CN112662694A - Maltose binding protein, maltose binding protein expression vector, recombinant engineering bacteria and application thereof - Google Patents

Abstract

The invention provides maltose binding protein, a maltose binding protein expression vector, recombinant engineering bacteria and application thereof, and belongs to the technical field of maltose binding protein preparation. The maltose-binding protein expression vector of the invention takes pMAL-C2 as an original vector, and a gene containing a termination code is inserted behind a malE gene. According to the invention, a gene containing a termination code is inserted into a multienzyme cutting site region behind a pMAL-C2 carrier malE gene, so that the expression of a downstream LacZ alpha gene can be avoided, a process of enzyme digestion for removing LacZ alpha is not needed in the process of preparing maltose-binding protein based on a maltose-binding protein expression carrier, MBP (43.12KD) can be directly obtained through affinity chromatography, and the preparation process of the MBP (43.12KD) can be simplified.

Description

Maltose binding protein, maltose binding protein expression vector, recombinant engineering bacteria and application thereof

Technical Field

The invention relates to the technical field of maltose binding protein preparation, and in particular relates to maltose binding protein, a maltose binding protein expression vector, recombinant engineering bacteria and application thereof.

Background

Coli Maltose Binding Protein (MBP) is encoded by the e.coli malE gene and is primarily responsible for e.coli transport and carbohydrate metabolism. It has been considered to have no biological activity or low biological activity, and is often used as a labeled protein in modern molecular biology research. Currently, there is increasing interest in the study of the immunopotentiating activity of MBP.

The existing research finds that MBP can induce the activation of Th1 in vitro, enhance the killing activity of NK cells and the phagocytic function of macrophages, and indicates that the MBP has the function of enhancing immunity. MBP combined with BCG can synergistically induce the activation of Th1 and increase the secretion level of IFN-gamma as a vaccine adjuvant, and further research proves that the MBP combined with BCG can directly act on CD4+ T cells in vitro, and the related molecular mechanism is related to TLR2/TLR4/TLR9 and a signal path between the TLR and the TLR 4. And subsequent studies show that the MBP combined with BCG can induce DC cell activation and maturation through TLR2 and TLR4, thereby indirectly promoting Th1 cell activation. Based on the above research results, it is possible that MBP is used as an immunopotentiator for vaccine development, and a simple and efficient process for preparing maltose binding protein is required for further research on the immune effect of MBP.

The pMAL-C2 system is a high-efficiency protein fusion expression and purification system. The pMAL-C2 vector contains an E.coli malE gene encoding MBP, and a downstream multiple cloning site thereof facilitates the insertion of a target gene, thereby expressing a fusion protein with MBP at the N-terminal. The vector can make cloned genes obtain high-efficiency expression through a 'tac' strong promoter and malE translation initiation signals, and further utilizes the affinity of MBP to maltose to achieve the further affinity purification of the fusion protein by using an Amylose column. The vector contains an Xa factor enzyme cutting site (IEGRIEF, enzyme cutting between R and I) between malE and a multi-enzyme cutting site, a target gene fragment is inserted behind the Xa factor enzyme cutting site, and MBP and a target protein can be separated through enzyme cutting.

The pMAL-C2 vector contains an Escherichia coli malE gene for coding MBP, can be used for expressing and purifying the MBP under the condition of not inserting a target gene, but the expressed protein has a downstream LacZ alpha sequence, and the LacZ alpha needs to be removed by enzyme digestion of an Xa factor enzyme digestion site, and the preparation process is complex. If a gene fragment containing a termination codon is inserted into the multiple cloning site behind the malE gene of the pMAL-C2 vector, the expression of the LacZ alpha gene can be avoided, and MBP (43.12KD) can be directly obtained by affinity chromatography.

Disclosure of Invention

The invention aims to provide maltose binding protein, a maltose binding protein expression vector, recombinant engineering bacteria and application thereof. The maltose-binding protein can be simply and efficiently prepared by utilizing the maltose-binding protein expression vector or the recombinant engineering bacteria.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a maltose-binding protein expression vector, which takes pMAL-C2 as an original vector and inserts a gene containing a termination code behind a malE gene.

Preferably, the gene containing the termination codon is inserted between the EcoRI and BamHI sites on pMAL-C2.

The invention also provides a recombinant engineering bacterium comprising the maltose-binding protein expression vector.

The invention also provides an application of the maltose binding protein expression vector or the recombinant engineering bacteria in preparation of maltose binding protein.

The invention also provides a maltose-binding protein expression vector based on the scheme or a maltose-binding protein prepared by the recombinant engineering bacteria.

The invention also provides a vaccine immunopotentiator, which comprises CPG-ODN and maltose-binding protein described in the scheme.

The invention also provides application of the immunopotentiator in the scheme in preparation of immunopotentiating drugs.

Preferably, the immunopotentiator is used as a vaccine adjuvant.

Preferably, the maltose binding protein and CPG-ODN enhance vaccine immunity by promoting lymphocyte proliferation, inducing lymphocyte proliferation and differentiation to Th1 cells, increasing DC proportion, increasing cDC1 proportion, increasing cDC2 proportion and up-regulating DC maturation.

The invention provides a maltose-binding protein expression vector, which takes pMAL-C2 as an original vector and inserts a gene containing a termination code behind a malE gene. The maltose-binding protein expression vector of the invention takes pMAL-C2 as an original vector, and a gene containing a termination code is inserted behind a malE gene. According to the invention, a gene containing a termination code is inserted into a multienzyme cutting site region behind a pMAL-C2 carrier malE gene, so that the expression of a downstream LacZ alpha gene can be avoided, a process of enzyme digestion for removing LacZ alpha is not needed in the process of preparing maltose-binding protein based on a maltose-binding protein expression carrier, MBP (43.12KD) can be directly obtained through affinity chromatography, and the preparation process of the MBP (43.12KD) can be simplified.

