CN113372452A

CN113372452A - Echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162 and application thereof

Info

Publication number: CN113372452A
Application number: CN202110637671.9A
Authority: CN
Inventors: 周晓涛; 丁剑冰; 孔慧芳; 曹春宝; 李玉娇
Original assignee: Xinjiang Medical University
Current assignee: Xinjiang Medical University
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2021-09-10
Anticipated expiration: 2041-06-08
Also published as: CN113372452B

Abstract

The invention discloses an echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162, and an immune experiment is carried out on a mouse, and the result shows that the recombinant protein has a better immune protection effect on the mouse, can generate a stable antibody and has the potential of developing an echinococcus granulosus vaccine.

Description

Echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162 and application thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to an echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162 and application thereof.

Background

Echinococcosis, also known as echinococcosis, is widely distributed around the world, is mainly prevalent in livestock areas, and is a serious zoonosis caused by echinococcosis larvae of echinococcus granulosus parasitizing in human body and some organs such as liver and lung of animals. The serious injury of the disease to the patients such as the farmers and the herdsmen is one of the main factors of the farmers and the herdsmen caused by the disease and the return to poverty due to the disease. The echinococcosis cystectomy is the first choice of treatment for echinococcosis patients at present; however, the injury caused by the operation to the human body is large and the recurrence is possible, and the chemotherapy drugs can generate some serious adverse reactions. Therefore, there is a need to develop more effective solutions, and vaccine prevention is an effective measure to prevent its prevalence.

The egG1Y162 antigen gene is cloned from echinococcus granulosus by the subgroup Caochidion and the like. The results show that the egG1Y162 gene, either in gene sequence or in amino acid sequence and protein structure, has high similarity to the candidate vaccine emG1Y162 developed by foreign researchers for protection of the terminal host, and is likely to be a protective antigen of the terminal host (see patent CN 101475938A). In addition, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is mainly expressed on activated T lymphocytes, and the monomer molecule consists of three parts, namely an Ig V extracellular region, a transmembrane region and a cytoplasmic tail region. The Ig V extracellular region can be efficiently combined with B7 molecules on an anti-presenting cell, and the immunogenicity of the recombinant vaccine can be enhanced by promoting the recognition and presentation of a fusion antigen by an antigen presenting cell, so that strong humoral and cellular immune responses are induced. How to improve the immunogenicity and antigen presentation effect of echinococcus granulosus egG1Y162 gene by using the Ig V extracellular region of host CTLA-4, thereby finally improving the prevention effect of the vaccine is a pending problem in the field.

Disclosure of Invention

The invention aims to provide a recombinant protein which has a good immune protection effect on mice, can generate stable antibodies and can be used as a candidate vaccine against echinococcus granulosus;

the invention also aims to provide the application of the recombinant protein or the coding sequence thereof in preparing a vaccine for preventing Echinococcus granulosus;

another object of the present invention is to provide a vaccine for preventing Echinococcus granulosus, which is prepared using the above recombinant protein.

The invention is realized by the following technical scheme:

an Echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162, which comprises Echinococcus granulosus protein EgG1Y162 and cytotoxic T lymphocyte-associated antigen 4 extracellular domain protein CTLA 4-IgV.

The amino acid sequence of the recombinant protein is shown in a sequence table SEQ ID NO. 1.

The nucleotide sequence of the recombinant protein is shown in a sequence table SEQ ID NO. 2.

The obtained recombinant protein is adopted to immunize mice, so that a higher immune protection effect is obtained.

The recombinant protein is used for immunizing mice, and experimental animals adopt healthy female Balb/C mice which are randomly divided into 4 groups: group A: saline NS placebo (n-20); group B: adjuvant control group (n ═ 20); group C: EgG1Y162 group (n ═ 20); group D: CTLA4-IgV-EgG1Y162 group (n ═ 20); A. b, C, D group is administered by back subcutaneous multiple injection of recombinant NS, adjuvant, EgG1Y162 and CTLA4-IgV-EgG1Y162 at dose of 100 ug/mouse/dose for 3 times, each time with 2 weeks interval, and collecting blood serum adjacent to the mice before each immunization. One week after the 3 rd immunization, each mouse was inoculated with about 1000 secondary infections of Echinococcus granulosus metacercaria. At 70 days after infection, blood is collected, mice are sacrificed and subjected to autopsy, and various immune index detections are carried out, wherein the immune index detections comprise vesicle wet weight inhibition rate, vesicle number and gross changes, ELISA detects specific antibody level changes and cytokine changes in mouse serum, and the immune protection effect is evaluated through statistical analysis of results. Compared with the control group, the group D remarkably improves the resistance of the mice to the Echinococcus granulosus, and the inhibition rate reaches 68.5 percent (P < 0.01).

