CN118255901A

CN118255901A - Recombinant protein of norovirus P particle chimeric LcR protein, preparation method and application thereof

Info

Publication number: CN118255901A
Application number: CN202410340844.4A
Authority: CN
Inventors: 谭志坚; 尚志祥; 邝春曼; 林瑞庆; 王新秋; 刘丽丹; 翁亚彪
Original assignee: Foshan Standard Bio Tech Co Ltd
Current assignee: Foshan Standard Bio Tech Co Ltd
Priority date: 2024-03-22
Filing date: 2024-03-22
Publication date: 2024-06-28

Abstract

The invention provides a recombinant protein of a norovirus P particle chimeric LcR protein, a preparation method and application thereof, wherein the recombinant protein comprises a norovirus P domain and a Calf leucocyte worm R7 protein, the amino acid sequence of the norovirus P domain is shown as SEQ ID No.3, and the amino acid sequence of the Calf leucocyte worm R7 protein is shown as SEQ ID No. 4. The recombinant protein of the chimeric LcR protein of the norovirus P particles can be used as a Karsch leucocytozoonosis vaccine for preventing and treating animal Karsch leucozoonosis, can maintain high antibody level for a long time, and provides a new method for preventing and controlling the Karsch leucozoonosis.

Description

Recombinant protein of norovirus P particle chimeric LcR protein, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant protein of a norovirus P particle chimeric Carpesium calycarpa R7 (LcR) protein, a preparation method and application thereof.

Background

Chicken Karsch's leucozoonosis (Leucocytozoonosis) is a serious poultry disease caused by zoonosis blood spore of Acidozoon, chicken is the only host, chickens of different varieties, ages and sexes can be infected, the disease pathogen is Karsch leucozoonosis (Leucocytozoon caulleryi), is the genus Leucozoonosis of the order Leucomatoda of the subclass Acidozoon, mainly invades the blood system, reproductive system and digestive system of the chicken, and is frequently generated in chickens of 3-6 weeks old, the disease is serious, and dysplasia leads to death. The infection rate of the backup chicken is higher than that of the chicks, but the death rate is not high; adult chickens are most susceptible, the laying rate of the laying hens is seriously reduced, and huge economic loss is caused to the chicken industry.

At present, chicken Ka's leukocytozoosis has no commercial vaccine, the treatment effect of the disease-causing chicken is poor, and the disease incidence rate can only be reduced by blocking the transmission path, namely eliminating transmission media (Culy and gnat) and improving the feeding management level of a farm. The scholars find that after the immune chickens are inoculated by using the sporozoites of the white blood cells of the Carpesium, the immune chickens only have complete protection, and soluble antigens and antibodies can be detected in all the serum of the surviving chickens, but the chickens can certainly see diseases after being inoculated with the sporozoites of the white blood cells, the production performance is reduced, the clinical symptoms are consistent with natural infection, and the parasitosis and the death rate are also improved. The specific antibody can be stimulated to generate by the chicken by inoculating the inactivated/non-inactivated tissue suspension vaccine containing the second generation merozoites, so that a certain immune protection effect is obtained, and the level of the antibody resisting the second generation merozoites in serum is proved to be positively correlated with the protection effect formed by the chicken.

The scholars found a highly immunogenic protein associated with the outer membrane of the second generation merozoites (second-generation schizont,2 GS): the level of anti-2 GS antibodies in the blood of chickens can reach a significant level after immunization of chickens with the genetically engineered subunit vaccine using LcR R7 protein as antigen (hereinafter referred to as "Karsch white blood cell insect R7 protein" or "LcR protein"), and chickens with high antibody titers can completely protect them from attack by Karsch sporozoites, while chickens with lower antibody titers can only produce partial protection, cannot prevent parasitemia, as well as the results of field experiments confirm the conclusion. However, the antibody level of LcR protein oil adjuvant genetically engineered vaccine after single immunization can be kept in the protective range only briefly (about 6 weeks), and high antibody titer cannot be formed for a long time (2-4 months) after the immunization is enhanced.

At present, the immune mechanism of the Karsch leucocyte worm R7 is not completely elucidated, although intracellular parasites are mainly used for cell immunity, a 2GS antibody generated after LcR protein vaccine immunization reacts with Karsch leucocyte worm 2GS membrane proteins in blood tissues to cause 2GS membrane denaturation, and finally the life cycle of the Karsch leucocyte worm is blocked, and the link humoral immunity is also important, which is consistent with the result that high antibody titer forms high protective force after LcR protein inoculation, so that providing higher and longer antibody titer is a better way for optimizing R7 genetic engineering vaccine and is the best method for controlling the popularity of the Karsch leucocyte worm.

Disclosure of Invention

In one aspect, the invention provides a recombinant protein of the P particle chimeric LcR protein of the norovirus, and after inoculation of the recombinant protein, chicken blood can keep high antibody titer for a long time. The recombinant protein comprises a norovirus P domain and a Karsch leucocyte worm R7 protein, wherein the amino acid sequence of the Karsch leucocyte worm R7 protein is shown as SEQ ID No.2, and the amino acid sequence of the norovirus P domain is shown as SEQ ID No. 4.

In another preferred example, the amino acid sequence of the recombinant protein is shown as SEQ ID No.6 or SEQ ID No.8, or the amino acid sequence of the recombinant protein has more than 85% sequence identity with the sequence shown as SEQ ID No.6 or SEQ ID No.8

In another aspect, the invention provides a polynucleotide encoding a recombinant protein of the aforementioned norovirus P particle chimeric LcR protein.

