacctcctgc 3720

tgctcctgcc tgaagggctg ctgctcttgt ggttcttgct gcaagttcga cgaggacgac 3780

tccgagcctg tgctcaaggg tgtcaagctg cactacacct aa 3822

<210> 6

<211> 216

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaacactc aatttgaggc cgttggaagg gaatttaata acttagaaag gagaatagag 60

aatttaaaca agaaaatgga agactgttgc ctagatgtct ggacttataa tgctgaactt 120

ctagttctca tggaaaatga gagaactcta gatttccatg actcaaatgt caagaacctt 180

tacgataaag tccgactaca gcttagggac aatgca 216

<210> 7

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgtgtgcagc ccaccgagtc catcgtgcgt ttccctaaca tcaccaactt gtgccccttc 60

ggcgaggtgt tcaacgctac tcgtttcgct tccgtgtacg cttggaaccg caagcgcatc 120

tctaactgcg tggccgacta ctccgtcctg tacaactccg cttccttcag caccttcaag 180

tgctacggtg tctcccctac caagctgaac gacctgtgct tcaccaacgt ctacgctgac 240

tccttcgtga tccgtggcga cgaagtgcgt cagatcgctc ctggtcaaac cggcaagatc 300

gctgactaca actacaagct gcccgacgac ttcaccggtt gcgtgatcgc ctggaactcc 360

aacaacctgg actctaaagt cggcggcaac tacaattacc tgtaccgtct gttccgcaag 420

tccaacctga agcctttcga gcgtgacatc tctaccgaga tctaccaggc tggttctacc 480

ccttgcaacg gtgtcgaggg tttcaactgc tacttcccac tgcagtccta cggtttccag 540

cctaccaacg gcgtcggtta ccagccttac cgtgtggtgg tgctgtcctt cgaactgctg 600

cacgctcctg ctactgtgtg cggtcccaag aaatccacca acctggtcaa gaacaaatgc 660

gtgaacttc 669

<210> 8

<211> 1695

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgaacactc aaatcctggt attcgctctg attgcgatca ttccaacaaa tgcagacaaa 60

atctgcctcg gacatcatgc cgtgtcaaac ggaaccaaag taaacacatt aactgaaaga 120

ggagtggaag tcgtcaatgc aactgaaaca gtggaacgaa caaacacccc caggatctgc 180

tcaaaaggga aaaggacagt tgacctcggt caatgtggac tcctggggac aatcactgga 240

ccacctcaat gtgaccaatt cctagaattt tcggccgatt taattattga gaggcgagaa 300

ggaagtgatg tctgttatcc tggaaaattc gtgaatgaag aagctttgag gcaaattctc 360

agagaatcag gcggaattga caaggaaccc atgggattca catacaatgg aataagaact 420

aatggggtga ccagtgcatg taggagatca ggatcttcat tctatgcaga aatgaaatgg 480

ctcctgtcaa acacagataa tgctgcattc ccgcagatga ctaagtcata taaaaataca 540

aaagaaagcc cagctataat agtatggggg atccatcatt ccgtttcaac tgcagagcaa 600

accaagctat atgggagtgg aaacaagctg gtgacagttg ggagttctaa ttatcaacaa 660

tctttcgtac cgagtccagg agcaagacca caagttaatg gtcaatctgg aagaattgac 720

tttcattggc taatactaaa tcccaatgat acagtcactt tcagtttcaa tggggctttc 780

atagctccag accgtgcaag cttcctgaga ggaaaatcta tgggaatcca gagtagagta 840

caggttgatg ccaattgtga aggggactgc tatcatagtg gagggacaat aataagtaac 900

ttgccatttc agaacataga tagcagggca gttggaaaat gtccgagata tgttaagcaa 960

aggagtcttc tgctggcaac agggatgaag aatgttcctg aggttccaaa gagaaaacgg 1020

actgcgagag gcctatttgg tgctatagcg ggtttcattg aaaatggatg ggaaggccta 1080

attgatggtt ggtatggttt cagacaccag aatgcacagg gagagggaac tgctgcagat 1140

tacaaaagca ctcaatcggc aattgatcaa ataacaggga aattaaaccg gcttatagca 1200

aaaaccaacc aacaatttaa gttgatagac aatgagttta atgaggtaga gaagcaaatc 1260

ggtaatgtga taaattggac cagagattct ataacagaag tatggtcata caatgctgaa 1320

ctcttggtgg caatggagaa ccagcataca attgatctgg ctgattcaga aatggacaaa 1380

ctgtacgaac gagtgaaaag acagctgaga gagaatgctg aagaagacgg cacgggttgc 1440

tttgaaatat ttcacaagtg tgatgatgac tgtatggcca gtattagaaa taacacctat 1500

gatcacagaa aatacagaga agaggcaatg caaaatagaa tacagattga cccagtcaaa 1560

ctaagcagcg gctacaaaga tgtgatactt tggtttagct tcggggcatc atgtttcata 1620

cttctagcca ttgtaatggg ccttgtcttc atatgtgtga agaatggaaa catgcggtgc 1680

actatttgta tataa 1695

<210> 9

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttaaacaaga aaatggaaga ctgttgccta gatgtctgga cttataatg 49