Based on the above, the invention provides the application of the recombinant protein or the coding sequence thereof in preparing the vaccine for preventing the Echinococcus granulosus.

Meanwhile, according to the application and the specific test result of the invention, the invention also provides a vaccine for preventing Echinococcus granulosus, which comprises the recombinant protein and the immunologic adjuvant.

The invention has the advantages and beneficial effects that:

the echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162 is prepared, and an immune experiment is performed on a mouse, and the result shows that the recombinant protein has a good immune protection effect on the mouse, can generate a stable antibody, and has the potential of developing an echinococcus granulosus vaccine.

Drawings

FIG. 1 shows the construction and identification results of prokaryotic expression plasmid pET30a-CTLA-4 IgV; a: map of Con A-stimulated lymphocytes (200 ×); b: total RNA electrophoretogram; c: performing gel electrophoresis on the PCR product; d: after the constructed CTLA-4IgV gene segment pMD19-T is transformed into competent cells DH5a, positive clones are screened by blue white spots; e plasmid BamHI and Kpn I double enzyme cutting, gel electrophoresis.

FIG. 2 shows the result of multiple sequence alignment of the CTLA-4IgV protein sequence of Xinjiang sheep using ClustalX software.

FIG. 3 shows the identification result of recombinant plasmid pET30a-CTLA-4 IgV; a, gel electrophoresis after double enzyme digestion of pET30a, M: DNA maker5000(bp), 1: the target band after plasmid digestion; b: pMD19-T-CTLA-4IgV double-enzyme digestion electrophoresis, M: DNA maker5000(bp), 1: the target band after plasmid digestion; c: plasmid PCR gel electrophoresis, M: DNA maker5000(bp) 1: PCR band of interest.

FIG. 4 shows the expression and identification of protein CTLA-4 IgV; analyzing the result of the recombinant protein CTLA-4IgV in the supernatant by SDS-PAGE; a, analyzing the result of the recombinant protein CTLA-4IgV in the precipitate by SDS-PAGE; the molecular weight standard of the M protein is 1-4, respectively, pET30a-CTLA-4IgV is induced for 0h at the concentration of 0.2mmol/L IPTG, 4h at the temperature of 28 ℃, 2h at the temperature of 28 ℃ plus 2h at the temperature of 37 ℃ and 4h at the temperature of 37 ℃. 5-8 respectively pET30a-CTLA-4IgV is induced for 0h at 28 ℃ for 4h, 2h at 28 ℃ for 2h, 37 ℃ for 2h and 4h at 37 ℃ at IPTG concentration of 0.5 mmol/L. 9-12 respectively pET30a-CTLA-4IgV is induced for 0h at the concentration of 0.8mmol/L of IPTG, 4h at 28 ℃, 2h at 37 ℃ and 4h at 37 ℃. C: western blot analysis of CTLA-4IgV recombinant protein: m: protein molecular mass standard; 1-2: CTLA-4IgV proteins.

FIG. 5 is a structural display diagram of the recombinant protein CTLA4-IgV-EgG1Y 162.

FIG. 6 shows the result of SDS-PAGE analyzing the expression of the recombinant protein CTLA-4IgV-EgG1Y 162; a: expressing the recombinant protein CTLA-4IgV-EgG1Y162 in supernatant; b: the expression of the recombinant protein CTLA-4IgV-EgG1Y162 in the precipitate and the molecular mass standard of M protein are 1-4 respectively, namely pET30a-CTLA-4IgV-EgG1Y162, and the expression is induced for 0h at the concentration of 0.2mmol/L, 4h at 28 ℃, 4h at 37 ℃ and 2h at 28 ℃ plus 2h at 37 ℃. 5-8 respectively pET30a-CTLA-4IgV-EgG1Y162 at IPTG concentration of 0.5mmol/L for 0h, 4h at 28 ℃, 4h at 37 ℃, 2h at 28 ℃ and 2h at 37 ℃. 9-12 respectively pET30a-CTLA-4IgV-EgG1Y162 at IPTG concentration of 0.8mmol/L for induction of 0h, 4h at 28 ℃, 4h at 37 ℃, 2h at 28 ℃ and 2h at 37 ℃.