In another preferred embodiment, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO:5 or SEQ ID No.7, or the nucleotide sequence of said polynucleotide is identical to the nucleotide sequence shown in SEQ ID No. 7: 5 or SEQ ID No.7 has a sequence identity of more than 85%. For example, 86%, 88%, 90%, 92%, 93%, 94%, 99% or more sequence identity.

In another aspect, the present invention provides a recombinant vector comprising the polynucleotide described above, or a recombinant bacterium having the polynucleotide of claim 1 or 2 integrated into its genome.

In another preferred embodiment, the vector used to construct the recombinant is pGEX4T-1 or pET28a.

In another aspect, the invention provides a recombinant bacterium comprising the recombinant vector or the recombinant bacterium having the polynucleotide integrated in its genome.

In another preferred embodiment, the recombinant bacterium is E.coli BL21 (DE 3).

In another aspect, the invention provides a calicheating leukocyte worm vaccine comprising the recombinant protein of the aforementioned norovirus P particle chimeric LcR protein.

In another aspect, the invention provides a pharmaceutical composition comprising the above mentioned vaccine against leucocyte worm; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method for preparing the recombinant protein of the nodavirus P particle chimeric LcR protein, which comprises the following steps:

(1) Constructing a recombinant vector containing a nucleic acid molecule encoding a recombinant protein of the aforementioned norovirus P particle chimeric LcR protein;

(2) Transferring the recombinant vector obtained in the step (1) into competent cells, screening positive clones, culturing, and lysing the cells to obtain lysate;

(3) And (3) extracting and purifying the protein from the lysate obtained in the step (2) to obtain the recombinant protein of the norovirus P particle chimeric LcR protein.

In another preferred embodiment, in step (1), the method for constructing a recombinant vector comprises the steps of:

The recombinant plasmid pET28a-MBP-P-LcR is constructed by inserting the sequence of the recombinant protein encoding the norovirus P particle chimeric LcR protein between the HindIII and Xho I cleavage sites of the pET28a-MBP vector.

In another preferred embodiment, in step (2), the competent cells are BL21 competent cells.

In another preferred embodiment, in step (3), the extraction and purification are performed using the following steps:

Centrifuging the lysate to collect supernatant, filtering the supernatant, adding the filtered supernatant into resin for incubation, eluting protein, and collecting target protein.

In a further aspect, the invention provides the use of the recombinant protein of the norovirus P particle chimeric LcR protein, the polynucleotide, the recombinant vector, the recombinant bacterium, the calicheating leukocyte worm vaccine or the pharmaceutical composition in the preparation of a medicament or vaccine for treating or preventing calicheating leukocyte worm diseases.

Drawings

FIG. 1 shows the norovirus VP1 and P domains, with the isolated P domains forming P particles.

Figure 2 shows 3 epitope loops on norovirus P particles and P dimers.

FIG. 3 is a diagram of recombinant plasmid pGEX4T-1-P-LcR 7.

FIG. 4 is a diagram showing the PCR identification result of the recombinant plasmid pGEX4T-1-P-LCR7 of the example of the present invention, wherein lane M: 2000 LADDER MARKER; lanes 1-4: a null PP amplified fragment; lane 5: P-LCR7 amplified fragment; n lanes: negative control. The hollow PP amplified fragment refers to the original norovirus P domain amplified sequence without inserted genes.

FIG. 5 shows SDS-PAGE induced by P-LCR7 protein according to the present invention, wherein lanes M1 and M2: protein marker; lane 1: inducing the recombinant whole bacteria (0.5 mM IPTG) for 18h at 16 ℃; lane 2: non-induced recombinant whole bacteria; lane 3: inducing the empty whole bacteria (0.5 mM IPTG) for 18h at 16 ℃; lane 4: uninduced empty whole bacteria; lane 5: cell lysis supernatant (0.5 mM IPTG) induced at 16℃for 18 h; lane 6: cell lysis pellet (0.5 mM IPTG) was induced for 18h at 16 ℃.

FIG. 6 shows SDS-PAGE of P-LCR7 protein purification results in an example of the present invention, wherein M lanes: protein marker; lane 1: ultrasonic supernatant; lane 2: loading sample flowing liquid; lane 3: a washing liquid; lane 4: a first eluent; lane 5: and eluting the second time.

FIG. 7 is a diagram showing the result of SDS-PAGE (left) Western blot (right) identification of the purified monomers and polymers of the P-LCR7 protein according to the embodiment of the present invention; wherein, the upper and lower arrows indicate the electrophoresis bands of multimeric P-LCR7 and monomeric P-LCR7 proteins, respectively.

FIG. 8 is a 24-mer protein map identified by the P-LCR7 protein electron microscope in the example of the present invention; from this figure it can be seen that the P-LCR7 protein successfully self-assembles in vitro into 24-mer VLPs with a diameter of about 20 nm.

FIG. 9 is a diagram showing the result of SDS-PAGE (left) Western blot (right) identification of the single and multiple polymers purified from the P-LcR 7: 7 ₁₂ protein according to the example of the present invention, wherein the upper and lower arrows indicate the electrophoresis bands of the multiple polymer P-LCR7 ₁₂ and single polymer P-LCR7 ₁₂ proteins, respectively; the Western blot electrophoretogram can see the single polymer and multiple polymer bands of the P-LcR 7: 7 ₁₂ protein, which indicates that the P-LcR 7: 7 ₁₂ can be self-assembled into the multiple VLPs in vitro.

FIG. 10 shows the antibody level curves of chicken blood after immunization with LCR7 protein, P-LCR7 protein and P-LCR7 ₁₂ protein, from which it can be seen that the P-LCR7 protein and P-LCR7 ₁₂ protein immunized groups have better immune effect, faster rise of antibody level, higher peak value and longer duration than the R7 protein immunized groups.

