Preparation and use of a combination of eg95 / ec95 proteins of echinococcus granulosus and echinococcus canadensis

Abstract

The application discloses a preparation and application of a fine granular echinococcus and a Canadian echinococcus EG95 / EC95 protein combination; the recombinant protein combination comprises dEG95 and dEC95 amino acid sequences; the dEG95 amino acid sequence is a modified amino acid sequence obtained by deleting a signal peptide region at an N terminal and a transmembrane region at a C terminal of EG95 of narrow fine granular echinococcus; the EG95 gene sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the dEC95 amino acid sequence is a modified amino acid sequence obtained by deleting a signal peptide region at an N terminal and a transmembrane region at a C terminal of EC95 of Canadian echinococcus; the EC95 gene sequence is shown as SEQ ID NO. 4, and the amino acid sequence is shown as SEQ ID NO. 5. The method has the advantages of high protein expression amount, simple process, low production cost and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to preparation and application of a combination of EG95 / EC95 proteins of Echinococcus granulosus and E. canadensis; in particular, to a modification method of EG95 / EC95 protein sequences of Echinococcus granulosus and E. canadensis, and a method for inducing expression of recombinant E. coli engineering bacteria prepared by using the modified protein sequences. BACKGROUND

[0002] Echinococcosis is a serious parasitic disease caused by the larvae of Echinococcus tapeworm, i.e., hydatid cyst, parasitizing in the lungs, liver and other tissues and organs of humans and animals. Echinococcosis is widely prevalent and has a worldwide distribution. The World Organization for Animal Health (OIE) classifies echinococcosis as a globally reported infectious disease and a multi-species zoonosis. The World Health Organization (WHO) lists echinococcosis as one of the diseases for which early warning systems are prioritized for prediction and emergency response. Echinococcosis is also one of the five parasitic diseases planned for prevention and control by the Ministry of Health of China.

[0003] According to the differences in the morphology of lesions and the infectious pathogens, echinococcosis is mainly divided into cystic echinococcosis (CE) and alveolar echinococcosis. CE is the most widely distributed and has the largest number of patients. The pathogenic agent of CE is currently composed of several Echinococcus granulosus complex species: E. granulosus sensu stricto (E. g. sensu stricto), E. canadensis (E. c. canadensis), E. equinus, and E. oligarthrus. CE caused by E. g. sensu stricto G1 accounts for more than 90%, and CE caused by E. c. canadensis G6 accounts for more than 7%. Echinococcus granulosus Echinococcus canadensis Eg Among them, CE caused by E. g. sensu stricto G1 accounts for more than 90%, and CE caused by E. c. canadensis G6 accounts for more than 7%.

[0004] Current studies show that the control of the prevalence of echinococcosis is mainly achieved by breaking the development cycle of Echinococcus tapeworm, controlling the infection of intermediate hosts such as humans and animals with echinococcosis, preventing or treating the terminal hosts such as dogs, and blocking the wide dissemination of eggs. Among them, vaccination of intermediate hosts can effectively control the prevalence of cystic echinococcosis. Lightowlers et al. found that EG95 is present in the oncosphere of E. g. sensu stricto and E. c. canadensis, and can induce a strong immune response in the intermediate host. EG95 ​One of the natural oncosphere antigens in Echinococcus granulosus and the most effective protective antigen among the screened proteins, a vaccine against echinococcosis granulosus in sheep has been successfully developed. However, the existing recombinant EG95 protein vaccine is prepared from E. coli inclusion bodies, has low purity and is difficult to maintain the spatial structure of the protein, and has no obvious cross-protection with Echinococcus canadensis G6, thus having certain limitations. SUMMARY

[0005] In view of the defects in the prior art described above, the purpose of the present application is to provide a preparation and application of EG95 / EC95 protein combination of Echinococcus granulosus and Echinococcus canadensis; in particular, the dEG95 and dEC95 in Echinococcus granulosus and Echinococcus canadensis are modified, and a recombinant expression plasmid, a recombinant E. coli engineering bacteria, and a fusion protein expressed by the recombinant E. coli engineering bacteria have the advantages of high purity, good solubility, convenient purification, and high biological activity. EC95 EG95 and Echinococcus canadensis are modified, and a recombinant expression plasmid, a recombinant E. coli engineering bacteria, and a fusion protein expressed by the recombinant E. coli engineering bacteria have the advantages of high purity, good solubility, convenient purification, and high biological activity.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In the first aspect, the present application relates to a recombinant protein combination, which comprises a dEG95 amino acid sequence and a dEC95 amino acid sequence.

[0008] The dEG95 amino acid sequence is a modified amino acid sequence of Echinococcus granulosus EG95. EG95 The dEG95 amino acid sequence is a modified amino acid sequence of Echinococcus granulosus EG95. E. coli The gene sequence of the dEG95 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

[0009] The dEC95 amino acid sequence is a modified amino acid sequence of Echinococcus canadensis EC95.

[0010] As an embodiment of the present application, the N-terminal of the EG95 amino acid sequence is truncated by 13, 14, 15, or 16 amino acids, and the C-terminal is truncated by 20, 22, 24, or 26 amino acids.

[0011] As an embodiment of the present application, the N-terminal of the EG95 amino acid sequence is truncated by 14 amino acids, and the C-terminal is truncated by 24 amino acids. The modified EG95 amino acid sequence, i.e. the dEG95 amino acid sequence, is shown as SEQ ID NO. 3.

[0012] ​As one embodiment of the present invention, the N-terminus of the EC95 amino acid sequence is truncated by 13, 14, 15 or 16 amino acids; and the C-terminus is truncated by 20, 22, 24 or 26 amino acids.

[0013] In one embodiment of the present invention, the N-terminus of the EC95 amino acid sequence is truncated by 14 amino acids, and the C-terminus is truncated by 24 amino acids. The modified EC95 amino acid sequence, i.e., the dEC95 amino acid sequence, is shown in SEQ ID NO. 6.

[0014] As one embodiment of the present invention, the amino acid sequence of dEG95 is shown in SEQ ID NO.3; the amino acid sequence of dEC95 is shown in SEQ ID NO.6.

[0015] Secondly, the present invention relates to a method for preparing a recombinant protein combination, the method comprising the following steps:

[0016] S1. The coding genes of dEG95 and dEC95 were cloned into a prokaryotic expression vector to obtain recombinant expression plasmids;

[0017] S2. Transfect prokaryotic expression strains with recombinant expression plasmids, screen single clones, ferment and culture, and induce expression to obtain the recombinant protein.

[0018] Specifically, as one embodiment of the present invention, step S1 includes the following steps:

[0019] A1. The dEG95 and dEC95 gene sequences, respectively optimized with gene synthesis codons and suitable for expression in Escherichia coli; the amino acid sequence of dEG95 is shown in SEQ ID NO.3, and the amino acid sequence of dEC95 is shown in SEQ ID NO.6;

[0020] Alternatively, two dEG95 amino acid sequences and two dEC95 amino acid sequences are linked together using a linker to form 2dEG95 amino acid sequences and 2dEC95 amino acid sequences, respectively; these are 2dEG95 and 2dEC95 gene sequences with codon optimization suitable for expression in E. coli.

[0021] Alternatively, the dEG95 amino acid sequence and the dEC95 amino acid sequence can be linked together by a linker to form the dEG95-dEC95 amino acid sequence or the dEC95-dEG95 amino acid sequence; respectively, the gene synthesis codons are optimized to create dEG95-dEC95 or dEC95-dEG95 gene sequences suitable for expression in E. coli.

[0022] The recombinant protein combination of this invention is original; and because the protein structures of EG95 and EC95 have not been resolved, their functional studies are quite difficult. Different truncations can cause coordination problems between the two monomers in the heterodimer, easily leading to misfolding or loss of activity of the heterodimer. This invention predicts their spatial structure through molecular simulation, molecular dynamics, and other methods, thus making the correct truncation choice, enabling soluble protein expression, and maintaining a stable spatial structure and conformation.

[0023] A2. The dEG95 and dEC95 genes were cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-dEG95-dEC95 or pETDuet-dEC95-dEG95 plasmids.

[0024] Alternatively, the 2dEG95 and 2dEC95 genes can be cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-2dEG95-2dEC95 or pETDuet-2dEC95-2dEG95 plasmids.