Drawings

FIG. 1 is a schematic representation of the engineering of the pMAL-C2 vector;

FIG. 2 shows the result of enzyme-cutting electrophoresis identification of the modified pMAL-inserted gene vector, wherein 1 is marker, 2 is pMAL-C2 vector, 3 is enzyme-cutting pMAL-C2 vector, 4 is modified pMAL-inserted gene vector, and 5 is enzyme-cutting modified pMAL-inserted gene vector;

FIG. 3 shows the results of high-pressure SDS assay of dust;

FIG. 4 shows the SDS-based identification of purified proteins, where 1 and 16 are markers; 2 is an effluent liquid; 3 and 4 are impurity washing liquid; 5-15 and 17-20 are eluents;

FIG. 5 shows the detection results of the content of bacterial endotoxin in MBP protein after 2 times of ultrafiltration and centrifugation, wherein the No. 1 negative result is negative, the No. 2 positive result is positive, the No. 3 MBP protein is negative, and the No. 4 test sample is positive, and the endotoxin is qualified;

FIG. 6 shows the result of WST1 on the proliferation of splenic lymphocytes in mice (1.5h), p < 0.05, p < 0.01;

FIG. 7 shows the results of ELISA assay of IFN-. gamma.p < 0.05 and P <0.01 in the culture supernatant of splenic lymphocytes from mice;

FIG. 8 shows the effect of MBP in combination with CpG2006 in vitro stimulation on DC subsets in splenocytes, where A is a gating strategy; b is a representative flow scattergram of each DC subgroup in the mouse splenocytes; c is the proportion of the total DC group to XCR1+ DC subgroup; d is the ratio of cDC2 to pDC subgroup; p < 0.05, p < 0.01;

FIG. 9 shows the effect of MBP in combination with CpG2006 in vitro stimulation on DC maturation in splenocytes, where A is a representative flow scatter plot of markers of maturation on DCs in mouse splenocytes; b is the expression level of the maturation marker on DC in the mouse splenocytes; p < 0.05, p < 0.01;

FIG. 10 shows the synergistic anti-tumor effect of MBP in combination with CpG2006 in mice, where A is the tumor weight of 10 mice per group; b is tumor inhibition rate (%) × 100% (1-experimental/control tumor weight); p < 0.05, p < 0.01.

FIG. 11 shows the effect of MBP in combination with CpG2006 as a vaccine immunopotentiator on DC subpopulations within mouse dLN, where A is a gating strategy; b is a representative flow scattergram of each subset of DCs in mouse dLN; c is the proportion of each subgroup of DCs in mouse dLN cells; p < 0.05, p < 0.01;

FIG. 12 shows the effect of MBP in combination with CpG2006 as a vaccine immunopotentiator on DC maturation within mouse dLN, where A is a representative flow scatter plot of maturation markers on DCs in mouse dLN; b is the expression level of the maturation marker on DCs in mouse dLN; p < 0.05, p < 0.01.

FIG. 13 shows the effect of MBP in combination with CpG2006 as a vaccine adjuvant on tumor suppression rate; wherein a is the tumor weight of 10 mice per group; b is tumor inhibition rate (%) × 100% (1-experimental/control tumor weight); p < 0.05, p < 0.01.

FIG. 14 shows the effect of MBP in combination with CpG2006 as a vaccine adjuvant on humoral immunity and Th cell immune responses, where A is the level of MUC 1-specific antibodies in serum; b is the ratio of IgG2c/IgG 1; c is the secretion of spleen cell IFN-gamma specific to MUC1 detected by ELISA; d is ELISA for detecting the IL-4 secretion of the MUC1 specific splenocytes; p < 0.05, p < 0.01.

FIG. 15 shows that MBP (43.12KD) synergistically increases CTL killing activity in combination with CpG2006, where A is a dynamic curve for monitoring CTL killing by the RTCA method (E: T is 50: 1); b is MUC 1-specific CTL killing activity (calculated 72 hours after addition of effector cells); MUC 1-specific CTL killing activity (%) [1-CI (experimental group)/CI (NS group) ] × 100%;

Detailed Description

The invention provides a maltose-binding protein expression vector, which takes pMAL-C2 as an original vector, and inserts a gene containing a termination code behind a malE gene; the nucleotide sequence of the gene containing the termination code is preferably as shown in SEQ ID NO: 1, wherein 4-6 are termination codes.

According to the invention, a gene containing a termination code is inserted into a multienzyme cutting site region behind a pMAL-C2 carrier malE gene, so that the expression of a downstream LacZ alpha gene can be avoided, a process of enzyme digestion for removing LacZ alpha is not needed in the process of preparing maltose-binding protein based on a maltose-binding protein expression carrier, MBP (43.12KD) can be directly obtained through affinity chromatography, and the preparation process of the MBP (43.12KD) can be simplified.

In the present invention, the gene containing the termination codon is inserted between EcoRI and BamHI sites on pMAL-C2; the nucleotide sequence of the maltose-binding protein expression vector is shown as SEQ ID NO: 2, wherein 2695-2700 are EcoRI sequences, and 3103-3108 are BamHI sequences; 1528 to 2706 are malE gene sequences; 2701-3702 are inserted genes; 2704 to 2706 are termination codes tag.

The invention also provides a recombinant engineering bacterium comprising the maltose-binding protein expression vector. In the present invention, the recombinant bacterium preferably uses Escherichia coli as a starting bacterium.

The invention also provides an application of the maltose binding protein expression vector or the recombinant engineering bacteria in preparation of maltose binding protein.

