Preparation and application of EC95 protein of canadian echinococcus

Abstract

The application discloses preparation and application of a Canadian echinococcus EC95 protein; the recombinant protein comprises a dEC95 amino acid sequence; 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 an EC95 of Canadian echinococcus; the EC95 gene sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. The application modifies the EC95 in the Canadian echinococcus, and prepares a recombinant expression plasmid and a recombinant E. coli engineering bacteria; the fusion protein expressed by the recombinant E. coli engineering bacteria has the advantages of high purity, good solubility, convenient purification and high biological activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation and application of Echinococcus canadensis EC95 protein; in particular to the modification method of Echinococcus canadensis EC95 protein sequence, and the preparation of recombinant expression plasmid, recombinant E. coli engineering bacteria, and the induction expression method of E. coli engineering bacteria using the modified protein sequence. BACKGROUND

[0002] Echinococcosis is a serious parasitic disease caused by the larvae of Echinococcus tapeworm, hydatid cyst, parasitizing in the lung, 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-animal zoonosis. The World Health Organization (WHO) lists echinococcosis as one of the diseases for which the global early warning system prioritizes 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 morphology of the lesion and the difference in the infecting pathogen, echinococcosis is mainly divided into cystic echinococcosis (CE) and multilocular echinococcosis. CE is the most widely distributed and has the largest number of patients. The pathogen of CE is currently composed of several Echinococcus granulosus complex species: Echinococcus granulosus, Echinococcus canadensis, Echinococcus equinus, and Echinococcus oligarthrus. CE caused by Echinococcus granulosus G1 type accounts for more than 90%, and CE caused by Echinococcus canadensis G6 type accounts for more than 7%.

[0004] Current research indicates that controlling the prevalence of echinococcosis primarily involves interrupting the developmental stages of Echinococcus tapeworm, controlling infection of intermediate hosts such as humans and animals, preventing or treating definitive hosts like dogs, and blocking the widespread dissemination of eggs. Vaccination of intermediate hosts is particularly effective in controlling the prevalence of Echinococcus granulosus. Lightowlers et al. discovered that EG95 is one of the naturally occurring oncocytosial antigens found in E. coli and is the most effective protective antigen among the many proteins screened. A vaccine against Echinococcus granulosus in sheep has already been successfully developed. However, existing recombinant EG95 protein vaccines are prepared by refolding inclusion bodies from E. coli, resulting in low purity and difficulty in maintaining the protein's spatial structure. Furthermore, they lack significant cross-protection against Echinococcus cantonensis G6, thus presenting certain limitations. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the purpose of this invention is to provide a preparation and application of Echinococcus cantonensis EC95 protein; specifically, it involves modifying EC95 in Echinococcus cantonensis and preparing a recombinant expression plasmid, a recombinant Escherichia coli engineered bacterium, and a fusion protein expressed by this recombinant Escherichia coli engineered bacterium, which has the advantages of high purity, good solubility, convenient purification, and high biological activity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention relates to a recombinant protein of Echinococcus caninus EC95, said recombinant protein comprising the dEC95 amino acid sequence;

[0008] The dEC95 amino acid sequence is the modified amino acid sequence of EC95 of Echinococcus caninus, obtained by deleting the N-terminal signal peptide region and the C-terminal transmembrane region; the EC95 gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] 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.

[0010] 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.3.

[0011] Secondly, the present invention relates to a method for preparing a recombinant protein of Echinococcus canis EC95, the method comprising the following steps:

[0012] S1. The coding gene of dEC95 was cloned into a prokaryotic expression vector to obtain a recombinant expression plasmid;

[0013] S2. Transfect prokaryotic expression strains with recombinant expression plasmids, screen single clones, ferment and culture, and induce expression to obtain the recombinant protein.

[0014] As one embodiment of the present invention, step S1 specifically includes the following steps:

[0015] A1. The dEC95 gene sequence optimized for expression in Escherichia coli using gene synthesis codons; the amino acid sequence of dEC95 is shown in SEQ ID NO.3;

[0016] Alternatively, two dEC95 amino acid sequences can be linked together via a linker to form a 2dEC95 amino acid sequence; the gene synthesis codon optimized for expression in E. coli;

[0017] The recombinant protein combination of this invention is original; and because the protein structure of EC95 has not been resolved, its functional study is quite difficult. Different truncations can cause coordination problems between the two monomers in the dimer, easily leading to misfolding or loss of activity of the heterodimer. This invention predicts the 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.