FIG. 7 shows the SDS-PAGE analysis of the purified recombinant protein CTLA-4IgV-EgG1Y 162; m protein molecular mass standard; 1, a crude protein; 2, passing through the column for the first time and passing through the column for the second time 3; 420mM imidazole; 540mM imidazole; 680mM imidazole; 7100mM imidazole; 8200mM imidazole; 9300mM imidazole; 10400mM imidazole; 11500mM imidazole.

FIG. 8 shows the result of Western blot analysis of recombinant protein CTLA-4IgV-EgG1Y 162; m: protein molecular mass standard; 1-2: CTLA-4IgV-EgG1Y162 recombinant protein.

FIG. 9 shows the Western Blot assay of immune sera of hydatid patients; a: western Blot detection of serum from cystic hydatid patients: the results of 14 cases of human serum of cystic echinococcosis are shown in the No. 1-7 and No. 20-26 bands respectively; b: western Blot assay of sera from uninfected hydatid patients: bands No. 1-6 are the results of normal human serum, respectively.

FIG. 10 shows the Western Blot detection result of the immunization serum of the hydatid mouse; a: western Blot detection of hydatid mouse serum: 1-4 and 23-27 are the results of the serum of 9 cases of infected hydatid cyst type mice respectively; b: western Blot assay of uninfected hydatid mouse serum: bands No. 1-6 are serum results of 6 normal mice, respectively.

FIG. 11 shows the result of detecting the binding ability of CTLA-4IgV-EgG1Y162 recombinant protein to dendritic cells.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

Detailed Description

The technical scheme of the invention is further explained by combining specific examples.

Example 1 construction of prokaryotic expression plasmid pET30a-CTLA-4IgV

1. Amplification of Xinjiang sheep CTLA-IgV gene

The amplification primer sequences are as follows:

CTLA4-V F: 5'-ACGGATCCTAATGTGACCCAGCCTCCAG-3', as shown in SEQ ID NO. 3 of the sequence list.

CTLA4-V R: 5'-TAGGTACCATCAGAATCCGGGCATGGTTC-3', as shown in SEQ ID NO. 4 of the sequence table.

After the Xinjiang sheep peripheral blood lymphocytes are induced by ConA for 48 hours, the cell volume is increased, the cell nucleus has obvious nuclear fission, and the cells proliferate to form clones, which is shown in figure 1A. Total RNA from the sheep lymphocytes after ConA stimulation was extracted by Trizol method, and cDNA was synthesized according to the Invitrogen reverse transcription kit (see FIG. 1B). Adding a PCR reaction system, wherein the reaction procedure is as follows: 94 ℃ for 5 min; 30s at 94 ℃; 30s at 60 ℃; at 72 ℃, 45s, and 35 cycles; finally 72 ℃ for 10 min. The DNA fragments recovered as dices are shown in FIG. 1C. The overnight pMD19-T-CTLA-4IgV was transformed into E.coli DH5a competent cells by ligase insertion into the pMD19-T vector and screened by blue-white screening, see FIG. 1D. White single colonies were picked and the recombinant plasmid pMD19-T-CTLA-4IgV was digested with restriction enzymes BamHI and KpnI as shown in FIG. 1E. And (3) detecting positive plasmids, sending the positive plasmids to a Kuntaili biotechnology limited company for Sanger sequencing, and comparing the sequencing result with BLAST of NCBI (national center for Biotechnology information) to obtain a comparison result, wherein the comparison result shows that the positive plasmids have the highest homology with the base sequence of the CTLA-4 extracellular region of the sheep, but are not completely the same. Therefore, the new gene CTLA-4 of Xinjiang sheep is successfully cloned, and the recombinant plasmid pMD19-T-CTLA-4IgV is constructed.