Detailed Description

The inventor of the present application has conducted intensive studies to find that the use of an immune vector to present LcR protein can provide higher and longer antibody titers, and is a way to optimize R7 genetic engineering vaccines.

Norovirus was originally isolated from fecal samples during the outbreak of gastroenteritis in the Russian Norwalk population in 1929. The norovirus has a simple structure, a rough surface and no envelope structure on the surface, the diameter of the virus is about 27-32 nm (KAPIKIAN ET al., 1972), the main structural protein VP1 of the norovirus forms a virus shell, the main structural protein is divided into an S domain and a P domain, the S domain participates in forming a shell polyhedral structure, and the P domain protrudes from the shell to form a P dimer of an external area of the shell. The P domain comprises a receptor binding site and the individual P domains are capable of self-assembly in vitro into structurally stable fully functional VLP particles. Three complex forms can be formed by expressing P region proteins modified with different ends in vitro: dimer, 12-mer P particles, and 24-mer P particles (TAN MING ET al.,2011;Bereszczak et al, 2012) (see fig. 1). Each P protein end of the P particle dimer has three outwardly protruding loop structures, loop1, loop2 and Loop3 (see FIG. 2). Norovirus P particle protein is a good immune carrier. By inserting foreign antigen sequences into the gene sequences of the P particle loop structure, the P particle chimeric protein with the antigen can be formed by prokaryotic expression, and the antigen protein can be displayed on the surface of the P particle. The P particles produced by prokaryotic expression are highly immunogenic and stable, are stable at low temperatures, frozen, lyophilized and ambient temperatures, and since each P monomer has three surface loops, insertion of foreign antigen into these loops results in 24 to 72 copies of antigen on the surface of the P particles, which can greatly enhance the antigenicity and immunogenicity of the antigen. Therefore, the norovirus P particles have wide prospect as antigen presenting carrier platforms.

However, the inventors have found in previous studies that norovirus P particle vectors are not suitable for all antigens, for example, the inventors have tried to chimeric norovirus P particles with clostridium perfringens alpha toxin gene alpha, but the results are not ideal, although soluble chimeric proteins can be obtained, the solubility ratio is extremely low, and the chimeric proteins after concentration are found to have no 20nm diameter particle proteins by transmission electron microscopy, and the intended purpose is not achieved. Moreover, the prior art only reports that norovirus P particle vectors are used for antigen binding of short sequence epitopes and short gene sequences with simple spatial structures, but cannot be purified as soluble proteins for antigens with complex structures and long sequences, and VLP particles cannot be easily formed, resulting in difficulty in purification and application. Inclusion body proteins are less immunogenic than soluble proteins. Compared with the P-EG95 chimeric recombinant protein made before the subject group of the inventor, the solubilizing label is MBP (40 kDa), pGEX4T-1 expression plasmid is selected, and the solubilizing label on the plasmid is GST label, so that the solubilizing effect is consistent with that of MBP, but the molecular weight is smaller (26 kDa), thereby reducing the molecular weight of the monomeric protein formed after long-sequence antigen insertion, being beneficial to the formation of 24-polymer macromolecular protein and the research of the gene immunity enhancing effect of longer sequences.

The inventors surprisingly found that combining the norovirus P particles with the R7 protein antigen of the card white blood cell worm, i.e., integrating the R7 protein antigen into the epitope loop of the norovirus P domain, resulted in recombinant proteins of the norovirus P chimeric LcR protein, which were not only highly expressed, well water-soluble, and easily produced fusion proteins, but also maintained high antibody levels for long periods of time when the recombinant proteins were immunized against chickens. In the research of the Karsch white blood cell genetic engineering vaccine, the antibody level is proportional to the protective force after vaccine immunization, and the higher the antibody level is maintained for longer, the time and the efficacy of the protective force are also increased. The P particles have high immunogenicity and stability, are very stable in low temperature, freezing, freeze-drying and environment temperature states, and can greatly enhance the antigenicity and immunogenicity of antigens due to the fact that each P monomer is provided with three surface rings, so that the norovirus P particle carrier is very in line with the optimization requirement of the R7 genetic engineering vaccine of the white blood cell insect, and the recombinant chimeric protein can provide better protection effect.

In the invention, NCBI is used for obtaining the antigen sequence of R7 protein of the leucocyte worm, analyzing the sequence, analyzing sequence information by using Signal P-5.0 and TMHMM, removing polypeptide sequences of Signal peptide and transmembrane structural domain, and optimizing the sequence according to the codon preference of escherichia coli. The nucleotide sequence of LcR protein antigen after colibacillus codon optimization is shown as SEQ ID No. 1. When the LcR protein is expressed by using escherichia coli in a heterologous way, the localization effect of the signal peptide is not needed, the redundant signal peptide and the transmembrane domain cannot be recognized and cut by the escherichia coli, the correct folding and immunogenicity of the LcR protein can be possibly affected, and the probability of misfolding of the LcR protein in the heterologous way can be reduced by removing the signal peptide and the transmembrane domain.

SEQ ID No.1：

The amino acid sequence of LcR protein of the invention is shown as SEQ ID No. 2.

SEQ ID No2：

In the invention, the nucleotide sequence of the norovirus (NoV) P particles after the optimization of the escherichia coli codons is shown as SEQ ID No. 3.

SEQ ID No.3：

The amino acid sequence of NoV P particles of the invention is shown as SEQ ID No. 4.

SEQ ID No.4：

In the invention, the nucleotide sequence of a recombinant protein (P-LCR 7 recombinant protein) of LcR protein embedded in the norovirus P particle is shown as SEQ ID No. 5.