In the present invention, when the original bacterium of the recombinant engineered bacterium is escherichia coli, the application preferably comprises the following steps:

1) inoculating the recombinant bacteria including the recombinant vector to an LB liquid culture medium, carrying out amplification culture to obtain a bacterial liquid, adding IPTG (isopropyl-beta-thiogalactoside) into the bacterial liquid, carrying out induction culture to obtain an induction liquid, centrifuging the induction liquid, and collecting precipitates to obtain bacterial dust;

2) dissolving the bacterial dust in a washing liquor buffer, homogenizing and crushing to obtain a homogeneous liquid, centrifuging the homogeneous liquid, and collecting a supernatant;

3) and carrying out Amylose affinity chromatography purification on the supernatant to obtain an eluent containing maltose binding protein.

The method comprises the steps of firstly inoculating recombinant bacteria comprising a recombinant vector to an LB liquid culture medium, carrying out amplification culture to obtain a bacterial liquid, adding IPTG (isopropyl-beta-thiogalactoside) into the bacterial liquid, carrying out induction culture to obtain an induction liquid, centrifuging the induction liquid, and collecting precipitates to obtain bacterial dust.

In the present invention, the LB liquid medium contains AMP; the concentration of AMP is preferably 0.1 mg/ml; the temperature of the amplification culture is preferably 37 ℃; the mode of the amplification culture is preferably shaking culture; the rotation speed of the shaking culture is 220 rpm; the amplification culture is preferably carried out twice, the time of the first amplification culture is preferably 12-16h, after the first amplification culture, the cultured bacterial liquid is transferred to a new LB liquid culture medium for the second amplification culture, the time of the second amplification culture is preferably 2-3 h, the OD value of the bacterial liquid is preferably 0.8-1.2, the OD value of the bacterial liquid is further preferably 1, and the bacterial growth is shown to be in a logarithmic growth phase. In the present invention, the final concentration of IPTG is preferably 1 mol/mL; the time for induction culture is preferably 4-5 h; the temperature of the induction culture is preferably 37 ℃. In the invention, the rotating speed of the centrifugation is preferably 8000-10000 rpm; the time for centrifugation is preferably 15-20 min.

After bacterial dust is obtained, the bacterial dust is dissolved in a washing liquor buffer, homogenization and crushing are carried out to obtain a homogeneous liquid, the homogeneous liquid is centrifuged, and a supernatant is collected; the homogenizing time is preferably 4 times, and the pressures of the homogenization 1 to the homogenization 4 are preferably 200bar, 850bar and 850ba respectively; after four times of homogenization, the bacterial dust is completely broken and the protein is completely released. In the present invention, the washing solution buffer is 1L, and preferably comprises the following components: 20ml of 1M Tris-HCl, 0.5M EDTA2ml, 11.6889g of NaCl, 700. mu.l of beta-mercaptoethanol and the balance of ultrapure water.

After obtaining the supernatant, the invention carries out the purification of the Amylose affinity chromatography to the supernatant to obtain the eluent containing the maltose binding protein. In the present invention, the conditions of the Amylose affinity chromatography preferably include: the diameter of the chromatographic column is 1.6 cm; the chromatography medium is Amylose resin; the eluent is a washing liquid buffer containing Maltose, and the proportion of Maltose to the washing liquid buffer is 0.18105 g: 50 ml; the flow rate of the eluent was 8 s/drop. In the present invention, the formulation of the lotion buffer is as described above, and is not described herein.

After the eluent is obtained, the method preferably further comprises the steps of carrying out first ultrafiltration concentration on the eluent by adopting a 30kD ultrafiltration tube to obtain a first concentrated solution, and carrying out second concentration on the first concentrated solution by adopting a 100kD ultrafiltration tube to obtain a concentrated solution containing maltose binding protein. In the specific implementation process of the invention, a 100kD ultrafiltration tube is soaked in 0.5M NaOH for 4h, washed with double distilled water for 40 times, and centrifuged at 4000rpm for 15min to remove endotoxin in MBP protein; the number of times of centrifugation is preferably 2.

The invention also provides a maltose-binding protein expression vector based on the scheme or a maltose-binding protein prepared by the recombinant engineering bacteria.

In the present invention, the amino acid sequence of the maltose binding protein is shown in SEQ ID NO3。

The invention also provides an immunopotentiator, which comprises CPG-ODN and maltose-binding protein described in the scheme.

In the present invention, the concentration of CpG-ODN is preferably 10 μ g/ml, the concentration of MBP is preferably 10 μ g/ml, and the incubation time is preferably 48h in the in vitro assay of CPG-ODN and maltose binding protein.

In the present invention, the concentration of CpG-ODN is preferably 6.25. mu.g/mouse, the concentration of MBP is preferably 50. mu.g/mouse, the number of immunizations is preferably 5, and the interval between immunizations is preferably 1 week in the in vivo mouse assay of CPG-ODN and maltose-binding protein.

In the present invention, the maltose binding protein and CPG-ODN preferably achieve immune enhancing effect by promoting proliferation of lymphocytes, inducing proliferation and differentiation of lymphocytes into Th1 cells, increasing the ratio of DCs 1 and DCs 2, and up-regulating DC maturation.

The invention also provides application of the immunopotentiator in the scheme in preparation of immunopotentiating drugs. In the present invention, the medicament includes a vaccine.

In the present invention, the immunopotentiator is used as a vaccine adjuvant. In the invention, the immunopotentiator is used as a vaccine adjuvant in an in-vivo experiment of mice, the concentration of CpG-ODN is preferably 50 mug/mouse, the concentration of MBP is preferably 50 mug/mouse, the concentration of MUC1 is preferably 50 mug/mouse, the immunization frequency is preferably 5 times, and the immunization time interval is preferably 1 week.