2) Multi-sequence alignment result of Xinjiang sheep CTLA-4IgV protein sequence by using ClustalX software

The protein sequence of the CTLA4-V region of Xinjiang sheep is as follows:

NVTQPPVVPASSRGVASFTCEYESSGKADEVRVTVLRKAGIQVTEVCAGTYMVED ELTFLDDSSCIGTSRGNKVNLTIQGLRAMDTGLYVCKVELMYPPPYYMGEGNGTQIYV IDPEPCPDSD, as shown in SEQ ID NO. 5 of the sequence list.

The protein sequences of all registered goat CTLA-4IgV (AAD04380.1, P001009214.1, XP027819684.1) downloaded from NCBI were aligned with the protein sequence of Xinjiang goat CTLA-4IgV cloned in this example (CTLA-4IgVXINJIANG) using Clustal X software, and it can be seen from FIG. 2 that the CTLA-4IgV protein sequence of Xinjiang goat is unique. Wherein the 9 th amino acid is P, and the diversity of species is also described. The molecular evolution tree can obtain the CTLA-4IgV protein of Xinjiang sheep in relative independence.

3) Construction and identification of prokaryotic expression plasmid pET30a-CTLA-4IgV

The prokaryotic expression vector pET30a is subjected to double enzyme digestion by Bgl II and Kpn I, and gel electrophoresis is carried out to obtain linear pET30a (figure 3A); plasmid pMD19-T-CTLA-4IgV was digested with BamHI and Kpn I, and after gel electrophoresis, a linear CTLA-4IgV fragment was obtained at 369bp (FIG. 3B). Connecting the CTLA-4IgV fragment with a linear pET30a band to construct a plasmid pET30a-CTLA-4IgV, transforming Escherichia coli DH5a competent cells by the constructed plasmid pET30a-CTLA-4IgV, picking a single colony, adding the single colony into 5mL LB liquid culture medium (containing 100g/mL ampicillin (Amp)), extracting plasmid DNA, using the recombinant plasmid as a template, amplifying by PCR, displaying that the amplified product is about 360bp by 1% agarose gel electrophoresis, and the size is consistent with the expected Gene fragment (FIG. 3C), sequencing the recombinant plasmid pET30a-CTLA-4IgV, wherein the sequencing result is consistent with the Gene sequence published on Gene-Bank, and the sequencing verification successfully constructs the recombinant pET30a-CTLA-4IgV, and sequencing, cloning and identifying the clone is correct.

4) Induction expression and identification of CTLA-4IgV protein

After pET30a-CTLA-4IgV is transformed into Ecoli. BL21(DE3) competent cells, IPTG induction expression at different concentrations and time, ultrasonication, centrifugation at 4000rpm for 15min, and SDS-PAGE electrophoresis identification of the supernatant and the precipitate respectively, it can be seen from FIGS. 4A and 4B that CTLA-4IgV protein has an expression band at about 21kDa, which is consistent with the expectation, and the protein expression level in the supernatant is obviously higher than that in the precipitate. The recombinant protein has the highest induction expression quantity of the recombinant protein in the supernatant under the conditions of 28 ℃, 0.2mmol/L IPTG concentration and 4h of induction. Westernblot identification is carried out by using a His antibody, and the result of figure 4C shows that an obvious band appears at the position of about 21kDa of the induced bacteria, and the band is consistent with the position of a band specifically expressed in SDS-PAGE electrophoresis, which preliminarily shows that the recombinant CTLA-4IgV protein is successfully expressed.

Example 2 construction of prokaryotic expression plasmid pET30a-CTLA-4IgV-EgG1Y162

The amino acid sequence of the recombinant protein CTLA4-IgV-EgG1Y162 is as follows:

NVTQPPVVPASSRGVASFTCEYESSGKADEVRVTVLRKAGIQVTEVCAGTYMVED ELTFLDDSSCIGTSRGNKVNLTIQGLRAMDTGLYVCKVELMYPPPYYMGEGNGTQIYV IDPEPCPDSDGTDDDDKAMADIGSEFVDPELMAKLTKELKTTLPEHFRWIHVGSRSLEL GWNATGLANLHADHIKLTANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAF KGGSQVFKYTGFIRTLAPGEDGADRASGF, see SEQ ID NO:1 of the sequence Listing.