SEQ ID No.5

The amino acid sequence of the P-LCR7 recombinant protein is shown as SEQ ID No. 6.

SEQ ID No.6

In the invention, the R7 protein gene fragment is simultaneously inserted into a first epitope loop and a second epitope loop of NoV P structural domains, the obtained chimeric P structural domain containing two sections of R7 protein gene sequences, the recombinant P structural domain is called P-LcR7 ₁₂, the recombinant plasmid is called pGEX4T-1-P-LcR7 ₁₂ plasmid, and the recombinant protein obtained by expression is called P-LcR7 ₁₂ recombinant protein (namely, one norovirus P particle chimeric two LcR protein recombinant proteins).

The nucleotide sequence of the recombinant protein of the P-LcR ₁₂ is shown as SEQ ID No. 7.

SEQ ID No.7

The amino acid sequence of the P-LcR ₁₂ recombinant protein is shown as SEQ ID No. 8.

SEQ ID No.8

In the description of the invention, "room temperature" means 0 to 40℃and more preferably 20 to 30 ℃.

Compared with the prior art, the recombinant protein of the chimeric LcR protein of the norovirus P particles has the following advantages:

1. The invention uses the R7 protein of the card white blood cell worm as a candidate antigen for preparing subunit vaccine, has good immunogenicity, uses NoV P granule protein as an immune carrier, and the R7 protein of the card white blood cell worm is embedded in the P granule of the norovirus, thus being capable of being fully presented.

2. The recombinant protein of the chimeric LcR protein of the norovirus P particle provided by the invention utilizes NoV P particles as an immune carrier, has the stability of NoV P particles and the good antigenicity of LcR protein, and can provide higher and longer antibody titer. Can only form immune protection for 2-3 months for immunized chickens, and the antibody level rises faster in the early stage of immunization, and can provide earlier immune protection effect compared with single LcR protein immunization.

3. The recombinant protein of the chimeric LcR protein of the norovirus P particle can be self-assembled into 24-mer VLP in vitro after purification, and the concentration is higher, and the proportion of 24-mer protein is higher.

For further detailed description of the present invention, the following description is given by way of specific embodiments and the accompanying drawings, with the understanding that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1

Preparation of recombinant vectors

1) Gene search and analysis of the R7 protein of the calipers white blood cell:

The gene sequence of the R7 protein of the leucocyte worm is obtained through GenBank, sequence information is analyzed by utilizing Signal P-5.0 and TMHMM, polypeptide sequences of Signal peptide and transmembrane structural domain are removed, and the nucleotide sequence shown as SEQ ID No.1 is obtained according to the optimal sequence of the codon preference of escherichia coli under the condition of not changing the amino acid sequence.

2) Construction of pGEX4T-1-P-LcR recombinant plasmid:

The NoV P structural domain cDNA sequence is obtained from NCBI gene database, and the nucleotide sequence shown in SEQ ID No.3 is obtained according to the optimal sequence of the codon preference of the escherichia coli under the condition of not changing the amino acid sequence. The optimized nucleotide sequence of LcR protein was integrated into loop2 of NoV P domain (length: 1644bp, GC%: 49%) and was synthesized by Hua Dacron gene company to obtain polynucleotide encoding recombinant protein of R7 protein of North Buddha's virus P particle chimeric leukocyte (P-LcR recombinant protein) (shown in SEQ ID NO: 5). Then, the polynucleotide encoding the P-LcR7 recombinant protein is inserted into pGEX4T-1 vector to construct recombinant plasmid pGEX4T-1-P-LcR7, the structure of which is shown in FIG. 3.

3) Construction and identification of recombinant vector pGEX 4T-1-P-LcR:

3.1 Double digestion was performed on the recombinant protein P-LcR and pGEX4T-1 plasmid (both containing BamHI and XhoI sites) respectively, and the double digestion system was as shown in Table 1.

Table 1 double enzyme digestion System

The reaction is carried out for more than 4 hours in a water bath kettle at 37 ℃ by taking 5 pipes respectively and 100 mu L.

3.2 P-LcR and pGEX4T-1 were purified:

(A) The weight of the gel was determined by weighing 1.5mL of clean centrifuge tube and then placing the gel containing the enzyme-digested product therein. Assuming a gel density of 1g/mL, the gel volume is given by: gel sheets having a mass of 0.3g had a volume of 0.3 mL. The gel was mixed with binding buffer (XP 2) in a volume ratio of 1:1 at 50-60℃until the gel was completely melted. Shaking every 2-3 min.

(B) The DNA Mini Column was placed in a 2mL collection tube.

(C) The completely melted DNA agarose solution (not more than 700. Mu.L) from step (A) was added to DNA MiniColumn. Centrifuge at 10,000Xg for 1min at room temperature. The filtrate was discarded and the collection tube reused. This step is repeated.

(D) Add 300 μl binding buffer (XP 2), centrifuge at maximum speed (. Gtoreq.13,000 g) for 1min at room temperature, discard filtrate and reuse the collection tube, add 700 μl SPW wash buffer, centrifuge at maximum speed for 1min at room temperature, discard filtrate and reuse the collection tube.

(E) HiBind DNA Mini Column was centrifuged at maximum speed for 2min to dryness. Note that: drying HiBind DNA Mini Column of the matrix prior to elution is important because residual ethanol may interfere with the operations described below.

(F) HiBind DNA Mini Column was transferred to a clean 1.5mL microcentrifuge tube. 15-30. Mu.L of elution buffer or deionized water was added directly to the column center membrane. Note that: the efficiency of DNA elution from HiBind DNA Mini Column is pH dependent. If the DNA is eluted with deionized water, a pH of around 8.5 should be ensured.