In the present invention, the immunopotentiator preferably enhances the anti-tumor effect of the vaccine by increasing the Th1 type immune response in mice and increasing the CTL killing activity in mice as an adjuvant. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental example 1 transformation of pMAL-C2 vector

1. Materials main experimental apparatus and reagents: a gene containing a termination code (synthesized by Biotechnology, Ltd.); pMAL-C2 vector (Changchun Kangyue Biotech Co., Ltd.); DH5 alpha allelopathic bacteria (Changchun Kangyue Biotech Co., Ltd.); EcoR I (TAKARA, 1040A); BamHI (TAKARA, 1010A); t4 DNALigase (TAKARA; 2011A); plasmid miniprep kit (Tiangen); DL2000 DNAmaker (TAKARA, D501A); GelGreen (Kaiyi organisms, KGMO 25G-1); IPTG (Sigma, 0.1mol/ml)

2. Method of producing a composite material

2.1 construction of plasmids

The pMAL-C2 vector was digested with EcoRI/BamHI and ligated to the gene fragment to be inserted via T4 DNAligase.

In this example, a schematic representation of the pMAL-C2 vector engineering is shown in FIG. 1.

(1) The pMAL-C2 vector is subjected to EcoR I and BamH I double enzyme digestion, and the enzyme digestion reaction system is as follows.

pMAL-C2 vector	21μl
		10×buffer	3μl
BamHI	1μl
		EcoRI	1μl
ddH2O	4μl
		Total reaction system	30μl

Enzyme cutting conditions are as follows: water bath at 37 ℃ for 2 h.

(2) Agarose gel electrophoresis and DNA recovery: the DNA fragment was separated by electrophoresis on a 1.5% agarose gel, and the vector fragment pMAL-C2 and the pseudoinserted gene fragment were recovered by freeze-thawing.

(3) Ligation of the insert to the vector: the inserted gene fragment was ligated with the pMAL-C2 vector under the action of T4 ligase as follows.

Connection conditions are as follows: the reaction was carried out at 16 ℃ overnight.

2.2 transformation and screening of recombinant plasmids:

the transformed pMAL-C2 plasmid is transformed into competent cells of Escherichia coli DH5 alpha, and AMP drug resistance screening is carried out.

(1) 3 laboratory-prepared DH 5. alpha. competent cells (100. mu.l) were lysed on ice.

(2) Adding 210 μ l of the extracted and transformed pMAL-C into 100 μ l of DH5 alpha competent cells, mixing uniformly, and ice-cooling for 30 min.

(3) The mixture was subjected to water bath shock at 42 ℃ for 90s, ice-cooled for 3min, 900. mu.l of LB medium without AMP was added to each tube, and shake-cultured in a shaker at 37 ℃ for 45 min.

(4) Centrifuge at 3000rpm for 5min at 4 deg.C, discard 600. mu.l of supernatant, and leave 400. mu.l of resuspended cells.

(5) And (3) paving the residual bacterial liquid on an LB culture plate containing AMP, standing for half an hour, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing for 24 hours at 37 ℃.

(6) Monoclonal colonies were picked and inoculated into 5ml liquid medium containing AMP, shaken for 16h with parameters set to: 37 ℃ and 210 rpm.

(7) After the bacteria shaking is finished, extracting plasmids, carrying out recombinant plasmid identification, and preserving strains.

2.3 extraction and identification of recombinant plasmid:

(1) plasmid extraction: the recombinant plasmid DNA was extracted according to the plasmid extraction kit instructions and the plasmid concentration was determined.

(2) And extracting 1 mu g of recombinant plasmid, and performing DNA sequencing identification.

(3) Enzyme cutting electrophoresis identification of recombinant plasmid: the recombinant expression vector was double digested with EcoR I and BamH I, and the digested products were identified by 1.5% agarose gel electrophoresis, the results are shown in FIG. 2.

3. As a result, we succeeded in constructing pMAL-inserted gene vector.

EXAMPLE 2 purification of MBP (43.12KD)

1. Materials main experimental apparatus and reagents: a high pressure homogenizer (AH-1500, ATS); AKTA (

KTA^TMpure 150L, GE, usa); gel imaging analysis system (Tanon 2500R, Shanghai Tianneng technologies, Inc.); β -mercaptoethanol (GENVIEW); amylose resins (BioLabs); bacterial endotoxin working standards (incorporated Andos Bio Inc. of Zhanjiang); limulus reagent (Zhenjiang Andos biology Co., Ltd.)

2. Method of producing a composite material

2.1 engineering bacteria culture

3.5ml of the engineering bacteria (transformed with the transformed plasmid pMAL-C2 Escherichia coli DH5 alpha) was pipetted into 350ml of LB medium containing AMP (AMP concentration 0.1mg/ml, 1: 1000), shaken in a shaker, and the parameters were set: the temperature is 37 ℃, the oscillation frequency is 220rpm, and the time is 12-16 h. The next day, 350ml of the inoculum was evenly distributed into seven large conical flasks with 500ml LB (with AMP) medium, 50ml each, in a total volume of 550 ml. Then, the cells were placed in an incubator shaker and shaken for 2.5 hours (shaking times 220 rpm). And detecting the OD value of the bacterial liquid to 1.0 by using an ultraviolet spectrophotometer. Adding an inducer IPTG (final concentration is 0.1-1 mol/mL) into the LB culture medium, and continuing shaking for 5 h. Centrifuging at 8000rpm for 20min to obtain bacterial dust.

2.2 high-pressure homogenization of the crushed dust

Dissolving the fungus dust by using a washing liquor buffer, filtering by using an autoclave gauze with four layers, homogenizing for four times by using a homogenizer under the pressures of 200bar, 850bar, 850bar and 850bar respectively, collecting the homogenized bacterium liquid for 4 times, identifying the fungus dust crushing condition under different times of homogenization by using SDS-PAGE electrophoresis, and obtaining the electrophoresis result after the 4 th time of homogenization as shown in figure 3.