[0018] A2. Clone the dEC95 or 2dEC95 gene into the pET24a plasmid vector to construct the pET24a-dEC95 or pET24a-2dEC95 plasmid.

[0019] In one embodiment of the present invention, in step A2, the two modified EC95 amino acid sequences are linked using a linker rich in glycine and serine. Preferably, the two modified EC95 amino acid sequences are linked using GGGSGGGS, and the 2dEC95 amino acid sequence is shown in SEQ ID NO.4.

[0020] In one embodiment of the present invention, in step S2, pET24a-dEC95 and pET24a-2dEC95 are transformed into Escherichia coli, respectively. After screening for single clones, the corresponding Escherichia coli strains are named 24a-dEC95 and 24a-2dEC95, respectively.

[0021] Furthermore, the *E. coli* strain 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.

[0022] As one embodiment of the present invention, step S2 specifically includes the following steps:

[0023] B1, pET24a-dEC95, and pET24a-2dEC95 were transformed into E. coli, and single clones were screened to obtain E. coli 24a-dEC95 and 24a-2dEC95.

[0024] B2. Strains 24a-dEC95 and 24a-2dEC95 were inoculated into 500 mL of LB medium containing kanamycin sulfate and cultured with shaking at 37°C 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. The recombinant protein was then induced, expressed, and purified.

[0025] Furthermore, in step B2, protein purification is performed 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.

[0026] Thirdly, the present invention relates to a recombinant expression plasmid comprising a coding gene of the recombinant protein combination.

[0027] As one embodiment of the present invention, the recombinant expression plasmid is pET24a-dEC95 or pET24a-2dEC95 plasmid.

[0028] Among the recombinant expression plasmids mentioned above, the pET24a-dEC95 plasmid was obtained by PCR amplification of the gene fragment dEC95 using dEC95 as a template through upstream primer Nde IF and downstream primer Xho IR; it was then ligated into the pET24a prokaryotic expression vector, the ligation product was transformed into E. coli DH5α competent cells, the plasmid was extracted, and the result was verified by sequencing.

[0029] The pET24a-2dEC95 plasmid was obtained by PCR amplification of the gene fragment 2dEC95 using 2dEC95 as a template, through upstream primer Nde IF and downstream primer Xho IR; ligated into the pET24a prokaryotic expression vector; the ligation product was transformed into E. coli DH5α competent cells; the plasmid was extracted and verified by sequencing.

[0030] Fourthly, the present invention relates to a recombinant engineered Escherichia coli strain obtained by transforming E. coli with the aforementioned recombinant expression plasmid. In some embodiments, it is obtained by transforming E. coli BL21(DE3) competent cells.

[0031] The engineered bacterium is 24a-dEC95 or 24a-2dEC95. In step B2, both strains 24a-dEC95 and 24a-2dEC95 can express the EC95 protein; preferably, strain 24a-2dEC95 will be used. Therefore, this invention also relates to a recombinant engineered Escherichia coli strain, namely Escherichia coli 24a-2dEC95, with accession number CCTCC NO: M2021749.

[0032] Fifthly, the present invention relates to the use of the aforementioned recombinant Echinococcus rubescens EC95 protein in the preparation of a medicament for treating echinococcosis infection.

[0033] The drugs and / or vaccines are administered to sheep, cattle, and camels.

[0034] In this invention, Escherichia coli 24a-2dEC95 was deposited with the China Center for Type Culture Collection on June 23, 2021, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2021749.

[0035] The present invention has the following beneficial effects:

[0036] This invention proposes a method for preparing Echinococcus canis EC95 protein, which has the advantages of high protein expression level, simple process and low production cost. Attached Figure Description

[0037] 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:

[0038] Figure 1SDS-PAGE images were generated for expression validation. Lane M represents the standard molecular weight of the protein. Lane 1: Uninduced whole bacterial sample (24a-dEC95); Lane 2: Induced whole bacterial sample (24a-dEC95); Lane 3: Precipitated sample (24a-dEC95); Lane 4: Supernatant (24a-dEC95); Lane 5: Precipitated sample (24a-2dEC95); Lane 6: Supernatant (24a-2dEC95); Lane 7: Induced whole bacterial sample (24a-2dEC95); Lane 8: Uninduced whole bacterial sample (24a-2dEC95).