The gene sequence of the recombinant protein CTLA4-IgV-EgG1Y162 is as follows:

AATGTGACCCAGCCTCCAGTGGTGCTGGCTAGCAGCCGGGGTGTTGCCAGCTT CACATGTGAATATGAGTCTTCAGGCAAAGCTGATGAGGTCCGGGTGACAGTGCTG CGGAAGGCAGGCATCCAGGTGACCGAAGTCTGTGCTGGGACCTACATGGTGGAGG ATGAGCTAACCTTCCTGGATGATTCCAGTTGCATTGGCACCTCCAGAGGAAACAAA GTGAACCTCACCATCCAAGGGCTGAGGGCCATGGACACTGGGCTCTATGTCTGCA AAGTGGAGCTCATGTACCCGCCGCCCTACTACATGGGCGAGGGCAATGGAACCCA GATTTATGTCATTGATCCAGAACCATGCCCGGATTCTGATGGTACCGACGACGACG ACAAGGCCATGGCTGATATCGGATCCGAATTCGTAGACCCAGAGCTAATGGCAAA GTTGACAAAGGAACTAAAGACCACACTGCCAGAACACTTCCGATGGATTCACGTG GGTTCCCGCTCCCTTGAATTGGGTTGGAATGCCACTGGTTTAGCCAATCTCCACGC AGACCACATTAAACTGACTGCAAACCTTTATACAACTTACGTTACCTTCAAGTACA GAAATGTTCCTATCGAACGTCAGAAACTCACTCTTGAGGGACTAAAGCCCAGTAC ATTCTACGAAGTGGTTGTGCAAGCATTTAAAGGAGGTTCCCAAGTTTTTAAATACA CTGGATTCATTAGAACACTGGCTCCAGGGGAAGATGGCGCTGACAGAGCTAGCGG ATTC, see SEQ ID NO. 2 of the sequence Listing.

1) Structure display of recombinant protein CTLA4-IgV-EgG1Y162

In prokaryotic expression plasmid pET30a (+), after the correct reading frame for protein expression is noticed, the recombinant protein CTLA4-IgV-EgG1Y162 expressed by the constructed plasmid pET30a-CTLA4-IgV-EgG1Y162 is composed of 6 parts shown in FIG. 5: starting from the N-terminus, there were first a His-Tag of 6 histidines, followed by a proteolytic site of thrombomin, an S-Tag, a CTLA4-IgV protein, a linker sequence (pET30a itself, containing 18 amino acids including an enterokinase proteolytic site), and finally EgG1Y 162. The structure of the recombinant protein CTLA4-IgV-EgG1Y162-2, and the end protein at the C terminal is EgG1Y 162-2.

2) Construction of recombinant plasmid pET30a-CTLA-4IgV-EgG1Y162

The successfully constructed plasmids are cut for 1h at 37 ℃ by EcoR I and Hind III respectively by taking pET30a-CTLA-4IgV and pET30a-EgG1Y162 which are successfully constructed as templates in a laboratory, and are subjected to electrophoresis by 1% agarose gel, gel cutting, recovery and purification of the cut products. The purified cleavage products were ligated overnight at 16 ℃. And transforming 5 mu L of the ligation product into 50 mu LDH5 alpha competent cells, shaking bacteria and plating to select positive clones, identifying the positive clones by bacteria liquid PCR, and sending the identified positive clones to a company for sequencing.

3) Induced expression of recombinant protein HIS-CTLA-4IgV-EgG1Y162

After Pet30a-CTLA-4IgV-EgG1Y162 was transformed into Ecoli. BL21(DE3) competent cells, at 28 ℃ for 4 hours or 28 ℃ for 2 hours and 37 ℃ for 2 hours at a final concentration of IPTG of 0.5 mmol/L; when the final concentration of IPTG was 0.8mmol/L, the protein expression level in the supernatant was the highest under the induction condition of 4 hours at 28 ℃ (see FIG. 6). When the final concentration of IPTG is 0.2mmol/L, the protein expression level in the precipitated inclusion bodies is highest under the induction condition of 2 hours at 28 ℃ and 2 hours at 37 ℃ for 2 hours.