2.2 Amylose affinity chromatography purification of proteins

A chromatographic column with the diameter of 1.6cm is added with 6ml of new Amylose resin medium, namely the height of 3cm, and the column is flushed for 10 times by using sterilized ultrapure water and filled with full flow rate; the column was flushed twice with washing solution buffer at full flow rate. Sucking out the medium, mixing the medium with the supernatant of the crushed and centrifuged bacteria solution, and shaking and mixing for 45min at 4 deg.C (on ice). The mixed liquid of the supernatant and the medium was put into a column and allowed to stand for 20 min. After the resin in the column had completely precipitated, the effluent was collected at a flow rate of 6 s/drop. The wash solution was collected 3 times using 20ml of wash buffer (prepared by placing on ice beforehand) at flow rates of 6 s/drop, 6 s/drop and 10 s/drop, respectively. 7ml of eluent (Maltose 0.18105g + Wash buffer 50ml) was added and the eluates (15 vials in total, 1 ml/vial) were collected at a flow rate of 8 s/drop. SDS-PAGE protein identification was performed on each vial sample (as shown in FIG. 4).

2.3 MBP protein concentration and endotoxin removal

MBP protein was concentrated using a 30kD ultrafiltration tube and centrifuged for 15min at 4000 rpm. The concentration of MBP protein was determined by the Lowry method. Diluted to about 0.2mg/ml with 0.9% NACL based on MBP protein concentration. Soaking 100kD ultrafilter tube in 0.5M NaOH solution for 4h, washing with double distilled water for 40 times, centrifuging at 4000rpm for 15min to remove endotoxin in MBP protein, and centrifuging for 2 times. The content of endotoxin in the purified MBP protein is identified by using a limulus reagent, is less than 0.125EU/ml, and meets the requirement required by the experiment.

3. As a result, it was found that the bacterial dust was completely broken and the protein was completely released after four times of homogenization, and the result of the degree of breakage of the bacterial dust is shown in FIG. 3. The SDS-PAGE electrophoresis identification result of the purified protein by the Amylose affinity chromatography is shown in figure 4, the target band of the eluent is single and has no miscellaneous band and is obvious, and the purification condition is proved to be suitable, so that the target protein is obtained.

Detection results of the content of toxins in MBP protein bacteria after the first ultrafiltration centrifugation of the 100kD ultrafiltration tube (not shown) are as follows: the No. 1 negative result is negative; the No. 2 positive result is positive; the positive MBP protein No. 3 and the positive test sample No. 4 indicate that the endotoxin detection is unqualified, and prompt that the endotoxin cannot be removed by one-time ultrafiltration. And (3) detecting the content of the bacterial endotoxin after the second ultrafiltration centrifugation: the No. 1 negative result is negative; the No. 2 positive result is positive; MBP protein No. 3 is negative, and test sample No. 4 is positive (figure 5), which indicates that 2 times of ultrafiltration and centrifugation can ensure that endotoxin in the purified protein sample is qualified for detection.

EXAMPLE 3 MBP (43.12KD) in combination with CPG-ODN synergistically promotes lymphocyte proliferation

1. Materials main experimental apparatus, animal and reagent: microplate readers (Epoch, Botrm instruments, Inc., USA); c57 BL/6 mice (SPF grade, female, 6-8 weeks old, weight 18-22 g) were purchased from Shenyang Biotechnology Ltd; WST-1(Roche, 30307600); CpG2006 (Ongzhou Ribo Biotech Co., Ltd., 4.368 mg/ml); MBP (Changchun kang Yue Biotech Co., Ltd., 0.147 mg/ml); IMDM culture medium (gibco), fetal bovine serum (MRC), streptomycin mixed solution 100 × (Solarbio)

2. Method of producing a composite material

2.1 mouse splenocyte suspension preparation

The mice were sacrificed, spleens were aseptically taken, the spleens were placed in gauze soaked in 5ml of IMDM medium, ground, spleen cell suspensions were prepared, counted, cell concentration was calculated, and cell concentration was adjusted to 7 × 10⁶One per ml.

2.2 drug-stimulated culture of splenocytes

Dividing the test sample into 6 groups according to the stimulation drug, wherein the groups are respectively negative control group (NC), MBP group (10 μ g/ml), CpG2006 (10 μ g/ml) and MBP (10 μ g/ml) + CpG2006 (10 μ g/ml), setting 3 multiple wells, laying 96-well plate, 7 × 10⁵One cell/well, total volume 200. mu.l/well (drug 100. mu.l/well, cell suspension 100. mu.l/well), placed in CO₂In incubator, 5% CO₂And cultured at 37 ℃ for 48 h.

2.3 WST-1 method for detecting cell proliferation

After spleen cells are cultured for 48h, 100 mu l/well supernatant is sucked, 3 multiple wells are collected together for subsequent cytokine detection, WST-110 mu l/well is added into the cells, and the mixture is respectively heated at 37 ℃ and 5% CO₂And (3) incubating for 1h, 1.5h and 2h under the condition, and detecting the absorbance at the wavelength of 450nm/630nm by using a microplate reader. The results of the experiment are expressed as the stimulation index ═ stimulation (c ═ stimulation: (a)Experimental group₄₅₀Experimental group₆₃₀) /(control group)₄₅₀Control group₆₃₀)

3. Results CpG2006, MBP + CpG2006 groups had higher stimulation indices than NC groups (p <0.01), suggesting that lymphocyte proliferation was promoted. The stimulation index of the MBP + CpG2006 group is obviously higher than that of the MBP or CpG2006 group (p is less than 0.01), and the combined use of the MBP and the CpG2006 is suggested to have the effect of synergistically promoting the proliferation of lymphocytes (figure 6).