[0025] Alternatively, the dEG95-dEC95 or dEC95-dEG95 gene can be cloned into the pET24a plasmid vector to construct the pET24a-dEG95-dEC95 or pET24a-dEC95-dEG95 plasmid.

[0026] In one embodiment of the present invention, in step A1, the linker is a linker rich in glycine and serine. Preferably, it is a GGGSGGGS linker.

[0027] In one embodiment of the present invention, in step S2, the prokaryotic expression strain is *Escherichia coli*. The *Escherichia coli* strain is commercially available, and examples, but not limited to, include: BL21(DE3), B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, and GI698. BL21(DE3) is preferred.

[0028] As one embodiment of the present invention, in step S2, the...

[0029] The recombinant expression plasmids pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pET24a-dEG95-dEC95, pET24a-dEC95-dEG95, pETDuet-2dEG95-2dEC95, or pETDuet-2dEC95-2dEG95 were transformed into *E. coli* BL21(DE3). Single clones were screened to obtain recombinant engineered *E. coli* strains Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, or Duet-2dEC95-2dEG95.

[0030] In one embodiment of the present invention, in step S2, the Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, or Duet-2dEC95-2dEG95 strains are inoculated into 500 mL of LB medium containing the corresponding antibiotics and cultured with shaking at 37°C until the OD value reaches 1.2-1.5. The seed culture is then inoculated into a fermenter for fermentation. When the OD value of the cells reaches 20-25, IPTG is added to a final concentration of 0.4 mM and induced for 12-14 h to obtain the recombinant protein complex.

[0031] As one embodiment of the present invention, step S2 further includes a step of purifying the protein using chromatographic chromatography, including but not limited to: ion exchange chromatography (e.g., cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (e.g., hydroxyapatite chromatography), gel filtration (gel size exclusion) chromatography, affinity chromatography, and molecular sieve chromatography. Affinity chromatography is preferred.

[0032] As one embodiment of the present invention, the amino acid sequence of 2dEG95 is shown in SEQ ID NO.7; the amino acid sequence of 2dEC95 is shown in SEQ ID NO.8; the amino acid sequence of dEG95-dEC95 is shown in SEQ ID NO.9; and the amino acid sequence of dEC95-dEG95 is shown in SEQ ID NO.10.

[0033] Thirdly, the present invention relates to a recombinant expression plasmid comprising a coding gene of the recombinant protein combination.

[0034] As one embodiment of the present invention, the recombinant expression plasmid is pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pET24a-dEG95-dEC95, pET24a-dEC95-dEG95, pETDuet-2dEG95-2dEC95, or pETDuet-2dEC95-2dEG95.

[0035] In the above recombinant expression plasmids, the pETDuet-dEG95-dEC95 plasmid used dEC95 as a template. The gene fragment dEC95 was amplified by PCR using the upstream primer dEC95-Nde IF and the downstream primer dEC95-Xho IR, then ligated into pETDuet-dEG95. The ligation product was then transformed. E. coli Plasmids were extracted from DH5α competent cells and verified by sequencing. Using dEG95 as a template, the gene fragment dEG95 was amplified by PCR using the upstream primer dEG95-BamH IF and the downstream primer dEG95-Hind III-R. This fragment was then ligated into the pETDuet-1 prokaryotic expression vector to obtain pETDuet-dEG95.

[0036] The pETDuet-dEC95-dEG95 plasmid was prepared using dEG95 as a template. The gene fragment dEG95 was amplified by PCR using the upstream primer dEG95-Nde IF and the downstream primer dEG95-Xho IR. This fragment was then ligated into pETDuet-dEC95, and the ligation product was transformed. E. coli Plasmids were extracted from DH5α competent cells and verified by sequencing. Using dEC95 as a template, the gene fragment dEC95 was amplified by PCR using the upstream primer dEC95-BamH IF and the downstream primer dEC95-Hind III-R. This fragment was then ligated into the pETDuet-1 prokaryotic expression vector to obtain pETDuet-dEC95.

[0037] pET24a-dEG95-dEC95 was developed using dEG95-dEC95 as a template. The gene fragment dEG95-dEC95 was amplified by PCR using the upstream primer dEG95-Nde IF and the downstream primer dEC95-Xho IR. This fragment was then ligated into the pET24a prokaryotic expression vector, and the ligation product was transformed. E. coli DH5α competent cells were used to extract plasmids, which were then verified by sequencing.

[0038] pET24a-dEC95-dEG95 was amplified by PCR using dEC95-dEG95 as a template, with upstream primer dEC95-Nde IF and downstream primer dEG95-Xho IR. The resulting gene fragment dEC95-dEG95 was then ligated into the pET24a prokaryotic expression vector, and the ligation product was transformed. E. coli DH5α competent cells were used to extract plasmids, which were then verified by sequencing.

[0039] pETDuet-2dEG95-2dEC95 uses 2dEC95 as a template. The gene fragment 2dEC95 was amplified by PCR using the upstream primer dEC95-Nde IF and the downstream primer dEC95-Xho IR. This fragment was then ligated into pETDuet-2dEG95, and the ligation product was transformed. E. coli Plasmids were extracted from DH5α competent cells and verified by sequencing. Using 2dEG95 as a template, the gene fragment 2dEG95 was amplified by PCR using the upstream primer dEG95-BamH IF and the downstream primer dEG95-Hind III-R. This fragment was then ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamH I and Hind III to obtain pETDuet-2dEG95.

[0040] pETDuet-2dEC95-2dEG95 is a gene fragment amplified by PCR using 2dEG95 as a template and dEG95-Nde IF upstream primer and dEG95-Xho IR downstream primer. This fragment is then ligated into pETDuet-2dEC95, and the ligation product is transformed. E. coli Plasmids were extracted from DH5α competent cells and verified by sequencing. Using 2dEC95 as a template, the gene fragment 2dEC95 was amplified by PCR using the upstream primer dEC95-BamH IF and the downstream primer dEC95-Hind III-R. This fragment was then ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamH I and Hind III to obtain pETDuet-2dEC95.

[0041] Fourthly, the present invention relates to a recombinant engineered Escherichia coli strain, which is transformed from the aforementioned recombinant expression plasmid. E. coli And thus obtained. In some embodiments, it is a transformation. Escherichia coli It is obtained from BL21 (DE3) competent cells.

[0042] The engineered bacteria are Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, or Duet-2dEC95-2dEG95.

[0043] Six strains—Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, and Duet-2dEC95-2dEG95—can all express the EG95 / EC95 protein combination. Preferably, strains 24a-dEG95-dEC95 and Duet-2dEG95-2dEC95 will be used; even more preferably, strain 24a-dEG95-dEC95 will be used. This strain exhibits better expression levels and immunogenicity.

[0044] Therefore, the present invention also relates to a recombinant engineered Escherichia coli strain, wherein the engineered strain is Escherichia coli. Escherichia coli 24a-dEG95-dEC95, accession number CCTCC NO: M2021750.

[0045] Fifthly, the present invention relates to the use of the aforementioned recombinant protein combination in the preparation of pharmaceuticals and / or vaccines against echinococcosis infection.

[0046] The drugs and / or vaccines are administered to sheep, cattle, and camels.

[0047] The Escherichia coli 24a-dEG95-dEC95 involved in this invention ( Figure 1 24a-dEG95-dEC95 was deposited with the China Center for Type Culture Collection (CCTCC) on June 23, 2021. The deposit address is Wuhan University, Wuhan, China, and the accession number is CCTCC NO:M 2021750.

[0048] The present invention has the following beneficial effects:

[0049] 1) This invention proposes a method for preparing the combination of fine-grained Echinococcus larvae and Echinococcus cantonensis EG95 / EC95 proteins through structural biology analysis based on molecular simulation and molecular dynamics. This method achieves soluble and efficient expression of the target protein and has the advantages of simple process and low production cost. The prepared target protein has outstanding immunogenicity.