[0039] Figure 2 The results are for protein purification; lane M: protein standard molecular weight; lane 1: protein sample 2dEC95; lane 2: protein sample dEC95. Detailed Implementation

[0040] 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.

[0041] The present invention adopts the following technical solution:

[0042] S1: The EC95 gene sequence of Echinococcus caninata 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 dEC95.

[0043] S2: Two dEC95 amino acid sequences are linked together by a linker to form a 2dEC95 amino acid sequence;

[0044] S3: Two gene synthesis codons optimized for expression in E. coli: dEC95 and 2dEC95 sequences;

[0045] S4: The dEC95 gene and 2dEC95 were cloned into the pET24a plasmid vector, respectively, to construct the pET24a-dEC95 and pET24a-2dEC95 plasmids.

[0046] S5: pET24a-dEC95 and pET24a-2dEC95 were transformed into Escherichia coli, and after screening for single clones, the corresponding E. coli were named 24a-dEC95 and 24a-2dEC95, respectively.

[0047] S6: Strains 24a-dEC95 and 24a-2dEC95 were inoculated into 500 mL of LB medium containing kanamycin sulfate and cultured with shaking at 37°C 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. The recombinant protein was then induced, expressed, and purified.

[0048] In step S1 above, 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 EC95 amino acid sequence is shown in SEQ ID NO.3.

[0049] In step S2 above, the two modified EC95 amino acid sequences are linked by a linker rich in glycine and serine; preferably, the two modified EC95 amino acid sequences are linked by GGGSGGGS, and the amino acid sequences are shown in SEQ ID NO.4.

[0050] The Escherichia coli used in step S5 above 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.

[0051] In step S6 above, both strains 24a-dEC95 and 24a-2dEC95 can express the EC95 protein; preferably, strain 24a-2dEC95, strain preservation number CCTCC M 2021749, will be used.

[0052] In step S6 above, protein purification is performed 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.

[0053] See the following examples for details:

[0054] Example 1: Optimization and synthesis of gene sequences.

[0055] Using *E. coli* as the host bacterium, this invention optimized the codons encoding the recombinant proteins dEC95 and 2dEC95. The optimized sequences were synthesized by Nanjing GenScript Biotech Co., Ltd. Specifically, 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. The two modified EC95 amino acid sequences were linked together using the "GGGSGGGS" formula to construct the single-stranded homodimer 2dEC95.

[0056] Simultaneously, using *E. coli* as the host bacterium, this invention optimized the codons encoding the recombinant protein aEC95. The optimized sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. The aEC95 amino acid sequence is an N-terminal truncated 8 amino acids from the EC95 protein amino acid sequence, as shown in SEQ ID NO.7. Two modified EC95 amino acid sequences, aEC95, were linked together using the "GGGSGGGS" formula to construct the single-stranded homodimer 2aEC95.

[0057] Example 2: Construction of recombinant expression vector.

[0058] 2.1 Construction of the recombinant expression vector pET24a-dEC95.

[0059] (1) Using dEC95 as a template, upstream primer Nde IF and downstream primer 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 Table 1 and Table 2.

[0060] Table 1: PCR primer names and sequences

[0061]

[0062] Table 2: PCR reaction procedure

[0063]

[0064] (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 the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was transformed into E. coli DH5α competent cells and plated on plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and cultured in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. The plasmid was then extracted. The recombinant plasmid pET24a-dEC95 was obtained, and the recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0065] 2.2 Construction of the recombinant expression vector pET24a-2dEC95.

[0066] (1) Using 2dEC95 as a template, upstream primer Nde IF and downstream primer Xho IR were designed. PCR amplification yielded the gene fragment 2dEC95. The 5' end of the upstream primer contained a restriction endonuclease Nde I site and a protective base, with the Nde I site sequence being CATATG. The 5' end of the downstream primer contained a restriction endonuclease Xho I site, a stop codon, and a protective base, with the Xho I site sequence being CTCGAG. Primer sequences and PCR reaction procedures are shown in Tables 3 and 4.