4. It was concluded that MBP in combination with CpG2006 synergistically promoted lymphocyte proliferation.

Experimental example 3 MBP (43.12KD) in combination with CPG-ODN induces the proliferation and differentiation of lymphocytes into Th1 cells

1. Materials main experimental apparatus, animal and reagent: microplate reader (Epoch, Botryn instruments, USA). C57 BL/6 mice (SPF grade, female, 6-8 weeks old, weight 18-22 g) were purchased from Shenyang Biotechnology Ltd. IFN-. gamma.mouse Uncoated ELISAKit (invitrogen); CpG2006 (Ongzhou Ribo Biotech Co., Ltd., 4.368 mg/ml); MBP (Changchun kang Yue Biotech Co., Ltd., 0.147 mg/ml).

2. The method was performed according to the instructions of the ELISA test kit from Invitrogen. Briefly, a 96-well microplate was first coated with a coating antibody (anti-IFN-. gamma.antibody), 3 wells per sample, and overnight at 4 ℃. The plate was washed 5 times with PBS-0.05% Tween-20, incubated in a closed solution for 1 hour, washed 2 times, and then a standard (gradient: 2000, 1000, 500, 250, 125, 62.5, 32.5, 15.625pg/ml) and 2-fold diluted sample (sample is the supernatant of the splenocytes cultured with drug stimulation in experiment 2) were added, 100. mu.l/well and incubated at room temperature for 2 hours. After 5 times of washing, 100 mul/hole of detection antibody is added for incubation for 1h at room temperature, 5 times of washing are carried out, 100 mul/hole of enzyme-labeled antibody Avindin-HRP is added, and incubation is carried out for 30min at room temperature. Washing 7 times, adding TMB 100. mu.l/well, and keeping away from light for 15min at room temperature. The reaction was terminated by adding 100. mu.l/well of 2N sulfuric acid, and absorbance at a wavelength of 450nm was measured by a microplate reader. And calculating the concentration of each sample according to a standard curve drawn by the standard substance.

3. Results after different drugs stimulate mouse spleen lymphocytes for 48 hours, the content of IFN-gamma in culture supernatant is detected by ELISA. The results are shown in fig. 7, the content of IFN- γ in the culture supernatants of the CpG2006 group and the MBP-CpG 2006 group is significantly higher than that of the control group, and the differences have statistical significance. And the MBP + CpG2006 group has obviously higher IFN-gamma level than that of the MBP or CpG2006 group (p <0.01) alone, which suggests that the combined application of the MBP and the CpG2006 has the effect of synergistically promoting the polarization of lymphocytes to Th1 cells (figure 7).

4. Conclusion MBP (43.12KD) in combination with CPG-ODN synergistically induced lymphocyte polarization to Th1 cells.

EXAMPLE 4 MBP (43.12KD) in combination with CPG-ODN synergistically increased DC and cDC1, cDC2 ratio and upregulated DC maturation

1. Materials main experimental apparatus, animal and reagent: flow cytometry (FACS Arial II, BD). C57 BL/6 mice (SPF grade, female, 6-8 weeks old, weight 18-22 g) were purchased from Shenyang Biotechnology Ltd. Flow antibodies CD16/32, PE-PDCA1, APC-CD40, AF700-MHC I, PE-cy7-CCR7 are purchased from eBioscience, BV510-FVS510, PE-CF594-CD80, PE-CD86 is purchased from BD, BV421-CD11C, FITC-MHC II, APC/Cyanine7 XCR1, PerCP/Cyanine5.5CD172a, BV421-B220, FITC-CD86 are purchased from Biolegend. CpG2006 (Ongzhou Ribo Biotech Co., Ltd., 4.368 mg/ml); MBP (Changchun kang Yue Biotech Co., Ltd., 0.147 mg/ml).

2. Method of producing a composite material

2.1 mouse splenocyte suspension preparation

Killing mice, taking spleens aseptically, putting spleens into gauze soaked in 5ml of IMDM culture medium, grinding, collecting splenocyte suspension, performing erythrocyte lysis, washing with PBS for 2 times after erythrocyte lysis, adding IMDM culture medium for resuspension of cells, filtering with 300-mesh filter screen, counting cells, calculating concentration, and adjusting concentration to 1 × 10⁷One per ml.

2.2 spleen cell stimulation culture

Dividing into 4 groups according to the stimulation drug, wherein the groups are respectively negative control group (NC), MBP group (10 μ g/ml), CpG2006 (10 μ g/ml) and MBP (10 μ g/ml) + CpG2006 (10 μ g/ml), setting 5 multiple wells, spreading 24-well plate, and 5 × 10⁶One cell/well, total volume 1 ml/well (500. mu.l drug/well, 500. mu.l cell suspension/well) placed in CO₂In incubator, 5% CO₂And cultured at 37 ℃ for 48 h.

2.3 flow cytometry detection of DC cell subsets and DC cell maturation marker expression levels

Collecting splenocytes cultured for 48h in a stimulation mode, adding CD16/32 antibody to block Fc receptor, washing, adding FVS510 live and dead cell dye and surface antibody, incubating for 30min in the absence of light on ice, washing, adding 500 ul PBS for resuspension, filtering by a 300-mesh filter screen, and detecting by a flow cytometer.