(G) Standing at room temperature for 2min. Centrifuge at maximum speed for 1min. Note that: about 70% of the bound DNA is eluted in the first pass, while the remaining DNA is eluted after the second pass, but at a lower concentration. Finally, the DNA is stored at the temperature of minus 20 ℃ and the recovery concentration is calculated by an Epoch microplate spectrophotometer.

3.3 The recombinant protein P-LcR and pGEX4T-1 cleavage recovery product were ligated. The connection system is shown in table 2 below. After the reagents were applied, the reaction system was placed at 16℃overnight (12-16 h) in a gene amplification apparatus.

Table 2 connection system

3.4 Ligation product conversion:

Taking 100 mu L DH5 alpha competent cells at-80 ℃, melting on ice, adding 10 mu L of the ligation product of the last step, mixing uniformly, and carrying out ice bath for 30min. Water bath at 42 ℃ for 90s, and then ice is put for 2min. 400. Mu.L of LB liquid medium was added thereto, and the culture was continued at 37℃and 220rpm for 45 minutes with shaking, to restore the plasmid resistance. Centrifuging at 4000rpm at 4deg.C for 5min, removing 400 μl of supernatant, applying the rest bacterial liquid to ampicillin plate, culturing at 37deg.C for 30min (plate is put forward), and culturing the plate upside down for about 12 hr after bacterial liquid absorption is complete until single colony is visible.

3.5 Identification of ligation products:

Designing a primer according to the base sequence of the P-LcR recombinant protein, wherein the amplification length is 1644bp:

an upstream primer: 5'-3' TGCAACGGCCGTTGCTCAAG (SEQ ID NO: 9);

A downstream primer: 5'-3' TTAGCAACGGCCGTTGCATAATGC (SEQ ID NO: 10).

BL21 (DE 3) competent cells were taken out from the ultra-low temperature refrigerator, placed on ice to melt, added with plasmid pGEX4T-1-P-LcR (100 ng), fully mixed, ice-bath for 30min,42℃for 45s, ice-bath for 3min, added with 100. Mu.LLB medium, cultured for 60min at 37℃with shaking table at 200rpm, evenly spread on Amp ⁺ plates, and cultured overnight at 37℃with inversion. Colony PCR identification was performed the following day: single colonies were picked on a plate, inoculated on a new Amp ⁺ LB agar plate, and then a PCR reaction system as shown in Table 3 was configured as follows:

TABLE 3 PCR reaction system

The PCR reaction conditions were: pre-denaturation at 94℃for 5min; thermal denaturation at 94℃for 10s, annealing at 60℃for 10s, and elongation at 72℃for 80s for 25 cycles; final extension at 72℃for 10min. After the PCR was completed, agarose gel electrophoresis was performed for 30min at 120V, and analysis was performed on a gel imager. The PCR identification of recombinant vector pGEX4T-1-P-LcR is shown in FIG. 4, where lane M: 2000 LADDER MARKER; lanes 1-4: p particles amplified fragments; lane 5: P-LcR amplified fragment; n lanes: negative control. And sequencing by sending the PCR positive plasmid to a large gene company, comparing the sequencing result with P-LcR, and finally successfully constructing a recombinant (expression) vector pGEX4T-1-P-LcR7, wherein the sequencing result is correct.

Example 2

Preparation of norovirus P particle chimeric LcP.sup.7 recombinant protein

1) Recombinant plasmid transformation

The positive clone of the previous step is propagated, inoculated into 5mL of LB medium containing 100 mug/mL Amp ⁺, shake cultured for 16-20h at 37 ℃ and 200rpm, and plasmid extraction is carried out.

(A) 5mL of the bacterial liquid was packed in 5-branch separation tubes, and centrifuged at 10,000Xg for 1min.

(B) The medium was poured out, 250 μl of each suspension was added, and the mixture was blown down and mixed.

(C) After 250. Mu.L of lysate was added, the tube was gently shaken for 2-3min; vigorous mixing is avoided during processing, as this would cut the chromosomal DNA resulting in lower plasmid purity; and the cracking reaction is not allowed to proceed for more than 5min; the storage solution II should be sealed when not in use to avoid acidification of the carbon dioxide in the air.

(D) 350 μl of neutralization solution was added and immediately inverted several times until a flocculent white precipitate form was obtained, avoiding localized sedimentation.

(E) Centrifugation is carried out at maximum speed (> 13,000 g) for 10min.

(F) HiBind DNA Mini Column was inserted into a 2mL collection tube, the supernatant obtained in step (E) was carefully aspirated into a bottle, avoiding aspiration of particles and cell debris, and centrifuged at maximum speed for 1min.

(G) The filtrate was discarded, the collection tube was reused, 500. Mu.L of HBC Buffer was added, and the mixture was centrifuged at maximum speed for 1min.

(H) The filtrate was discarded and the collection tube was reused, 700 μ LDNAWash Buffer was added and centrifuged at maximum speed for 1min.

(I) The filtrate was discarded and the empty HiBind DNA Mini Column was centrifuged at maximum speed for 2min. Drying HiBind DNA Mini Column the matrix prior to elution is important because residual ethanol may interfere with subsequent operations.

(J) HiBind DNA Mini Column were placed in a 1.5mL microcentrifuge tube.

(K) 30. Mu.L of sterile deionized water was added to the center of the column.

(L) left standing at room temperature for 1min, and centrifuged at the highest speed for 1min. About 70% of the bound DNA is eluted in the first pass, while the remaining DNA is eluted after the second pass, but at a lower concentration.