4) Purification of recombinant protein CTLA-4IgV-EgG1Y162

Eluting with imidazole of different concentrations, and collecting the liquid after passing through the column. And (4) carrying out SDS-PAGE detection on the liquid after passing through the column, and analyzing the content and purity of the protein in the eluent with each concentration. The results in FIG. 7 show that the concentration of 200mM, 300mM imidazole was found to be the best eluent by SDS-PAGE analysis, under which conditions the protein component of interest was relatively pure. And under the condition, the protein is purified for a plurality of times, and the protein is collected so as to be used for the later animal immunity experiment.

5) Identification of recombinant protein HIS-CTLA-4IgV-EgG1Y162

Identification of His antibody: the Western blot result shows that the induced bacteria have obvious bands at the position of about 34.3kDa, which is consistent with the position of the band specifically expressed in SDS-PAGE electrophoresis, and the results preliminarily show that the recombinant protein CTLA-4LgV-EgG1Y162 is successfully expressed (see figure 8).

② identifying immune serum:

western Blot detection is carried out after primary antibody is used for diluting blood serum of a cystic echinococcosis patient and normal human serum by 1:50, and secondary antibody is used for diluting goat anti-human antibody by 1:3000, wherein 14 cases of blood serum of the cystic echinococcosis patient and 6 cases of normal human serum, wherein 14 cases of primary antibody of the cystic echinococcosis patient have reaction bands at 34.3kDa (shown in figure 9). 6 normal human sera were primary antibodies, and no protein band appeared (as shown in FIGS. 2-28). Here, the sensitivity ═ true yang/(true yang + false yang) x 100% ═ 14/(14+0) x 100%, calculated as 100%; specificity ═ true yin/(true yin + false yin) x 100% ═ 6/(6+0) x 100%, calculated as 100%

As a result: after primary antibody is diluted by 1:50 and secondary antibody is diluted by 1:2000 of mouse anti-His antibody, Western Blot detection is carried out, and 9 cases of serums of the cystic echinococcus and 6 cases of serums of normal mice are carried out, wherein 9 cases of serums of the cystic echinococcus have reaction bands at 34.3kDa (shown in figure 9). Serum from 6 normal mice was primary antibody, and no protein band was observed (as shown in FIG. 10). Here, the sensitivity ═ true yang/(true yang + false yang) x 100% ═ 9/(9+0) x 100%, calculated as 100%; the specificity (true yin/(true yin + false yin) x 100% ═ 6/(6+0) x 100%, calculated as 100%.

Example 3CTLA4-IgV promotes the binding of EgG1Y162 to dendritic cells

The method comprises the following steps: bone marrow hematopoietic stem cells from mice on day 6 of stimulus induction, cell suspensions were collected to obtain immature dendritic cells (imDCs). Culturing in 3 cell culture 6-well plates (named as control group, protein CTLA-4IgV-EgG1Y162 binding group and protein EgG1Y162 binding group) with a culture volume of 3 ml/well and 3 × 10⁶And (4) cells. Adding 500ng/ml protein CTLA-4IgV-EgG1Y162 into each well of immature dendritic cells in the protein CTLA-4IgV-EgG1Y162 binding group, adding protein EgG1Y162 with the same concentration into each well of the protein EgG1Y162 binding group, and culturing for 24h and 48h respectivelyThereafter, the cell suspension was collected. 1X 10⁶Flow tubes, three tubes per group. 2ml PBS was added to each tube and gently blown to mix well, then 1000rbp was centrifuged for 5min, the supernatant was discarded, 50ul PBS containing 1ul FITC-HiS antibody was added, and the mixture was incubated at 4 ℃ for 30min in the dark. The cells were filtered through a 70um filter, 300ul PBS was added to resuspend the cells, and cytokine expression was detected within 4 h. Simultaneously setting a blank tube and a single dyeing tube.

As a result: referring to FIG. 11, the percentage of DCs that bound EgG1Y162 was 0.740% at 12 hours of co-cultivation; the DC percentage of the binding protein CTLA-4IgV-EgG1Y162 is 1.32%; after 24 hours of co-incubation, the percentage of DCs bound to EgG1Y162 was 18.1%; the DC percentage of the binding protein CTLA-4IgV-EgG1Y162 is 28.0 percent and is obviously increased. Therefore, the CTLA-4IgV promotes the combination of protein EgG1Y162 and DC.