3. Results induction of antigen-specific CTL responses against exogenously delivered vaccine antigens is dependent on the presentation of these antigens, particularly the cross-presentation of dendritic cells. DCs are functionally heterogeneous and can be divided into two major subtypes: plasma cell-like dc (pdc) is primarily involved in antiviral immunity, whereas classical dc (cdc) is primarily associated with antigen presentation. Classical dc (cdc) can be further divided into two distinct subtypes: classical DC type 1 (cDC1, CD11 c) responsible for antigen presentation to CD8+ T through MHC I⁺CD26⁺XCR1^hiCD172a^loIRF8^hiIRF4^lo) And classical DC type 2 (cDC2, CD11 c) responsible for antigen presentation to CD4+ T through MHC II⁺CD26⁺XCR1^loCD172a^hiIRF8^loIRF4^hi). The cDC1 is a major cross-presentation subset recognized at present and plays a key role in the generation of tumor-specific CTL responses. Maturation of DCs is marked by the movement of MHC/peptide complexes to the cell membrane, upregulation of the costimulatory molecule CD80/CD86, and expression of cytokines that drive T-cell proliferation and differentiation (e.g., IL-12). These molecules are referred to as signals 1, 2 and 3, respectively, and are required for normal T cell activation.

We used MBP in combination with CpG2006 to stimulate mouse splenocytes in vitro and flow cytometric analysis of DC subsets and maturation therein. The results show that MBP in combination with CpG2006 synergistically increased the total DC, cDC1(XCR1+ DC) and cDC2 ratios. While pDC also showed the same trend, although not statistically significant (fig. 8). Whereas analysis of DC cell maturation markers showed: MBP in combination with CpG2006 up-regulated the expression of co-stimulatory molecules CD40, CD80, CD86 on DC, while increasing MHC I and MHC II levels on DC cell surfaces, whereas chemokine CCR7 associated with DC cell migration showed no significant difference (FIG. 9).

4. Conclusion MBP in combination with CpG2006 synergistically increased DC and ratio of cDC1 and cDC2, promoted DC cell maturation, up-regulated DC cell MHC molecule expression, and thereby enhanced DC antigen presentation capacity.

EXAMPLE 5 study of the antitumor Activity of MBP (43.12KD) in combination with CPG2006 in mice

1. Materials main experimental apparatus, animal and reagent: c57 BL/6 mice (SPF grade, female, 6-8 weeks old, weight 18-22 g) were purchased from Shenyang Biotechnology Ltd. CpG2006 (Ongzhou Ribo Biotech Co., Ltd., 4.368 mg/ml); MBP (Changchun kang Yue Biotech Co., Ltd., 0.147 mg/ml). Flow antibodies CD16/32, PE-PDCA1, APC-CD40, AF700-MHC I, PE-cy7-CCR7 are purchased from eBioscience, BV510-FVS510, PE-CF594-CD80, PE-CD86 are purchased from BD, BV421-CD11C, FITC-MHC II, APC/Cyanine7 XCR1, PerCP/Cyanine5.5CD172a, BV421-B220, FITC-CD86 are purchased from Biolegend.

2. Method of producing a composite material

2.1 immunization with mouse vaccine and tumor bearing

Mice were randomly divided into 4 groups of 10 mice each, namely saline control (NC), MBP (50 μ g/mouse), CpG2006 (6.25 μ g/mouse each of CpG 2006), MBP + CpG2006 (50 μ g/mouse and CpG20066.25 μ g/mouse) groups, and immunized subcutaneously once a week for a total of 5 times. Tumor bearing on day 4 after last immunization, right flank and back were injected subcutaneously with B16 melanoma cells at 5X 10⁵One/only.

2.2 MBP (43.12KD) combined with CPG2006 as adjuvant for the inhibition of mouse B16 melanoma

Mice were sacrificed 14 days after tumor loading, subcutaneous tumors were dissected and weighed, and tumor weight and tumor inhibition rate were calculated for each group. The formula is as follows: tumor inhibition rate (control tumor weight-experimental tumor weight)/control tumor weight × 100%.

2.3 flow cytometry detection of DC cell subset proportion and DC cell maturation marker expression level in mouse dLN

The mice are sacrificed 14 days after tumor loading, lymph nodes in a drainage area are taken, lymph node cell suspension is prepared, CD16/32 antibody is added to block Fc receptor, washing is carried out, FVS510 live dead cell dye and surface antibody are added to carry out incubation for 30min in the dark place on ice, washing is carried out, 500 mu l PBS is added to carry out heavy suspension, and the detection is carried out by a flow cytometer after filtering by a 300-mesh filter screen.

3. Results

3.1 MBP and CpG2006 have a synergistic antitumor effect in mice

Tumor weight and tumor inhibition rate results show that the MBP, CpG2006 and MBP + CpG2006 groups all show tumor inhibition effects, wherein the tumor weight of the CpG2006 and MBP + CpG2006 groups is statistically different from that of the NC group. The tumor inhibition rates of the three experimental groups of MBP, CpG2006 and MBP + CpG2006 are 3.87%, 36.80% and 58.17% respectively, and the tumor inhibition rate of the MBP + CpG2006 group is obviously higher than that of the MBP and CpG2006, which suggests that the MBP and CpG2006 have a synergistic anti-tumor effect in a mouse body (FIG. 10).

3.2 the combination of MBP and CpG2006 can synergistically increase the ratio of cDC1 and cDC2 in mouse dLN and up-regulate DC cell maturation

14 days after tumor loading, we performed flow cytometry analysis of DC subsets and maturation in lymph nodes in the mouse drainage area. The results show that immunization with MBP, CpG2006 and MBP + CpG2006 all increased the ratio of total DC, cDC1(XCR1+ DC) and cDC2 in lymph nodes in the draining region of mice, whereas MBP in combination with CpG2006 showed a synergistic effect (fig. 11). The pDC did not show the same trend among the groups and the results are not given. Whereas analysis of DC cell maturation markers showed: MBP, CpG2006 and MBP + CpG2006 can up-regulate the expression of co-stimulatory molecules CD40, CD80 and CD86 on DC in lymph nodes of a drainage region of a mouse, and can improve the MHC II level on the surface of a DC cell, and all DC in each group express MHC I without difference. The same trend was also shown for the chemokine CCR7 associated with DC cell migration (fig. 12).