2. Mu.L of the plasmid extracted in the above step was transformed into BL 21-expressing cells according to the method of step 3.4, and positive clones were identified using the identification primers.

2) Inducible expression of recombinant proteins and identifying inducible expression of recombinant proteins:

The positive clone in the step 1) is selected and inoculated into 4mL of LB culture medium containing 100 mug/mL Amp ⁺, shaking culture is carried out at 37 ℃ and 200rpm, when OD600 is between 0.4 and 0.6, IPTG with the final concentration of 0.5mM is respectively added into the test tubes of the positive clone and the empty bacterial liquid, the culture is carried out for 18 hours at 16 ℃, the other two test tubes are used as negative references of the positive clone and the empty bacterial liquid, and the expression and the solubility of the protein are detected by SDS-PAGE and Western blot.

The detection steps are as follows:

Sample preparation: taking 450 μl of the pellet after centrifugation, re-suspending in 300 μl of lysis buffer (50 mM Tris-HCl,150mM NaCl,5%glycerol,pH 8.0), and performing ultrasonic lysis for 1 min;

Whole-bacteria sample: mixing 100 μ LPBS with 50 μL loading buffer (5×), heating at 100deg.C for 10min, and centrifuging at 12000rpm for 5min.

Supernatant and inclusion body samples: 200 μl of lysate was centrifuged at 15000 rpm for 10min, and the supernatant and pellet were taken separately. Mu.l of loading buffer (6X) was mixed to 50. Mu.l of supernatant as a supernatant sample. The pellet was resuspended in 10. Mu.l loading buffer (6X) and 50. Mu.l PBS as inclusion body samples. After heating at 100℃for 10 minutes, centrifugation was carried out at 15000 rpm for 5 minutes, and the sample was loaded.

3) SDS-PAGE detection of recombinant proteins

Experiments are carried out according to the operation instructions of BeyoGel ^TM SDS-PAGE prefabricated gel (Tris-Gly, 4-20% and 12 holes) of Biyun Tian company, including the treatment of the prefabricated gel, the cleaning and sample loading of sample holes, and the like, then electrophoresis is carried out for 80V 30min and 120V 60min, a gel plate is pried off, and a target gel is cut for standby. Gel staining experiments were performed according to the instructions of coomassie brilliant blue staining kit (P0017A) from the company bi yun, including gel staining, incubation at room temperature for 1h, destaining overnight, gel imaging, etc. And (3) analyzing the solubility proportion of the recombinant protein by utilizing an SDS gray value of a chemiluminescent imaging system, detecting the total protein concentration of the crushed sample by utilizing a BCA protein concentration detection kit, and predicting the concentration of the target protein by utilizing the solubility proportion.

FIG. 5 shows the SDS-PAGE result of the recombinant protein P-LcR in the example of the present invention, wherein the M1 lane: protein marker; lane 1: inducing the recombinant whole bacteria (0.5 mM IPTG) for 18h at 16 ℃; lane 2: non-induced recombinant whole bacteria; lane 3: inducing the empty whole bacteria (0.5 mM IPTG) for 18h at 16 ℃; lane 4: uninduced empty whole bacteria; lane 5: cell lysis supernatant (0.5 mM IPTG) induced at 16℃for 18 h; lane 6: cell lysis pellet (0.5 mM IPTG) was induced for 18h at 16 ℃. The bands pointed by the arrows in this figure are the bands corresponding to the recombinant protein P-LcR. The figure illustrates that P-LcR7 was successfully expressed in the supernatant and that more soluble protein was present than inclusion body protein.

4) Western-blotting analysis of recombinant proteins

(A) Electrophoresis: the expressed target protein was used as a sample for SDS-PAGE electrophoresis, and 1 piece of PVDF membrane with a proper size and 4 pieces of filter paper with the same size were previously cut before the electrophoresis was completed. The PVDF membrane is soaked in methanol for 5min for activation, and then is immersed in electrotransport buffer for 10min. Slightly soaking the filter paper and the sponge in an electrotransport buffer solution;

(b) Transferring: and after electrophoresis is finished, cutting a needed gel part into an electrotransport buffer liquid bubble for 10min, putting the gel part into a negative plate, a sponge, 2 layers of filter paper, a membrane, a separation gel, 2 layers of filter paper, the sponge and a positive plate in sequence, and expelling bubbles possibly existing between the filter paper, the gel and the membrane by using a glass rod during the putting, otherwise, the bubble imprinting is visible on the membrane during the color development. Placing the film transfer plate into an electric transfer tank, adding an electric transfer buffer solution to the position of the upper edge of the film, and carrying out electric transfer for 60min at a constant voltage of 80V;

(c) Washing the film: washing PVDF membrane after membrane transfer with TBST or PBST buffer solution at 37deg.C for 3 times, each time for 5min;

(d) Closing: soaking the washed membrane in 5% skimmed milk powder sealing solution, shaking at 80rpm at room temperature for 1 hr or overnight at 4deg.C; washing the film: washing with TBST or PBST buffer solution at 37deg.C for 5min for 3 times;

(e) Incubation resistance: diluting HIS monoclonal antibody with TBST at a ratio of 1:5000, and reacting with PVDF membrane at 37deg.C for 1h or overnight at 4deg.C;

(f) Washing the film: washing with TBST or PBST buffer solution at 37deg.C for 5min for 3 times;

(g) Incubation of secondary antibody: diluting goat anti-mouse IgG polyclonal antibody by TBST (Tunnel boring st) at a ratio of 1:10000, soaking PVDF (polyvinylidene fluoride) membrane therein, and reacting for 1h at room temperature;

(h) Washing the film: washing with TBST or PBST buffer solution at 37deg.C for 5min for 3 times;

(i) Color development: the ECL color development is carried out for 2-3 min, then TBST is used for stopping the reaction, and a chemiluminescent imaging system is used for observing the specific band.