[0067] Table 3: PCR primer names and sequences

[0068] Primer name Sequence Nde I-F 5'-GGTCCATATGCATCACCATCATCACCACGACCTG-3' SEQ ID NO. 5 Xho I-R 5'-CCGCTCGAGTTA GACGGTAGATTCTTTTTTACCAGC-3' SEQ ID NO. 6

[0069] Table 4: PCR reaction procedure

[0070]

[0071] (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 the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was transformed into E. coli DH5α competent cells and plated on plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and cultured in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. The plasmid was then extracted. The recombinant plasmid pET24a-2dEC95 was obtained. The recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0072] 2.3 Construction of the recombinant expression vector pET24a-aEC95.

[0073] (1) Using aEC95 as a template, upstream primer a-Nde IF and downstream primer a-Xho IR were designed. The gene fragment 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 5 and 6.

[0074] Table 5: PCR primer names and sequences

[0075] Primer name Sequence a-Nde I-F 5'-GGTCCATATGCATCACCATCATCACCACCTGTTC-3' SEQ ID NO. 8 a-Xho I-R 5'-CCGCTCGAGTTA AGTCAGAACTACAACGATGC-3' SEQ ID NO. 9

[0076] Table 6: PCR reaction procedure

[0077]

[0078] (2) The amplified gene fragment 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 transformed into E. coli DH5α competent cells and plated on plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and cultured in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. The plasmid was then extracted. The recombinant plasmid pET24a-aEC95 was obtained, and the recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0079] 2.4 Construction of the recombinant expression vector pET24a-2aEC95.

[0080] (1) Using 2aEC95 as a template, upstream primer a-Nde IF and downstream primer a-Xho IR were designed. PCR amplification yielded the gene fragment 2aEC95. The upstream primer incorporated a restriction endonuclease Nde I site and a protective base at its 5' end, with the NdeI site sequence being CATATG. The downstream primer incorporated a restriction endonuclease Xho I site, a stop codon, and a protective base at its 5' end, with the Xho I site sequence being CTCGAG. Primer sequences and PCR reaction procedures are shown in Tables 7 and 8.

[0081] Table 7: PCR primer names and sequences

[0082] Primer name Sequence a-Nde I-F 5'-GGTCCATATGCATCACCATCATCACCACCTGTTC-3' SEQ ID NO. 8 a-Xho I-R 5'-CCGCTCGAGTTA AGTCAGAACTACAACGATGC-3' SEQ ID NO. 9

[0083] Table 8: PCR reaction procedure

[0084]

[0085] (2) The amplified gene fragment 2aEC95 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 transformed into E. coli DH5α competent cells and plated on plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and cultured in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. The plasmid was then extracted. The recombinant plasmid pET24a-2aEC95 was obtained. The recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0086] Example 3: Construction of recombinant bacteria.

[0087] The pET24a-dEC95, pET24a-2dEC95, pET24a-aEC95, and pET24a-2aEC95 strains were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 100 μg / ml kanamycin sulfate, and incubated at 37°C. When colonies were clearly visible on the plates, single colonies were picked and placed in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate, and incubated at 37°C. 1 ml of the bacterial culture was then added to a final concentration of 8% glycerol and frozen at -80°C to obtain recombinant engineered bacteria 24a-dEC95, 24a-2dEC95, 24a-aEC95, and 24a-2aEC95, which were used as seed cultures for subsequent experiments.

[0088] Example 4: Expression verification of recombinant bacteria.

[0089] 4.1 Remove the recombinant bacterial strain from -80℃, thaw it, and inoculate it into 40 ml of liquid LB medium containing 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.

[0090] 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).

[0091] 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.

[0092] 4.4 Centrifuge the entire lysis buffer at 14000g for 10 minutes to separate soluble and insoluble components.

[0093] 4.5 SDS-PAGE electrophoresis analysis of the distribution of the target protein in soluble and insoluble components, such as... Figure 1 As shown.

[0094] 4.6 Results analysis showed that the recombinant proteins 24a-dEC95 and 24a-2dEC95 were expressed in partially soluble form and partially in inclusion body form; pET24a-aEC95 and pET24a-2aEC95 were mostly expressed in inclusion body form.

[0095] Example 5: Fermentation of recombinant bacteria.