Example 4 immunization of recombinant vaccine CTLA4-IgV-EgG1Y162

1) Animal(s) production

The experimental animals were healthy female Balb/C mice, randomly divided into 4 groups: group A: saline NS placebo (n-20); group B: adjuvant control group (n ═ 20); group C: EgG1Y162 group (n ═ 20); group D: CTLA4-IgV-EgG1Y162 group (n ═ 20).

2) Method of producing a composite material

A. B, C, D group is administered by back subcutaneous multiple injection of recombinant NS, adjuvant, EgG1Y162 and CTLA4-IgV-EgG1Y162 at dose of 100 ug/mouse/dose for 3 times, each time with 2 weeks interval, and collecting blood serum adjacent to the mice before each immunization. One week after the 3 rd immunization, each mouse was inoculated with about 1000 secondary infections of Echinococcus granulosus metacercaria. At 70 days after infection, blood is collected, mice are sacrificed and subjected to autopsy, and various immune index detections are carried out, wherein the immune index detections comprise vesicle wet weight inhibition rate, vesicle number and gross changes, ELISA detects specific antibody level changes and cytokine changes in mouse serum, and the immune protection effect is evaluated through statistical analysis of results.

3) Results

Observing echinococcus granulosus substantially

The abdominal cavities of 20 mice in the NS control group and the adjuvant control group have echinococcus granulosus sacs growing, and the sac walls are smooth and transparent, high in tension and large in number. The NS control group was observed similarly to the adjuvant control group. 6 mice (6/20) in the EgG1Y162 group did not have hydatid cyst in abdominal cavity and liver surface, indicating that the 7 mice were completely protected, and the other 13 mice all had echinococcus cyst growth; in the CTLA4-IgV-EgG1Y162 group, 9 (9/20) mice have no hydatid cyst in the abdominal cavity and on the surface of the liver, which indicates that the 9 mice are completely protected, and 11 mice all have echinococcus cyst; in the two groups of vaccine protection, even though some of the mice had echinococcal sac growth, the number of sacs was reduced, the volume was reduced, some of the surface of the sacs was turbid, some of the wall of the sacs was collapsed, was detached from the abdominal cavity, or was attached to the mesenteric or hepatic surface, as compared to the NS control group.

Results of inhibition of bursa Wet weight

A. B, C, D Echinococcus granulosus cysts were removed from each group of mice and weighed, and the cyst wet weight inhibition rate was calculated, and the results are shown in the following table, in comparison with the NS control group, the cyst wet weight inhibition rate of the adjuvant control group was 11.4%, the cyst wet weight inhibition rate of the EgG1Y162 group was 60.8%, and the cyst wet weight inhibition rate of the CTLA4-IgV-EgG1Y162 group was 68.5%. The immune procedure adopted by the invention is safe, effective, simple and feasible.

TABLE 1 statistical table of the results of inhibition of bursa wet weight

Variance analysis showed that there was no statistical difference between the adjuvant and NS control groups (P > 0.05). The recombinant EgG1Y162 group, CTLA4-IgV-EgG1Y162 group and NS group have statistical significance (P < 0.01).

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

SEQUENCE LISTING

<110> Sinkiang university of medicine

<120> Echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162 and application thereof