<210> 10

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cattataagt ccagacatct aggcaacagt cttccatttt cttgtttaa 49

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgttcgtgt tcttggtgct g 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttaggtgtag tgcagcttga c 21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgtgtgcagc ccaccgagtc c 21

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gaagttcacg catttgttct tg 22

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgaacactc aaatcctgg 19

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttatatacaa atagtgcacc 20

Claims (9)

1. A heterotrimeric domain, characterized in that the heterotrimeric domain is formed by site-directed mutation of DNA bases of viral envelope glycoprotein domain polypeptide fragments, the N-terminal of each polypeptide in the heterotrimeric domain after the site-directed mutation contains a long a-helical fragment, the C-terminal contains an antiparallel short a-helical fragment, and the two helical fragments are connected by a flexible loop;

the site-directed mutation leads to the introduction of 2S-S covalent bond sites on the long alpha-helix segment;

the site of the directed mutation is G29C and F30C of the polypeptide fragment; the amino acid sequence of the heterotrimeric domain polypeptide is shown as SEQ ID NO. 1.

2. The heterotrimeric domain according to claim 1, wherein the heterotrimeric domain polypeptide has an amino acid sequence as shown in SEQ ID No.1 and a DNA sequence as shown in SEQ ID No. 6.

3. A heterotrimeric fusion protein composition, selected from the group consisting of an S-TD trimeric protein, an RBD-TD trimeric protein, or an H7-TD trimeric protein; wherein each of the trimeric proteins consists of (1) and (2):

(1) The polypeptide of the heterotrimeric domain portion of claim 1 or 2, having an amino acid sequence as set forth in SEQ ID No. 1;

(2) A fusion protein polypeptide of a non-domain to be trimerized linked to the N-terminus of the polypeptide of the heterotrimeric domain portion of claim 1 or 2;

the amino acid sequence of the fusion protein polypeptide of the non-structural domain to be trimerized of the S-TD trimeric protein is shown as SEQ ID NO. 2;

the amino acid sequence of the fusion protein polypeptide of the non-structural domain to be trimerized of the RBD-TD trimeric protein is shown in SEQ ID NO. 3;

the amino acid sequence of the fusion protein polypeptide of the non-structural domain to be trimerized of the H7-TD trimeric protein is shown as SEQ ID NO. 4.

4. A method of preparing a heterotrimeric fusion protein composition of claim 3, comprising the steps of:

(1) Carrying out PCR (polymerase chain reaction) site-specific mutagenesis on DNA bases of G29C and F30C sites of the domain polypeptide to obtain a trimerization domain DNA fragment; recovering a trimerization domain DNA fragment, cloning the DNA fragment to a plasmid vector, constructing a trimerization domain recombinant plasmid, and obtaining an amino acid sequence of a polypeptide of the mutated domain shown as SEQ ID NO. 1;

(2) Inserting the fusion protein polypeptide gene segment of the non-structural domain to be trimerized into a trimerization structural domain recombinant plasmid to form a heterotrimeric fusion protein recombinant plasmid; the nucleotide sequence of the fusion protein polypeptide gene segment of the non-structural domain to be trimerized is selected from the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO. 8;

3) Cloning the heterotrimeric fusion protein recombinant plasmid to an expression plasmid vector to obtain a recombinant expression plasmid vector of the heterotrimeric fusion protein;

(4) Transfecting host cells with the recombinant expression plasmid vector of the heterotrimeric fusion protein, recovering host cell culture supernatant, and extracting and purifying the heterotrimeric fusion protein composition.

5. The method of making a heterotrimeric fusion protein composition according to claim 4, wherein the host cell is selected from eukaryotic cells or prokaryotic cells.

6. The method of preparing a heterotrimeric fusion protein composition according to claim 5, wherein the host cell is selected from any one of insect cells, mammalian cells, E.coli cells, fungal cells.

7. A vaccine comprising the heterotrimeric fusion protein composition of claim 3.

8. The vaccine of claim 7, further comprising an adjuvant selected from at least one of an aluminum salt adjuvant, a CpG Toll-Like adjuvant, a water-in-oil-in-water adjuvant, a squalene adjuvant, a vegetable oil adjuvant, a liposome adjuvant, a nanoparticle adjuvant, or a Freund's adjuvant.

9. The use of the heterotrimeric fusion protein composition of claim 3 in the preparation of a pestivirus antibody detection formulation or a pestivirus pathogen monitoring formulation, wherein the formulation comprises any one of an ELISA detection kit, a colloidal gold test strip, a chemiluminescent detection kit or a fluorescent luminescent detection kit.

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