[0096] The strain was inoculated into 500 mL of LB medium containing 100 μg / mL kanamycin sulfate and cultured with shaking at 37°C until OD reached. 600 When the OD value is 1.2–1.5, inoculate the seed culture into a 5L fermenter at a 10% inoculation rate for fermentation culture. When the cell OD value reaches 1.2–1.5, the fermentation culture is carried out. 600 When the pH value reaches 20–25, lower the culture temperature to 28°C and add IPTG to a final concentration of 0.4 mM, inducing for 12–14 hours. Centrifuge to collect approximately 500 g of wet cell weight.

[0097] 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.

[0098] Example 6: Purification of recombinant protein.

[0099] 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.

[0100] 6.2 Centrifuge 28000g of lysis buffer for 40 minutes and collect the supernatant.

[0101] 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 6FF. 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.

[0102] 6.4 SDS-PAGE electrophoresis analysis of protein purification status, such as... Figure 2 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 obtained by one-step purification had a purity greater than 80% and a concentration of approximately 1 mg / ml.

[0103] Example 7: Immunogenicity analysis of recombinant protein.

[0104] 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.

[0105] 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 from 5-30 ml of rabbits on day 35 for ELISA detection; 8) Bloodletting of the experimental animals was performed on day 38.

[0106] 7.3 Antibody titer detection. The antibody titer of the immune serum was detected by ELISA. The results are shown in Table 9. The results indicate that the recombinant protein has good immunogenicity. The antibody titer produced by 2dEC95 protein is higher than that produced by dEC95 protein, indicating that the single-chain dimer form can improve the immunogenicity of the protein.

[0107] Table 9: Antibody titer test results.

[0108]

[0109]

[0110] 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. sequence list <110> Shenlian Biopharmaceutical (Shanghai) Co., Ltd. <120> Preparation and application of Echinococcus canis EC95 protein <130> DD15188 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 471 <212> DNA <213> Echinococcus. canadensis <400> 1 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> 2 <211> 156 <212> PRT <213> Echinococcus. canadensis <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 Ile Glu Thr Arg Thr Thr Glu Thr Pro 20 25 30 [[IDID=4]]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 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 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> 4 <211> 244 <212> PRT <213> Artificial Sequence <400> 4 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> 5 <211> 34 <212> DNA <213> Artificial Sequence <400> 5 ggtccatatg catcaccatc atcaccacct ggca 34 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <400> 6 ccgctcgagt tagacggtag attctttttt accagc 36 <210> 7 <211> 148 <212> PRT <213> Artificial Sequence <400> 7 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 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> 8 <211> 34 <212> DNA <213> Artificial Sequence <400> 8 ggtccatatg catcaccatc atcaccacct gttc 34 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <400> 9 ccgctcgagt taagtcagaa ctacaacgat gc 32

Claims

1. A recombinant protein from Echinococcus canis EC95, characterized in that, The recombinant protein is dEC95 with an amino acid sequence as shown in SEQ ID NO.3 or 2dEC95 with an amino acid sequence as shown in SEQ ID NO.4; The dEC95 amino acid sequence 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.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. A method for preparing the recombinant protein of Echinococcus canis EC95 as described in claim 1, characterized in that, The method includes the following steps: S1. The coding gene of dEC95 was 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 recombinant Echinococcus canis EC95 protein according to claim 2, characterized in that, Step S1 specifically includes the following steps: A1. The dEC95 gene sequence optimized for expression in Escherichia coli using gene synthesis codons; the amino acid sequence of dEC95 is shown in SEQ ID NO.3; Alternatively, two dEC95 amino acid sequences can be linked together via a linker to form a 2dEC95 amino acid sequence; the gene synthesis codon optimized for expression in E. coli; A2. Clone the dEC95 or 2dEC95 gene into the pET24a plasmid vector to construct the pET24a-dEC95 or pET24a-2dEC95 plasmid.

4. A recombinant expression plasmid comprising the encoding gene of the recombinant protein of Echinococcus canis EC95 as described in claim 1.

5. A recombinant engineered Escherichia coli strain, characterized in that, The engineered bacteria are transformed from the recombinant expression plasmid as described in claim 4. E. coli And thus.

6. A recombinant engineered Escherichia coli strain, characterized in that, The engineered bacteria is Escherichia coli. Escherichia coli 24a-2dEC95, accession number CCTCC NO: M2021749.

7. Use of the recombinant Echinococcus rubescens EC95 protein as described in claim 1 in the preparation of a pharmaceutical agent for treating echinococcosis infection.

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