The detection result proves that the P-LcR recombinant protein is indeed obtained.

Example 3

Purification of the recombinant protein of the norovirus P particle chimeric LcP7

The recombinant expression bacteria were amplified in large amounts at 10000rpm for 10min, the cells were collected, resuspended in 10mL of binding buffer, sonicated for 50min, then the supernatant was collected at 10000rpm for 20min, filtered through a 0.45 μm filter and purified using PurKine ^TM GST-Tag Purification Nickel Column kit. The purification comprises the following specific steps:

(i) And (3) column loading: resuspension medium, adding proper amount of medium into chromatographic column according to the amount of protein to be purified, and standing.

(Ii) Balance: 5-10 column volumes of equilibration buffer (140mM Nacl;2.7mM Kcl;10mM Na2HPO4;1.8mMKH2PO4;PH7.4) were added to the column to equilibrate the column until effluent conductance and pH were unchanged (consistent with equilibration solution).

(Iii) Loading: the treated protein extract samples (prepared by mixing the protein extract with an equal volume of Binding buffer) were added to the resin and incubated. To avoid clogging the column, the sample should be centrifuged or microfiltered (0.45 um).

(Iv) Washing: after the sample is loaded, the chromatographic column is washed by an equilibrium buffer solution with the volume of 5-10 times of the column volume, and effluent liquid is collected.

(V) Eluting: the solution was eluted with elution buffer (50 mM Tris-Cl pH 8.0, 10m M reduced glutathione. Glutathione concentration was adjusted appropriately depending on the binding force of the target protein. Glutathione was easily oxidized and was prepared in situ), and the effluent was collected and stored at-80 ℃.

(Vi) And (3) detection: the fractions containing the target protein were detected and identified by SDS-PAGE and Western Blot methods.

FIG. 6 shows SDS-PAGE results of purified P-LcR7 recombinant proteins obtained in the example of the present invention, wherein M lanes: protein marker; lane 1: ultrasonic supernatant; lane 2: loading sample flowing liquid; lane 3: a washing liquid; lane 4: a first eluent; lane 5: and eluting the second time. The bands pointed by the arrows in this figure are the bands corresponding to the recombinant protein P-LcR.

FIG. 7 is a diagram showing the results of SDS-PAGE (left) and Western blot (right) identification of the purified monomers and polymers of the P-LcR protein in the examples of the present invention; wherein lane M: protein marker; lane 1: the recombinant protein P-LcR is loaded. The bands pointed by the arrows in the figure are bands corresponding to the P-LcR recombinant protein, and the upper and lower arrows respectively indicate multimeric and monomeric recombinant proteins, which indicate that the P-LcR7 recombinant protein can self-assemble into multimeric protein particles in vitro.

6) Projection electron microscopy observation of P-LcR7 multimeric protein formation

The purified P-LcR protein of example 3 was removed, diluted to a protein concentration of 1mg/mL, and 10. Mu.L of the diluted protein sample was removed, and the solution was added dropwise to a copper mesh to precipitate for 1min, and the supernatant was removed by filtration. 10 mu L of uranyl acetate is added dropwise to the copper net to precipitate for 1min, the floating liquid is sucked by filter paper, and the copper net is dried for a plurality of minutes at normal temperature. And (3) carrying out electron microscope detection imaging under the voltage condition of 80-120kv, observing the morphology of protein particles, and photographing and storing the transmission electron microscope imaging result, wherein the result is shown in fig. 8.

Example 4

Preparation of P-LcR7 ₁₂ recombinant protein

The inventor also constructs and obtains a chimeric P domain containing two sections of R7 protein gene sequences by inserting the R7 protein gene fragments into a first epitope loop and a second epitope loop of NoV P domain simultaneously, wherein the recombinant P domain is called P-LcR7 ₁₂, and the recombinant plasmid is called pGEX4T-1-P-LcR7 ₁₂ plasmid.

1) Construction of pGEX4T-1-P-LcR ₁₂ recombinant plasmid

By homologous recombinationMultiS the kit links R7 to the loop1 on pGEX 4T-1-P-LcR.

(1) The primers were designed as shown in Table 5 below:

TABLE 5 pGEX4T-1-P-LcR ₁₂ primer

(2) Linearization amplification of pGEX4T-1-P-LcR ₁₂ plasmid the PCR system was configured as shown in Table 6 below and the amplification procedure is shown in Table 7.

Table 6 pGEX4T-1-P-LcR ₁₂ plasmid linearization system

Table 7 pGEX4T-1-P-LcR ₁₂ plasmid linearization amplification procedure.

(3) The target fragment R7 homology arm was amplified, and the PCR system was prepared as shown in Table 8, and the amplification procedure was as shown in Table 9.

TABLE 8 target fragment R7 homology arm amplification system

TABLE 9 target fragment R7 homology arm amplification procedure

(4) Ligation, transformation and identification

The connection system is prepared according to the table 10, and after being blown and evenly mixed, the mixture is reacted for 30min at 37 ℃, and after the completion, the mixture is stored at 4 ℃. Subsequently, 10. Mu.L of the recombinant product was mixed with 100. Mu.L of competent cells for transformation, and positive clones were identified as in step 3.6 and step 3.7 above.

Table 10 connection system

The positive clone was further cultured, treated, identified and purified to obtain P-LcR7 ₁₂ recombinant protein.