[0050] 2) This invention simplifies the operation by using heterodimers to allow the proteins to retain their original structures and functions. For the first time, the fusion expression of EG95 and EC95 proteins allows the fusion protein to have the activities of both components while being expressed in soluble form, laying a solid foundation for subsequent vaccine and drug development. Attached Figure Description

[0051] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0052] Figure 2 Electrophoresis diagram for protein expression verification; where, lane 1: protein molecular weight standard sample; lane 2: blank control; lane 3: Duet-dEG95-dEC95 supernatant; lane 4: Duet-dEG95-dEC95 precipitate; lane 5: Duet-dEC95-dEG95 supernatant; lane 6: Duet-dEC95-dEG95 precipitate; lane 7: 24a-dEG95-dEC95 supernatant; lane 8: 24a-dEG95-d EC95 precipitate; Lane 9: 24a-dEC95-dEG95 supernatant; Lane 10: 24a-dEC95-dEG95 precipitate; Lane 11: Duet-2dEG95-2dEC95 supernatant; Lane 12: Duet-2dEG95-2dEC95 precipitate; Lane 13: Duet-2dEC95-2dEG95 supernatant; Lane 14: Duet-2dEC95-2dEG95 precipitate; Lane 15: Blank control;

[0053] Figure 3 Electrophoresis results of recombinant protein purification; Lane 1: Protein molecular weight standard sample; Lane 2: Protein sample purified by Duet-dEG95-dEC95; Lane 3: Protein sample purified by Duet-dEC95-dEG95; Lane 4: Protein sample purified by 24a-dEG95-dEC95; Lane 5: Protein sample purified by 24a-dEC95-dEG95; Lane 6: Protein sample purified by Duet-2dEG95-2dEC95; Lane 7: Protein sample purified by Duet-2dEC95-2dEG95.

[0054] Figure 4 The results of ELISA testing are based on competition with the EG95 antigen.

[0055] EG95 Results of ELISA testing for EC95 antigen competition. Detailed Implementation

[0056] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0057] This invention relates to the preparation of a combination of fine-grained echinococcosis larvae and Echinococcus cantonensis EG95 / EC95 proteins, comprising the following steps:

[0058] B1: Echinococcus granulosus in the narrow sense E. coli The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. After amino acid sequence modification, the N-terminal signal peptide region and the C-terminal transmembrane region were deleted, and the modified amino acid sequence was obtained, which is called dEG95.

[0059] B2: The EC95 gene sequence of Echinococcus caninata is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5. After amino acid sequence modification, the N-terminal signal peptide region and the C-terminal transmembrane region were deleted, and the modified amino acid sequence was obtained, which is called dEC95.

[0060] B3: Two dEG95 amino acid sequences are linked together by a linker to form a 2dEG95 amino acid sequence;

[0061] B4: Two dEC95 amino acid sequences are linked together by a linker to form a 2dEC95 amino acid sequence;

[0062] B5: The dEG95 amino acid sequence and the dEC95 amino acid sequence are linked together in different orders by a linker to form the dEG95-dEC95 amino acid sequence and the dEC95-dEG95 amino acid sequence.

[0063] B6: Six sequences optimized for expression in E. coli, namely dEG95, 2dEG95, dEC95, 2dEC95, dEG95-dEC95, and dEC95-dEG95.

[0064] B7: The dEG95 and dEC95 genes were cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-dEG95-dEC95 and pETDuet-dEC95-dEG95 plasmids, respectively.

[0065] B8: The dEG95-dEC95 and dEC95-dEG95 genes were cloned into the pET24a plasmid vector, respectively, to construct the pET24a-dEG95-dEC95 and pET24a-dEC95-dEG95 plasmids.

[0066] B9: The 2dEG95 and 2dEC95 genes were cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-2dEG95-2dEC95 and pETDuet-2dEC95-2dEG95 plasmids, respectively.

[0067] B10: pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pET24a-dEG95-dEC95, pET24a-dEC95-dEG95, pETDuet-2dEG95-2dEC95, and pETDuet-2dEC95-2dEG95 were transformed into Escherichia coli BL21(DE3), and after screening for single clones, the corresponding E. coli strains were named Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, and Duet-2dEC95-2dEG95, respectively.

[0068] B11: The Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, and Duet-2dEC95-2dEG95 strains were inoculated into 500 mL of LB medium containing the corresponding antibiotics and cultured at 37°C with shaking until the OD value reached 1.2–1.5. The seed culture was then inoculated into a fermenter for fermentation. When the OD value of the cells reached 20–25, IPTG was added to a final concentration of 0.4 mM, and induction was performed for 12–14 h. Recombinant protein complexes were obtained through induced expression.

[0069] In step B1, the N-terminus of the EG95 amino acid sequence is truncated by 13, 14, 15, or 16 amino acids; the C-terminus is truncated by 20, 22, 24, or 26 amino acids; preferably, the N-terminus of the EG95 amino acid sequence is truncated by 14 amino acids and the C-terminus is truncated by 24 amino acids, and the modified dEG95 amino acid sequence is shown in SEQ ID NO.3.

[0070] In step B2, the N-terminus of the EC95 amino acid sequence is truncated by 13, 14, 15, or 16 amino acids; the C-terminus is truncated by 20, 22, 24, or 26 amino acids; preferably, the N-terminus of the EC95 amino acid sequence is truncated by 14 amino acids and the C-terminus is truncated by 24 amino acids, and the modified dEC95 amino acid sequence is shown in SEQ ID NO.6.

[0071] In step B3, the two dEG95 amino acid sequences are linked by a linker rich in glycine and serine; preferably, the two dEG95 amino acid sequences are linked by GGGSGGGS, and the amino acid sequences are shown in SEQ ID NO.7.

[0072] In step B4, the two dEC95 amino acid sequences are linked by a linker rich in glycine and serine; preferably, the two dEC95 amino acid sequences are linked by GGGSGGGS, and the amino acid sequences are shown in SEQ ID NO.8.

[0073] In step B5, the amino acid sequences of dEG95 and dEC95 are linked by a linker rich in glycine and serine; preferably, the amino acid sequences of dEG95 and dEC95 are linked by GGGSGGGS, and the different amino acid sequences according to the order of dEG95 and dEC95 are shown in SEQ ID NO.9 (dEG95-dEC95) and SEQ ID NO.10 (dEC95-dEG95).

[0074] The E. coli in step B10 is derived from commercially available sources, including, but not limited to, BL21(DE3), B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, and GI698. BL21(DE3) is preferred.

[0075] In step B11, all six strains—Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, and Duet-2dEC95-2dEG95—can express the EG95 / EC95 protein combination. Preferably, strains 24a-dEG95-dEC95 and Duet-2dEG95-2dEC95 will be used. More preferably, strain 24a-dEG95-dEC95 will be used.

[0076] Step B11 also includes a step of purifying the protein using chromatographic chromatography, including but not limited to: ion exchange chromatography (e.g., cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (e.g., hydroxyapatite chromatography), gel filtration (gel size exclusion) chromatography, affinity chromatography, and molecular sieve chromatography. Affinity chromatography is preferred.

[0077] Example 1: Optimization and Synthesis of Gene Sequences

[0078] by E. coli Using the host bacterium as a host bacterium, this invention optimized the codons of the base sequences encoding recombinant proteins dEG95, dEC95, 2dEG95, 2dEC95, dEG95-dEC95, and dEC95-dEG95. The optimized base sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. Specifically, the N-terminus of the EG95 amino acid sequence was truncated by 14 amino acids, and the C-terminus was truncated by 24 amino acids. Two modified EG95 amino acid sequences were linked together using the "GGGSGGGS" string to construct the single-chain homodimer 2dEG95. The N-terminus of the EC95 amino acid sequence was truncated by 14 amino acids, and the C-terminus was truncated by 24 amino acids. Two modified EC95 amino acid sequences were linked together using the "GGGSGGGS" string to construct the single-chain homodimer 2dEC95. The modified EG95 amino acid sequences and the modified EC95 amino acid sequences were linked together using the "GGGSGGGS" string in different orders to construct the single-chain heterodimers dEG95-dEC95 and dEC95-dEG95.