<130> /

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 259

<212> PRT

<213> Artificial Synthesis

<400> 1

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

1 5 10 15

Ser Phe Thr Cys Glu Tyr Glu Ser Ser Gly Lys Ala Asp Glu Val Arg

20 25 30

Val Thr Val Leu Arg Lys Ala Gly Ile Gln Val Thr Glu Val Cys Ala

35 40 45

Gly Thr Tyr Met Val Glu Asp Glu Leu Thr Phe Leu Asp Asp Ser Ser

50 55 60

Cys Ile Gly Thr Ser Arg Gly Asn Lys Val Asn Leu Thr Ile Gln Gly

65 70 75 80

Leu Arg Ala Met Asp Thr Gly Leu Tyr Val Cys Lys Val Glu Leu Met

85 90 95

Tyr Pro Pro Pro Tyr Tyr Met Gly Glu Gly Asn Gly Thr Gln Ile Tyr

100 105 110

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

115 120 125

Asp Lys Ala Met Ala Asp Ile Gly Ser Glu Phe Val Asp Pro Glu Leu

130 135 140

Met Ala Lys Leu Thr Lys Glu Leu Lys Thr Thr Leu Pro Glu His Phe

145 150 155 160

Arg Trp Ile His Val Gly Ser Arg Ser Leu Glu Leu Gly Trp Asn Ala

165 170 175

Thr Gly Leu Ala Asn Leu His Ala Asp His Ile Lys Leu Thr Ala Asn

180 185 190

Leu Tyr Thr Thr Tyr Val Thr Phe Lys Tyr Arg Asn Val Pro Ile Glu

195 200 205

Arg Gln Lys Leu Thr Leu Glu Gly Leu Lys Pro Ser Thr Phe Tyr Glu

210 215 220

Val Val Val Gln Ala Phe Lys Gly Gly Ser Gln Val Phe Lys Tyr Thr

225 230 235 240

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

245 250 255

Ser Gly Phe

<210> 2

<211> 777

<212> DNA

<213> Artificial Synthesis

<400> 2

aatgtgaccc agcctccagt ggtgctggct agcagccggg gtgttgccag cttcacatgt 60

gaatatgagt cttcaggcaa agctgatgag gtccgggtga cagtgctgcg gaaggcaggc 120

atccaggtga ccgaagtctg tgctgggacc tacatggtgg aggatgagct aaccttcctg 180

gatgattcca gttgcattgg cacctccaga ggaaacaaag tgaacctcac catccaaggg 240

ctgagggcca tggacactgg gctctatgtc tgcaaagtgg agctcatgta cccgccgccc 300

tactacatgg gcgagggcaa tggaacccag atttatgtca ttgatccaga accatgcccg 360

gattctgatg gtaccgacga cgacgacaag gccatggctg atatcggatc cgaattcgta 420

gacccagagc taatggcaaa gttgacaaag gaactaaaga ccacactgcc agaacacttc 480

cgatggattc acgtgggttc ccgctccctt gaattgggtt ggaatgccac tggtttagcc 540

aatctccacg cagaccacat taaactgact gcaaaccttt atacaactta cgttaccttc 600

aagtacagaa atgttcctat cgaacgtcag aaactcactc ttgagggact aaagcccagt 660

acattctacg aagtggttgt gcaagcattt aaaggaggtt cccaagtttt taaatacact 720

ggattcatta gaacactggc tccaggggaa gatggcgctg acagagctag cggattc 777

<210> 3

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 3

acggatccta atgtgaccca gcctccag 28

<210> 4

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 4

taggtaccat cagaatccgg gcatggttc 29

<210> 5

<211> 123

<212> PRT

<213> Sinkiang sheep

<400> 5

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

1 5 10 15

Ser Phe Thr Cys Glu Tyr Glu Ser Ser Gly Lys Ala Asp Glu Val Arg

20 25 30

Val Thr Val Leu Arg Lys Ala Gly Ile Gln Val Thr Glu Val Cys Ala

35 40 45

Gly Thr Tyr Met Val Glu Asp Glu Leu Thr Phe Leu Asp Asp Ser Ser

50 55 60

Cys Ile Gly Thr Ser Arg Gly Asn Lys Val Asn Leu Thr Ile Gln Gly

65 70 75 80

Leu Arg Ala Met Asp Thr Gly Leu Tyr Val Cys Lys Val Glu Leu Met

85 90 95

Tyr Pro Pro Pro Tyr Tyr Met Gly Glu Gly Asn Gly Thr Gln Ile Tyr

100 105 110

Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120

Claims

1. An echinococcus granulosus recombinant protein CTLA4-IgV-EgG1Y162, which is characterized in that: comprises echinococcus granulosus protein EgG1Y162 and cytotoxic T lymphocyte-associated antigen 4 extracellular domain protein CTLA 4-IgV.

2. The recombinant protein of claim 1, wherein: the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

3. The recombinant protein of claim 1, wherein: the nucleotide sequence of the recombinant protein is shown as SEQ ID NO. 2.

4. Use of the recombinant protein or the coding sequence thereof according to any one of claims 1 to 3 for the preparation of a vaccine for the prevention of Echinococcus granulosus.

5. A vaccine for the prevention of echinococcus granulosus, characterized by: comprising the combination protein of any one of claims 1-3 and an immunoadjuvant.