FIG. 9 is a diagram showing the results of SDS-PAGE (left) and Western blot (right) identification of the purified monomers and polymers of the P-LcR ₁₂ protein in the examples of the present invention; wherein lane M: protein marker; lane 1: the recombinant protein P-LcR7 ₁₂ was loaded, and the result shows that the single polymer and the multimeric band of the protein P-LcR7 ₁₂ can be seen in a Western blot electrophoresis chart, which shows that the P-LcR7 ₁₂ can be self-assembled into multimeric VLPs in vitro.

Example 5

Immunoprotection efficacy evaluation of norovirus P particle chimeric LcP recombinant protein

50 Mug of the purified P-LcR recombinant protein obtained in example 3 was mixed with a high-efficiency compound adjuvant B (white oil adjuvant, guangzhou Qiaoling Biotechnology Co., ltd.) in a ratio of 2:3, and the mixture was shaken and mixed until the oil drops drop by drop on the water surface were not spread (the mixture was used). The LcR protein and the P-LcR7 ₁₂ protein are taken to be 50 mug respectively, and the oil adjuvant vaccine is prepared according to the proportion and the process.

The prepared three oil adjuvant vaccines are respectively immunized on 5 spf chickens of 4 days old, the immunization is enhanced once in 14 days after the immunization, and serum is collected and separated weekly before and after the immunization to detect specific IgG antibodies.

The serum ELISA detection comprises the following specific steps:

① Coating: lc-R7 protein was diluted 1:50 times (to 0.1 ug/ml) with 1 Xcoating solution and added to the ELISA plate at 100 ul/well. Incubate at 37℃for 2 hours.

② Washing and drying: pouring out the liquid in the ELISA plate, beating to dry, washing for 3 times (plate washer), and beating to dry again.

③ Closing: after beating to dryness, the mixture was blocked with 5% skim milk at 200 ul/well. Overnight at 4 ℃.

④ Washing and drying: the same as in the step (2).

⑤ Adding serum to be detected: the serum to be tested was diluted 1:100 with 5% skim milk, 100 ul/well. Incubate at 37℃for 1 hour. (1 ul serum+100 ul skim milk)

⑥ Washing and drying: the same as in the step (2).

⑦ Adding a secondary antibody: sheep anti-chicken IgG-HRP was diluted 1:4000 with 5% skim milk at 100 ul/well. Incubate at 37℃for 0.5 hours.

⑧ Washing and drying: the same as in the step (2).

⑨ Color development: 100ul of color development solution was added to each well, incubated at 37℃for 15 minutes, and stop solution was added at 50 ul/well.

⑩ Reading the result: the results were read with OD 450. The data and plots were analyzed using GRAPHPAD PRISM and the results are shown in table 4 and fig. 10.

Table 4 results of specific antibody levels after immunization with LcR7, P-LcR and P-LcR7 ₁₂ proteins.

From Table 4 and FIG. 10, it can be seen that the oil adjuvant vaccinated group vaccinated with the recombinant proteins P-LcR7 and P-LcR7 ₁₂ resulted in higher levels of specific antibodies and higher efficiency in raising the antibody levels, faster increases in antibody levels, and longer duration, compared to the oil adjuvant vaccinated group vaccinated with LcR, and that the oil adjuvant vaccinated group vaccinated with the recombinant proteins P-LcR7 and P-LcR7 ₁₂ remained higher after 91 days (about 3 months).

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of this application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.

Claims

1. A recombinant protein of a chimeric LcR protein of a norovirus P particle, characterized in that the recombinant protein comprises a norovirus P domain and a Leucomatous R7 protein,

The amino acid sequence of the R7 protein of the Carpesium calycinum is shown as SEQ ID No.2

The amino acid sequence of the norovirus P domain is shown as SEQ ID No. 4.

2. The recombinant protein of the norovirus P particle chimeric LcR protein according to claim 1, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID No.6 or SEQ ID No.8, or the amino acid sequence of the recombinant protein has more than 85% sequence identity with the sequence shown as SEQ ID No.6 or SEQ ID No. 8.

3. A polynucleotide encoding a recombinant protein of the norovirus P particle chimeric LcR protein of claim 1 or 2.

4. A polynucleotide according to claim 3, wherein the nucleotide sequence of the polynucleotide is set forth in SEQ ID NO:5 or SEQ ID No.7, or

The nucleotide sequence of the polynucleotide is identical to the nucleotide sequence of SEQ ID NO:5 or SEQ ID No.7 has a sequence identity of more than 85%.

5. A recombinant vector comprising the polynucleotide of claim 3 or 4.

6. A recombinant bacterium comprising the recombinant vector of claim 5, or the polynucleotide of claim 1 or 2 integrated into the genome of the recombinant bacterium.

7. A calicheating leukocyte worm vaccine comprising a recombinant protein of the norovirus P particle chimeric LcR protein of claim 1 or 2.

8. A pharmaceutical composition comprising the calicheating leukocyte worm vaccine of claim 7; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

9. A method for preparing a recombinant protein of the norovirus P particle chimeric LcR protein according to claim 1 or 2, characterized in that the preparation method comprises the steps of:

(1) Constructing a recombinant vector comprising a nucleic acid molecule encoding a recombinant protein of the norovirus P particle chimeric LcR protein of claim 1 or 2;

10. Use of a recombinant protein of the norovirus P particle chimeric LcR protein of claim 1 or 2, a nucleic acid molecule of claim 3 or 4, a recombinant vector of claim 5, a recombinant bacterium of claim 6, a calicheating leukocyte worm vaccine of claim 7, or a pharmaceutical composition of claim 8 in the manufacture of a medicament or vaccine for the treatment or prevention of calicheating leukocyte worm disease.