[0079] Synchronous, with E. coli Using the host bacterium as a host, this invention optimized the codons of the base sequences encoding recombinant proteins aEG95 and aEC95. The optimized base sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. The aEG95 amino acid sequence is an 8-amino acid truncated from the N-terminus of the EG95 protein amino acid sequence, as shown in SEQ ID NO. 19; the aEC95 amino acid sequence is an 8-amino acid truncated from the N-terminus of the EC95 protein amino acid sequence, as shown in SEQ ID NO. 20. The aEG95 and aEC95 amino acid sequences were linked in different orders using the "GGGSGGGS" formula to construct single-stranded heterodimers aEG95-aEC95 and aEC95-aEG95.

[0080] Example 2: Construction of recombinant expression vector.

[0081] 2.1 Construction of the recombinant expression vector pETDuet-2dEG95-2dEC95.

[0082] 2.1.1 Construction of the pETDuet-2dEG95 vector

[0083] (1) Using 2dEG95 as a template, an upstream primer dEG95-BamH IF and a downstream primer dEG95-Hind III-R were designed. The gene fragment 2dEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease BamH I site and a protective base, wherein the BamH I site sequence is GGATCC; the 5' end of the downstream primer was introduced with a restriction endonuclease Hind III site, a stop codon and a protective base, wherein the Hind III site sequence is AAGCT. The primer sequences and PCR reaction procedures are shown in Table 1 and Table 2.

[0084] Table 1: Primer Names and Sequence Information

[0085]

[0086] Table 2: PCR reaction procedure

[0087]

[0088] (2) The amplified gene fragment 2dEG95 was digested with restriction enzymes BamHI and HindIII. The digested gene fragment was recovered and ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamHI and HindIII. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted to obtain the recombinant plasmid pETDuet-2dEG95.

[0089] 2.1.2 Construct the pETDuet-2dEG95-2dEC95 vector.

[0090] (1) Using 2dEC95 as a template, an upstream primer dEC95-Nde IF and a downstream primer dEC95-Xho IR were designed. The gene fragment 2dEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 3 and 4.

[0091] Table 3: Primer Names and Sequence Information

[0092]

[0093] Table 4: PCR reaction procedure

[0094]

[0095] (2) The amplified gene fragment 2dEC95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into pETDuet-2dEG95 treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted. The recombinant plasmid pETDuet-2dEG95-2dEC95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0096] 2.2 Construction of the recombinant expression vector pETDuet-2dEC95-2dEG95.

[0097] 2.2.1 Construction of the pETDuet-2dEC95 vector

[0098] (1) Using 2dEC95 as a template, an upstream primer dEC95-BamH IF and a downstream primer dEC95-Hind III-R were designed. The gene fragment 2dEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease BamH I site and a protective base, wherein the BamH I site sequence is GGATCC; the 5' end of the downstream primer was introduced with a restriction endonuclease Hind III site, a stop codon and a protective base, wherein the Hind III site sequence is AAGCT. The primer sequences and PCR reaction procedures are shown in Tables 5 and 6.

[0099] Table 5: Primer Names and Sequence Information

[0100]

[0101] Table 6: PCR reaction procedure

[0102]

[0103] (2) The amplified gene fragment 2dEC95 was digested with restriction enzymes BamHI and HindIII. The digested gene fragment was recovered and ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamHI and HindIII. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted to obtain the recombinant plasmid pETDuet-2dEC95.

[0104] 2.2.2 Construct the pETDuet-2dEC95-2dEG95 vector.

[0105] (1) Using 2dEG95 as a template, an upstream primer dEG95-Nde IF and a downstream primer dEG95-Xho IR were designed. The gene fragment 2dEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 7 and 8.

[0106] Table 7: Primer Names and Sequence Information

[0107]

[0108] Table 8: PCR reaction procedure

[0109]

[0110] (2) The amplified gene fragment 2dEG95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into pETDuet-2dEC95 treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted. The recombinant plasmid pETDuet-2dEC95-2dEG95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0111] 2.3 Construction of the recombinant expression vector pETDuet-dEG95-dEC95.

[0112] 2.3.1 Construction of the pETDuet-dEG95 vector

[0113] (1) Using dEG95 as a template, an upstream primer dEG95-BamH IF and a downstream primer dEG95-Hind III-R were designed. The gene fragment dEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease BamH I site and a protective base, wherein the BamH I site sequence is GGATCC; the 5' end of the downstream primer was introduced with a restriction endonuclease Hind III site, a stop codon and a protective base, wherein the Hind III site sequence is AAGCTT. The primer sequences and PCR reaction procedures are shown in Tables 9 and 10.

[0114] Table 9: Primer Names and Sequence Information

[0115]

[0116] Table 10: PCR reaction procedure

[0117]

[0118] (2) The amplified gene fragment dEG95 was digested with restriction enzymes BamHI and HindIII. The digested gene fragment was recovered and ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamHI and HindIII. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted to obtain the recombinant plasmid pETDuet-dEG95.

[0119] 2.3.2 Construct the pETDuet-dEG95-dEC95 vector.

[0120] (1) Using dEC95 as a template, upstream primer dEC95-Nde IF and downstream primer dEC95-Xho IR were designed. The gene fragment dEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 11 and 12.

[0121] Table 11: Primer Names and Sequence Information

[0122]

[0123] Table 12: PCR reaction procedure

[0124]

[0125] (2) The amplified gene fragment dEC95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into pETDuet-dEG95 treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted. The recombinant plasmid pETDuet-dEG95-dEC95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0126] 2.4 Construction of the recombinant expression vector pETDuet-dEC95-dEG95.

[0127] 2.4.1 Construction of the pETDuet-dEC95 vector

[0128] (1) Using dEC95 as a template, an upstream primer dEC95-BamH IF and a downstream primer dEC95-Hind III-R were designed. The gene fragment dEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease BamH I site and a protective base, wherein the BamH I site sequence is GGATCC; the 5' end of the downstream primer was introduced with a restriction endonuclease Hind III site, a stop codon and a protective base, wherein the Hind III site sequence is AAGCT. The primer sequences and PCR reaction procedures are shown in Tables 13 and 14.

[0129] Table 13: Primer Names and Sequence Information

[0130]

[0131] Table 14: PCR reaction procedure

[0132]

[0133] (2) The amplified gene fragment dEC95 was digested with restriction enzymes BamHI and HindIII. The digested gene fragment was recovered and ligated into the pETDuet-1 prokaryotic expression vector treated with the same restriction enzymes BamHI and HindIII. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted to obtain the recombinant plasmid pETDuet-dEC95.

[0134] 2.4.2 Construct the pETDuet-dEC95-dEG95 vector.

[0135] (1) Using dEG95 as a template, upstream primer dEG95-Nde IF and downstream primer dEG95-Xho IR were designed. The gene fragment dEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 15 and 16.

[0136] Table 15: Primer Names and Sequence Information

[0137]

[0138] Table 16: PCR reaction procedure

[0139]

[0140] (2) The amplified gene fragment dEG95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into pETDuet-dEC95 treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coliDH5α competent cells were plated on agar plates containing 50 μg / ml ampicillin and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 50 μg / ml ampicillin at 37°C. Plasmids were then extracted. The recombinant plasmid pETDuet-dEC95-dEG95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0141] 2.5 Construction of the recombinant expression vector pET24a-dEG95-dEC95.

[0142] (1) Using dEG95-dEC95 as a template, an upstream primer dEG95-Nde IF and a downstream primer dEC95-XhoI-R were designed. The gene fragment dEG95-dEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 17 and 18.

[0143] Table 17: Primer Names and Sequence Information

[0144]

[0145] Table 18: PCR reaction procedure

[0146]

[0147] (2) The amplified gene fragment dEG95-dEC95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 100 μg / ml kanamycin sulfate and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. Plasmids were then extracted. The recombinant plasmid pET24a-dEG95-dEC95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0148] 2.6 Construction of the recombinant expression vector pET24a-dEC95-dEG95.

[0149] (1) Using dEC95-dEG95 as a template, an upstream primer dEC95-Nde IF and a downstream primer dEG95-XhoI-R were designed. The gene fragment dEC95-dEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 19 and 20.

[0150] Table 19: Primer Names and Sequence Information

[0151]

[0152] Table 20: PCR reaction procedure

[0153]

[0154] (2) The amplified gene fragment dEC95-dEG95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 100 μg / ml kanamycin sulfate and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. Plasmids were then extracted. The recombinant plasmid pET24a-dEC95-dEG95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0155] 2.7 Construction of the recombinant expression vector pET24a-aEG95-aEC95.

[0156] (1) Using aEG95-aEC95 as a template, an upstream primer aEG95-Nde IF and a downstream primer aEC95-XhoI-R were designed. The gene fragment aEG95-aEC95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 21 and 22.

[0157] Table 21: Primer Names and Sequence Information

[0158]

[0159] Table 22: PCR reaction procedure

[0160]

[0161] (2) The amplified gene fragment aEG95-aEC95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. E. coli DH5α competent cells were plated on agar plates containing 100 μg / ml kanamycin sulfate and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. Plasmids were then extracted. The recombinant plasmid pET24a-aEG95-aEC95 was obtained, and sequencing confirmed its consistency with the target sequence.

[0162] 2.8 Construction of the recombinant expression vector pET24a-aEC95-aEG95.

[0163] (1) Using aEC95-aEG95 as a template, an upstream primer aEC95-Nde IF and a downstream primer aEG95-XhoI-R were designed. The gene fragment aEC95-aEG95 was amplified by PCR. The 5' end of the upstream primer was introduced with a restriction endonuclease Nde I site and a protective base, wherein the Nde I site sequence is CATATG; the 5' end of the downstream primer was introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Tables 23 and 24.

[0164] Table 23: Primer Names and Sequence Information

[0165]

[0166] Table 24: PCR reaction procedure

[0167]

[0168] (2) The amplified gene fragment aEC95-aEG95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was then transformed. Figure 1DH5α competent cells were plated on plates containing 100 μg / ml kanamycin sulfate and incubated at 37°C. Once colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. Plasmids were then extracted. The recombinant plasmid pET24a-aEC95-aEG95 was obtained, and sequencing confirmed its sequence consistency with the target sequence.

[0169] Example 3: Construction of recombinant bacteria.

[0170] Convert the above pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pET24a-dEG95-dEC95, pET24a-dEC95-dEG95, pETDuet-2dEG95-2dEC95, pETDuet-2dEC95-2dEG95, pET24a-aEG95-aEC95, and pET24a-aEC95-aEG95 respectively. Figure 2 BL21(DE3) competent cells were plated on LB agar plates containing the appropriate antibiotic (50 μg / ml ampicillin or 100 μg / ml kanamycin sulfate) and incubated at 37°C. Once colonies were clearly visible, a single colony was picked and placed in 3 ml of liquid medium containing the appropriate antibiotic (50 μg / ml ampicillin or 100 μg / ml kanamycin sulfate) and incubated at 37°C. 1 ml of this bacterial culture was then added to a final concentration of 8% glycerol. The recombinant engineered bacteria Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, Duet-2dEC95-2dEG95, 24a-aEG95-aEC95, and 24a-aEC95-aEG95 were obtained by freezing at 80℃ and used as seeds for subsequent experiments.

[0171] Example 4: Expression verification of recombinant bacteria.

[0172] 4.1 Remove the recombinant bacterial strain from -80℃, thaw it, and inoculate it into 40 ml of liquid LB medium containing the corresponding antibiotic (50 μg / ml ampicillin or 100 μg / ml kanamycin sulfate). Incubate at 37℃ until OD is reached. 600 When the value reached 0.6, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 12–14 h.

[0173] 4.2 Centrifuge at 10000g for 20 minutes in a pre-weighed centrifuge tube, discard the supernatant, and collect the cells. Resuspend the precipitate in 4 ml of ice-cold 20 mM Tris-HCl pH 7.5 to obtain a 10-fold concentration (40 ml culture medium to 4 ml buffer).

[0174] 4.3 Ultrasonic treatment. Ultrasonic treatment was performed on ice using 15% power, with a 2-second working period followed by a 2-second pause, for a total duration of 15 minutes.

[0175] 4.4 Centrifuge the entire lysis buffer at 14000g for 10 minutes to separate soluble and insoluble components.

[0176] 4.5 SDS-PAGE electrophoresis analysis of the distribution of the target protein in soluble and insoluble components, such as... Figure 3 As shown.

[0177] 4.6 Results analysis showed that the recombinant proteins Duet-dEG95-dEC95, Duet-dEC95-dEG95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, and Duet-2dEC95-2dEG95 were expressed in partially soluble form and partially in inclusion body form; 24a-aEG95-aEC95 and 24a-aEC95-aEG95 were mostly expressed in inclusion body form.

[0178] Example 5: Fermentation of recombinant bacteria.

[0179] The strain was inoculated into 500 mL of LB medium containing the appropriate antibiotic (50 μg / mL ampicillin or 100 μg / mL kanamycin sulfate) and cultured with shaking at 37°C until OD was reached. 600 When the OD value is around 1.2 to 1.5, inoculate the seed culture into a 5L fermenter at an inoculation rate of 10% for fermentation culture. 600 When the pH value reaches approximately 20–25, lower the culture temperature to 28°C and simultaneously add IPTG to a final concentration of 0.4 mM, inducing for 12–14 hours. Centrifuge to collect approximately 500 g of wet bacterial cells.

[0180] Resuspend the bacterial cells by adding 10 ml of resuspension buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl) per gram of wet bacterial cells.

[0181] Example 6: Purification of recombinant protein.

[0182] 6.1 The resuspended bacterial solution was broken up using a homogenizer at a pressure of 700 bar, and the process was repeated 4 times.

[0183] 6.2 Centrifuge the lysis buffer at 28000g for 40 minutes and collect the supernatant.

[0184] 6.3 Affinity chromatography was used for purification. The protein chromatography equipment was an AKTA pure 150m protein purifier, the packing material was Ni Sepharose 6 FF, the equilibration buffer was 20mM Tris-HCl pH 7.5 and 500mM NaCl, the washing buffer was 30mM imidazole, and the elution buffer was 500mM imidazole.

[0185] 6.4 SDS-PAGE electrophoresis analysis of protein purification status, such as... Figure 4 As shown, the results indicate that the target protein was completely bound to the chromatography column and eluted by the elution buffer. The target protein was obtained through one-step purification.

[0186] 6.5 Using grayscale analysis, the amount of recombinant protein purified per liter of culture medium was calculated to be 83 mg, 57 mg, 750 mg, 541 mg, 673 mg, 607 mg, 45 mg, and 38 mg for Duet-dEG95-dEC95, 24a-dEG95-dEC95, 24a-dEC95-dEG95, Duet-2dEG95-2dEC95, Duet-2dEC95-2dEG95, 24a-aEG95-aEC95, and 24a-aEC95-aEG95, respectively. Among these, 24a-dEG95-dEC95 yielded the highest amount of purified protein.

[0187] Example 7: Immunogenicity analysis of recombinant protein.

[0188] 7.1 Preparation of immunoassay samples. The purified recombinant protein was diluted with PBS to 100 μg / ml and emulsified with sterile Montanide ISA 50V adjuvant at a volume ratio of 1:1 to prepare the samples required for immunoassay.

[0189] 7.2 Rabbit Immunization Experiment. Two New Zealand rabbits were immunized in each group using the prepared samples. The immunization and blood collection process included: 1) Collecting approximately 5 ml of blood before immunization; 2) First immunization on day 1: 1 ml of blood was administered to each rabbit; 3) Blood was collected before the second immunization to prepare serum samples; 4) Second immunization on day 15: 1 ml of blood was administered to each rabbit; 5) Blood was collected before the third immunization to prepare serum samples; 6) Third immunization on day 29: 0.5 ml of blood was administered to each rabbit; 7) Blood was collected on day 35 for ELISA detection; 8) Bloodletting of the experimental animals was performed on day 38.

[0190] 7.3 Antibody titer detection. The antibody titer of the immune serum was detected by ELISA. The results are shown in Table 25. The antibody titer of 24a-dEG95-dEC95 was significantly higher than that of other groups.

[0191] Table 25: Results of antibody titer detection by enzyme-linked immunosorbent assay (ELISA)

[0192]

[0193] Example 8: Evaluation of the cross-protective effect of antiserum.

[0194] It has been reported that there is no cross-protection effect between Echinococcus granulosus G1 and Echinococcus cantonensis G6. Therefore, this invention designs a competitive ELISA experiment. To facilitate purification, the coating antigen is tagged with MBP and the immunization antigen is tagged with 6×HIS.

[0195] 8.1 Using the antigen MBP-2dEG95 as the coating antigen, anti-6×HIS-2dEG95 serum was incubated with the following antigens: 6×HIS-dEG95-dEC95, 6×HIS-2dEG95, and 6×HIS-2dEC95, respectively. The results are as follows... ​ This indicates that the bivalent antigen 6×HIS-dEG95-dEC95 can completely block the binding of anti-6×HIS-2dEG95 serum to MBP-2dEG95 antigen, further confirming that antigen 6×HIS-dEG95-dEC95 retains the complete antigenicity of 6×HIS-2dEG95.

[0196] 8.2 Using the antigen MBP-2dEC95 as the coating antigen, anti-6×HIS-2dEC95 serum was incubated with the following antigens: 6×HIS-dEG95-dEC95, 6×HIS-2dEC95, and 6×HIS-2dEG95, respectively. The results are as follows... ​ This indicates that the bivalent antigen 6×HIS-dEG95-dEC95 can completely block the binding of anti-6×HIS-2dEC95 serum to MBP-2dEC95 antigen, further confirming that antigen 6×HIS-dEG95-dEC95 retains the complete antigenicity of 6×HIS-2dEC95.

[0197] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

[0198] sequence list

[0199] <110> Shenlian Biopharmaceutical (Shanghai) Co., Ltd.

[0200] <120> Preparation and application of Echinococcus granulosus and Echinococcus cantonensis EG95 / EC95 protein combination

[0201] <130> DD15186

[0202] <160> twenty four

[0203] <170> SIPOSequenceListing 1.0

[0204] <210> 1

[0205] <211> 471

[0206] <212> DNA

[0207] <213> Echinococcus granulosus

[0208] <400> 1

[0209] atggcattcc agttatgtct cattttgttt gcgacttcag ttttggctca ggaatacaaa60

[0210] ggaatgggcg tagagacaag gacaacagag actccgctcc gtaaacactt caatttgact120

[0211] cctgtgggtt ctcagggcat tcgcttaagt tgggaagtcc aacacttgtc tgacctcaaa180

[0212] ggaacagata tttctctaaa agcggtgaat ccttctgacc cgttagtcta caaaagacaa240

[0213] actgcaaaat tctcagatgg acaactcact atcggcgaac tgaagccctc cacattatac300

[0214] aaaatgactg tggaagcagt gaaagcgaaa aagaccattt tgggattcac cgtagacatt360

[0215] gagacaccgc gcgctggcaa gaaggaaagc actgtaatga ctagtggatc cgccttaaca420

[0216] tccgcaatcg ctggttttgt attcagctgc atagtggttg tccttacttg a471

[0217] <210>2

[0218] <211>156

[0219] <212>PRT

[0220] <213>Echinococcus granulosus

[0221] <400>2

[0222] Met Ala Phe Gln Leu Cys Leu Ile Leu Phe Ala Thr Ser Val Leu Ala 151015

[0224] Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 2025 30

[0226] Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 354045

[0228] Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 50 5560

[0230] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 65707580

[0232] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 859095

[0234] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 100105110

[0236] Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 115120125

[0238] Glu Ser Thr Val Met Thr Ser Gly Ser Ala Leu Thr Ser Ala Ile Ala 130135140

[0240] Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr 145150155

[0242] <210>3

[0243] <211>118

[0244] <212>PRT

[0245] <213>Artificial Sequence

[0246] <400>3

[0247] Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 151015

[0249] Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 202530

[0251] Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 354045

[0253] Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0255] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0257] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0259] Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100 105110

[0261] Lys Lys Glu Ser Thr Val 115

[0263] <210>4

[0264] <211>471

[0265] <212>DNA

[0266] <213>Echinococcus canadensis

[0267] <400>4

[0268] atggcattcc agttatgtct cattttgttt gcgacttcag ttttggctca ggaatacaaa60

[0269] ggaatgggca tagagacaag gacaacagag actccgctcc gcaaacactt caatttgact120

[0270] cttgtgggtt ctcagggcat tcgcttaagt tgggatgtcc aacacttgtc tgacctcaaa180

[0271] ggaacaaata ttctctaaa agcggtgaat ccttccgacc cgttagtcta caaagacaa240

[0272] actgcaaat tctcagatgg acaactcact attggtgaac tgaagccctc cacattatac300

[0273] aaaatgactg tggagcagt gaagcgaaa aagaccattt tggaattcac cgtagacatt360

[0274] gagacaccgc ccgctggcaa gaggaagc actgtaatga ctagtggatc cgccttaaca420

[0275] tccacaatcg ctggtttcgt attcagctgc atagtggttg tccttacttg a471

[0276] <210> 5

[0277] <211> 156

[0278] <212> PRT

[0279] <213> Echinococcus. canadensis

[0280] <400> 5

[0281] Gln Leu Cys Leu Ile Leu Phe Ala Thr Ser Val Leu Ala 151015

[0283] Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 202530

[0285] Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 354045

[0287] Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 505560

[0289] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 65707580

[0291] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 859095

[0293] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 100105110

[0295] Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 115120125

[0297] Glu Ser Thr Val Met Thr Ser Gly Ser Ala Leu Thr Ser Thr Ile Ala 130135140

[0299] Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr 145150155

[0301] <210>6

[0302] <211>118

[0303] <212>PRT

[0304] <213>Artificial Sequence

[0305] <400>6

[0306] Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 151015

[0308] Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 202530

[0310] Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 354045

[0312] Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0314] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0316] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0318] Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100105110

[0320] Lys Lys Glu Ser Thr Val 115

[0322] <210>7

[0323] <211>244

[0324] <212>PRT

[0325] <213>Artificial Sequence

[0326] <400>7

[0327] Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 151015

[0329] Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 202530

[0331] Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 354045

[0333] Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0335] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0337] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0339] Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100105110

[0341] Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115120125

[0343] Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 130135140

[0345] Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 145150155160

[0347] Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 165170175

[0349] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180185190

[0351] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195200205

[0353] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210215220

[0355] Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 225230235240

[0357] Glu Ser Thr Val

[0358] <210>8

[0359] <211>244

[0360] <212>PRT [[ID=​​​​​​​​​​

[0365] Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 202530

[0367] Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 354045

[0369] Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0371] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0373] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0375] Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100105110

[0377] Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115120125

[0379] Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 130135140

[0381] Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 145150155160

[0383] Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 165170175

[0385] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180185190

[0387] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195200205

[0389] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210215220

[0391] Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 225230235240

[0393] Glu Ser Thr Val

[0394] <210>9

[0395] <211>244

[0396] <212>PRT

[0397] <213>Artificial Sequence

[0398] <400>9

[0399] Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 151015

[0401] Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 202530

[0403] Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 354045

[0405] Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0407] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0409] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0411] Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100105110

[0413] Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115120125

[0415] Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 130135140

[0417] Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 145150155160

[0419] Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 165170175

[0421] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180185190

[0423] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195200205

[0425] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210215220

[0427] Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 22​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 505560

[0443] Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65707580

[0445] Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 859095

[0447] Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100105110

[0449] Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115120125

[0451] Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 130135140

[0453] Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 145150155160

[0455] Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 165170175

[0457] Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180185190

[0459] Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195200205

[0461] Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210215220

[0463] Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 225230235240

[0465] Glu Ser Thr Val

[0466] <210>11

[0467] <211>29

[0468] <212>DNA

[0469] <213>Artificial Sequence

[0470] <400>11

[0471] cgcggatccc tggcgcaaga atacaaagg29

[0472] <210>12

[0473] <211>35

[0474] <212>DNA

[0475] <213>Artificial Sequence

[0476] <400>12

[0477] cccaagcttt tagacagtag attctttttt gcctg35

[0478] <210>13

[0479] <211>31

[0480] <212>DNA

[0481] <213> Artificial Sequence

[0482] <400> 13

[0483] ggtccatatg catcaccatc atcaccacct g31

[0484] <210> 14

[0485] <211> 36

[0486] <212> DNA

[0487] <213> Artificial Sequence

[0488] <400> 14

[0489] ccgctcgagt tagacggtag attctttttt accagc36

[0490] <210> 15

[0491] <211> 29

[0492] <212> DNA

[0493] <213> Artificial Sequence

[0494] <400> 15

[0495] cgcggatccc tggcacagga atacaaagg29

[0496] <210> 16

[0497] <211> 32

[0498] <212> DNA

[0499] <213> Artificial Sequence

[0500] <400> 16

[0501] cccaagcttt tagacggtag attctttttt ac32

[0502] <210> 17

[0503] <211> 33

[0504] <212> DNA

[0505] <213> Artificial Sequence

[0506] <400> 17

[0507] ggtccatatg catcaccatc atcaccacct ggc33

[0508] <210> 18

[0509] <211> 32

[0510] <212> DNA

[0511] <213> Artificial Sequence

[0512] <400> 18

[0513] ccgctcgagt tagacagtag attctttttt gc32

[0514] <210> 19

[0515] <211> 148

[0516] <212> PRT

[0517] <213> Artificial Sequence

[0518] <400> 19

[0519] Leu Phe Ala Thr Ser Val Leu Ala Gln Glu Tyr Lys Gly Met Gly Val 151015

[0521] Glu Thr Arg Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr 202530

[0523] Pro Val Gly Ser Gln Gly Ile Arg Leu Ser Trp Glu Val Gln His Leu 354045

[0525] Ser Asp Leu Lys Gly Thr Asp Ile Ser Leu Lys Ala Val Asn Pro Ser 505560

[0527] Asp Pro Leu Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln 65707580

[0529] Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val 859095

[0531] Glu Ala Val Lys Ala Lys Lys Thr Ile Leu Gly Phe Thr Val Asp Ile 100105110

[0533] Glu Thr Pro Arg Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly 115120125

[0535] Ser Ala Leu Thr Ser Ala Ile Ala Gly Phe Val Phe Ser Cys Ile Val 130135140

[0537] Val Val Leu Thr 145

[0539] <210>20

[0540] <211>148

[0541] <212>PRT

[0542] <21​​​​​​​​​​Glu Thr Arg Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr 202530

[0548] Leu Val Gly Ser Gln Gly Ile Arg Leu Ser Trp Asp Val Gln His Leu 354045

[0550] Ser Asp Leu Lys Gly Thr Asn Ile Ser Leu Lys Ala Val Asn Pro Ser 505560

[0552] Asp Pro Leu Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln 65707580

[0554] Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val 859095

[0556] Glu Ala Val Lys Ala Lys Lys Thr Ile Leu Glu Phe Thr Val Asp Ile 100105110

[0558] Glu Thr Pro Pro Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly 115120125

[0560] Ser Ala Leu Thr Ser Thr Ile Ala Gly Phe Val Phe Ser Cys Ile Val 130135140

[0562] Val Val Leu Thr 145

[0564] <210>21

[0565] <211>33

[0566] <212> DNA

[0567] <213> Artificial Sequence

[0568] <400> twenty one

[0569] ggtccatatg catcaccatc atcaccacct gtt33

[0570] <210> twenty two

[0571] <211> 32

[0572] <212> DNA

[0573] <213> Artificial Sequence

[0574] <400> twenty two

[0575] ccgctcgagt taagtcagaa ctacaacgat gc32

[0576] <210> twenty three

[0577] <211> 31

[0578] <212> DNA

[0579] <213> Artificial Sequence

[0580] <400> twenty three

[0581] ggtccatatg catcaccatc atcaccacct g31

[0582] <210> twenty four

[0583] <211> 32

[0584] <212> DNA

[0585] <213> Artificial Sequence

[0586] <400> twenty four

[0587] ccgctcgagt taggtcagaa caacaacgat gc32 sequence list <110> Shenlian Biopharmaceutical (Shanghai) Co., Ltd. <120> Preparation and application of Echinococcus granulosus and Echinococcus cantonensis EG95 / EC95 protein combination <130> DD15186 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 471 <212> DNA <213> Echinococcus granulosus <400> 1 atggcattcc agttatgtct cattttgttt gcgacttcag ttttggctca ggaatacaaa 60 ggaatgggcg tagagacaag gacaacagag actccgctcc gtaaacactt caatttgact 120 cctgtgggtt ctcagggcat tcgcttaagt tgggaagtcc aacacttgtc tgacctcaaa 180 ggaacagata tttctctaaa agcggtgaat ccttctgacc cgttagtcta caaaagacaa 240 actgcaaaat tctcagatgg acaactcact atcggcgaac tgaagccctc cacattatac 300 aaaatgactg tggaagcagt gaaagcgaaa aagaccattt tgggattcac cgtagacatt 360 gagacaccgc gcgctggcaa gaaggaaagc actgtaatga ctagtggatc cgccttaaca 420 tccgcaatcg ctggttttgt attcagctgc atagtggttg tccttacttg a 471 <210> 2 <211> 156 <212> PRT <213> Echinococcus granulosus <400> 2 Met Ala Phe Gln Leu Cys Leu Ile Leu Phe Ala Thr Ser Val Leu Ala 1 5 10 15 Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 20 25 30 Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 35 40 45 Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 50 55 60 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 65 70 75 80 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 85 90 95 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 100 105 110 Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 115 120 125 Glu Ser Thr Val Met Thr Ser Gly Ser Ala Leu Thr Ser Ala Ile Ala 130 135 140 Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr 145 150 155 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <400> 3 Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95<(...)>0001194Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val 115 <210> 4 <211> 471 <212> DNA (注:原文中 标签后的括号内容可能有误,推测为 ,已按此翻译,如果有误请根据实际情况调整。) <213> Echinococcus. canadensis <400> 4 atggcattcc agttatgtct cattttgttt gcgacttcag ttttggctca ggaatacaaa 60 ggaatgggca tagagacaag gacaacagag actccgctcc gcaaacactt caatttgact 120 cttgtgggtt ctcagggcat tcgcttaagt tgggatgtcc aacacttgtc tgacctcaaa 180 ggaacaaata tttctctaaa agcggtgaat ccttccgacc cgttagcta caaaagacaa 240 actgcaaaat tctcagatgg acaactcact attggtgaac tgaagccctc cacattatac 300 aaaatgactg tggaagcagt gaaagcgaaa aagaccatttt tggaattcac cgtagacatt 360 gagacaccgc ccgctggcaa gaaggaaagc actgtaatga ctagtggatc cgccttaaca 420 tccacaatcg ctggtttcgt attcagctgc atagtggttg tccttacttg a 471 <210> 5 <211> 156 <212> PRT <213> Echinococcus. canadensis <400> 5 Met Ala Phe Gln Leu Cys Leu Ile Leu Phe Ala Thr Ser Val Leu Ala 1 5 10 15 Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 20 25 30 Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 35 40 45 Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 50 55 60 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 65 70 75 80 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 85 90 95 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 100 105 110 Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 115 120 125 Glu Ser Thr Val Met Thr Ser Gly Ser Ala Leu Thr Ser Thr Ile Ala 130 135 140 [[ID=3'2]]Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr 145 150 155 <210> 6 <211> 118 <212> PRT <213> Artificial Sequence <400> 6 It should be noted that there seems to be a small error in the original text where "Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr" has an incorrect tag "ID=3'2" which was corrected to "ID=32" in the translation.Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val 115 <210> 7 <211> 244 <212> PRT <213> Artificial Sequence <400> 7 Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 225 230 235 240 Glu Ser Thr Val <210> 8 <211> 244 <212> PRT <213> Artificial Sequence <400> 8 Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 225 230 235 240 Glu Ser Thr Val <210> 9 <211> 244 <212> PRT <213> Artificial Sequence <400> 9 Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 225 230 235 240 ​​​​​​​​​​​​Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 225 230 235 240 Glu Ser Thr Val <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <400> 11 cgcggatccc tggcgcaaga atacaaagg 29 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <400> 12 cccaagcttt tagacagtag attctttttt gcctg 35 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <400> 13 ggtccatatg catcaccatc atcaccacct g 31 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <400> 14 ccgctcgagt tagacggtag attctttttt accagc 36 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <400> 15 cgcggatccc tggcacagga atacaaagg 29 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <400> 16 cccaagcttt tagacggtag attctttttt ac 32 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <400> 17 ggtccatatg catcaccatc atcaccacct ggc 33 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <400> 18 ccgctcgagt tagacagtag attctttttt gc 32 <210> 19 <211> 148 <212> PRT <213> Artificial Sequence <400> 19 Leu Phe Ala Thr Ser Val Leu Ala Gln Glu Tyr Lys Gly Met Gly Val 1 5 10 15 Glu Thr Arg Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr 20 25 30 Pro Val Gly Ser Gln Gly Ile Arg Leu Ser Trp Glu Val Gln His Leu 35 40 45 Ser Asp Leu Lys Gly Thr Asp Ile Ser Leu Lys Ala Val Asn Pro Ser 50 55 60 Asp Pro Leu Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln 65 70 75 80 Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val 85 90 95 Glu Ala Val Lys Ala Lys Lys Thr Ile Leu Gly Phe Thr Val Asp Ile 100 105 110 Glu Thr Pro Arg Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly 115 120 125 Ser Ala Leu Thr Ser Ala Ile Ala Gly Phe Val Phe Ser Cys Ile Val 130 135 140 Val Val Leu Thr 145 <210> 20 <211> 148 <212> PRT <213> Artificial Sequence <400> 20 Leu Phe Ala Thr Ser Val Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile 1 5 10 15 Glu Thr Arg Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr 20 25 30 Leu Val Gly Ser Gln Gly Ile Arg Leu Ser Trp Asp Val Gln His Leu 35 40 45 Ser Asp Leu Lys Gly Thr Asn Ile Ser Leu Lys Ala Val Asn Pro Ser 50 55 60 Asp Pro Leu Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln 65 70 75 80 Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val 85 90 95 Glu Ala Val Lys Ala Lys Lys Thr Ile Leu Glu Phe Thr Val Asp Ile 100 105 110 Glu Thr Pro Pro Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly<www.patentguru.com>115 120 125 Ser Ala Leu Thr Ser Thr Ile Ala Gly Phe Val Phe Ser Cys Ile Val 130 135 140 Val Val Leu Thr 145 <210> twenty one <211> 33 <212> DNA <213> Artificial Sequence <400> twenty one ggtccatatg catcaccatc atcaccacct gtt 33 <210> twenty two <211> 32 <212> DNA <213> Artificial Sequence <400> twenty two ccgctcgagt taagtcagaa ctacaacgat gc 32 <210> twenty three <211> 31 <212> DNA <213> Artificial Sequence <400> twenty three ggtccatatg catcaccatc atcaccacct g 31 <210> twenty four <211> 32 <212> DNA <213> Artificial Sequence <400> twenty four ccgctcgagt taggtcagaa caacaacgat gc 32

Claims

1. A fusion protein, characterized in that, dEG95 and dEC95 are linked together by a linker to form recombinant proteins dEG95-dEC95 or dEC95-dEG95. The amino acid sequence of dEG95 is the modified amino acid sequence obtained by deleting the N-terminal signal peptide region and the C-terminal transmembrane region of EG95 from Echinococcus granulosus; the EG95 gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the amino acid sequence of dEG95 is shown in SEQ ID NO.

3. The amino acid sequence of dEC95 is the modified amino acid sequence obtained by deleting the N-terminal signal peptide region and the C-terminal transmembrane region of EC95 from Echinococcus caninus; the EC95 gene sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5; the amino acid sequence of dEC95 is shown in SEQ ID NO.

6. The amino acid sequence of dEG95-dEC95 is shown in SEQ ID NO.9; The amino acid sequence of dEC95-dEG95 is shown in SEQ ID NO.

10.

2. A method for preparing the fusion protein as described in claim 1, characterized in that, Includes the following steps: S1. The gene encoding the dEG95-dEC95 amino acid sequence or the dEC95-dEG95 amino acid sequence is cloned into a prokaryotic expression vector to obtain a recombinant expression plasmid. S2. Transfect prokaryotic expression strains with recombinant expression plasmids, screen single clones, ferment and culture, and induce expression to obtain the recombinant protein.

3. The method for preparing the fusion protein as described in claim 2, characterized in that, Step S1 specifically includes the following steps: S11. Gene sequences of dEG95-dEC95 or dEC95-dEG95, respectively, optimized with gene synthesis codons and suitable for expression in E. coli. S12. The dEG95-dEC95 or dEC95-dEG95 gene is cloned into the pET24a plasmid vector to construct the pET24a-dEG95-dEC95 or pET24a-dEC95-dEG95 plasmid.

4. The method for preparing the fusion protein as described in claim 3, characterized in that, In step S2, the pET24a-dEG95-dEC95 or pET24a-dEC95-dEG95 recombinant expression plasmid is transformed into Escherichia coli BL21(DE3).

5. A recombinant expression plasmid comprising the encoding gene of the fusion protein as described in claim 1.

6. The recombinant expression plasmid according to claim 5, characterized in that, The recombinant expression plasmid is pET24a-dEG95-dEC95 or pET24a-dEC95-dEG95.

7. A recombinant engineered Escherichia coli strain, characterized in that, The engineered bacteria were obtained by transforming the recombinant expression plasmid as described in claim 6 into E.coli BL21(DE3) competent cells.

8. A recombinant engineered Escherichia coli strain, characterized in that, The engineered bacteria is *Escherichia coli* 24a-dEG95-dEC95, with accession number CCTCC NO: M2021750.

9. A protein composition, characterized in that, It includes two proteins: dEG95 and dEC95; Alternatively, it may include two dimer proteins, 2dEG95 and 2dEC95, wherein the 2dEG95 dimer is formed by two dEG95 proteins linked together by a linker, and the 2dEC95 dimer is formed by two dEC95 proteins linked together by a linker. The amino acid sequence of dEG95 is that of Echinococcus granulosus. EG95 The modified amino acid sequence was obtained by deleting the N-terminal signal peptide region and the C-terminal transmembrane region; EG95 The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the amino acid sequence of dEG95 is shown in SEQ ID NO.3; The amino acid sequence of dEC95 is the modified amino acid sequence obtained by deleting the N-terminal signal peptide region and the C-terminal transmembrane region of EC95 from Echinococcus caninus; the EC95 gene sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5; the amino acid sequence of dEC95 is shown in SEQ ID NO.

6. The amino acid sequence of the 2dEG95 is shown in SEQ ID NO.7; the amino acid sequence of the 2dEC95 is shown in SEQ ID NO.

8.

10. A method for preparing the protein composition as described in claim 9, characterized in that, The method includes the following steps: A1. The gene encoding the dEG95 amino acid sequence, dEC95 amino acid sequence, 2dEG95 amino acid sequence, or 2dEC95 amino acid sequence is cloned into a prokaryotic expression vector to obtain a recombinant expression plasmid. A2. Transfect prokaryotic expression strains with recombinant expression plasmids, screen single clones, ferment and culture, and induce expression to obtain the recombinant protein.

11. The method for preparing the protein composition according to claim 10, characterized in that, Step A1 specifically includes the following steps: A11. dEG95 and dEC95 gene sequences optimized for expression in E. coli, respectively, using gene synthesis codons. Alternatively, the 2dEG95 and 2dEC95 gene sequences, respectively, were optimized with gene synthesis codons to be suitable for expression in E. coli; A12. The dEG95 and dEC95 genes were cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-dEG95-dEC95 or pETDuet-dEC95-dEG95 plasmids. Alternatively, the 2dEG95 and 2dEC95 genes can be cloned into the pETDuet™-1 plasmid vector in different orders to construct the pETDuet-2dEG95-2dEC95 or pETDuet-2dEC95-2dEG95 plasmids.

12. The method for preparing the protein composition according to claim 11, characterized in that, In step A2, the recombinant expression plasmids pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pETDuet-2dEG95-2dEC95, or pETDuet-2dEC95-2dEG95 are transformed into Escherichia coli BL21(DE3).

13. A recombinant expression plasmid comprising the encoding gene of the protein composition as described in claim 9.

14. The recombinant expression plasmid according to claim 13, characterized in that, The recombinant expression plasmid is pETDuet-dEG95-dEC95, pETDuet-dEC95-dEG95, pETDuet-2dEG95-2dEC95, or pETDuet-2dEC95-2dEG95.

15. A recombinant engineered Escherichia coli strain, characterized in that, The engineered bacteria are transformed from the recombinant expression plasmid as described in claim 14. E. coli It is obtained from BL21 (DE3) competent cells.

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