4. Conclusion MBP synergizes with CpG2006 to increase cDC1 and cDC2 in mouse dLN and up-regulate DC cell maturation and have synergistic anti-tumor effects in mice.

EXAMPLE 6 MBP (43.12KD) in combination with CPG-ODN as adjuvant enhances vaccine efficacy in vivo

1. Materials main experimental apparatus, animal and reagent: microplate reader (Epoch, Botrg instruments, USA), flow cytometer (FACS Arial II, BD). C57 BL/6 mice (SPF grade, female, 6-8 weeks old, weight 18-22 g) were purchased from Shenyang Biotechnology Ltd. CpG2006 (Ongzhou Ribo Biotech Co., Ltd., 4.368 mg/ml); MUC1 polypeptide (synthesized by Shanghai purple zone Biotech Co., Ltd., 531883, 2 mg/ml); MBP (Changchun kang Yue Biotech Co., Ltd., 0.147 mg/ml). Goatanti-Mouse IgG2c-HRP (southern Biotech), HRP RatInti-Mouse IgG1-HRP (southern Biotech), and Peroxidase-conjugatedAffinipure Goatanti-Mouse IgG (H + L) (Proteintech).

2. Method of producing a composite material

2.1 immunization with mouse vaccine and tumor bearing

Mice were randomized into 4 groups of 10 mice each, saline control (NC), MUC + MBP (50 μ g/mouse of MUC1 and MBP), MUC1+ CpG2006 (50 μ g/mouse of MUC1 and CpG 2006), MUC1+ MBP + CpG2006(MUC1, 50 μ g/mouse of MBP and CpG 2006), immunized subcutaneously once a week for 5 total immunizations. The 4 th day after the last immunization, the right flank back is injected with melanoma cells B16-MUC15 x 10^5 which are over-expressed by MUC1 subcutaneously. Mice were sacrificed 14 days after tumor loading.

2.2 MBP (43.12KD) combined with CPG2006 as adjuvant for the inhibition of mouse B16-MUC1 melanoma

On day 14 after tumor loading, mice were sacrificed, subcutaneous tumors were dissected and weighed, and tumor weight and tumor inhibition rate were calculated for each group. The formula is as follows: tumor inhibition rate (control tumor weight-experimental tumor weight)/control tumor weight × 100%.

2.3 Effect of MBP (43.12KD) in combination with CPG2006 as adjuvant on the MUC 1-specific antibody subtype in mice

On day 14 after tumor loading, mice were sacrificed, eyeballs were removed and blood was taken, serum was extracted, and serum MUC 1-specific antibodies were detected by ELISA. Specifically, 96-well plates were coated with 0.5. mu.g/well of MUC1 polypeptide, incubated overnight at 4 ℃ and then blocked with 2% bovine serum albumin in PBS. Add 100. mu.l of 1: the serum samples diluted 500 were incubated at 37 ℃ for 1.5 hours. Washing and incubation with Goat Anti-Mouse IgG2c-HRP, HRP Rat Anti-Mouse IgG1-HRP, Peroxidase-conj μ gated affinity Goat Anti-Mouse IgG (H + L) for 2 hours at 37 ℃. Then washed and incubated with the substrate OPD at 37 ℃ for 10 minutes in the absence of light and 2N H was added₂SO ₄Termination reaction, useThe microplate reader measures absorbance at a wavelength of 490 nm.

2.4 Effect of MBP (43.12KD) in combination with CPG2006 as adjuvant on the level of proliferation and cytokine secretion by MUC 1-specific splenocytes

Laying a 96-well plate according to the cell counting result, arranging 3 wells, setting 1 multiplied by 10^6 cells/well, stimulating and culturing MUC1 polypeptide (20 mu g/ml), putting the plate in a CO2 incubator, and culturing at 37 ℃ in 5% CO 2. And (5) plating for the third day, carrying out half-amount liquid change, and continuing culturing. On the fifth day of culture, 100. mu.l/well of the supernatant was aspirated, 3 wells were pooled together, and the supernatants collected above were assayed for IFN-. gamma.and IL-4 levels by sandwich ELISA.

2.5 CTL killing experiment mice were sacrificed 14 days after tumor loading, spleens were taken, spleen cell suspensions were prepared, cultured for 5 days with stimulation of MUC1 polypeptide (20. mu.g/ml), cells were collected as effector cells, B16-MUC1 was cultured to 3 rd to 5 th generations as target cells, and CTL killing activity was measured by the RTCA method. First in E-Plate16 at 5X 10³Target cells were cultured at a cell/well density, and after the cells were observed to have entered a logarithmic growth phase (in this experiment, effector cells were added at about 16h of target cell culture), effector cells were added to kill the target cells (E: T25: 1 or 50: 1), and 72 hours were observed, and CTL killing activity was calculated, where CTL killing activity ═ 1-CI (experimental group)/CI (target cells)]×100％。

3. Results

3.1 MBP (43.12KD) combined with CpG2006 can improve the tumor inhibition effect of MUC1 cancer vaccine in mice

Tumor weight and tumor inhibition rate results show that the MUC1+ MBP, MUC1+ CpG2006 and MUC1+ MBP + CpG2006 groups all show tumor inhibition effects, wherein the MUC1+ CpG2006 and MUC1+ MBP + CpG2006 groups have statistical significance. The group MUC1+ MBP + CpG2006 has a significantly higher tumor suppression rate than MUC1+ MBP and MUC1+ CpG2006, which suggests that MBP and CpG2006 can synergistically enhance the tumor suppression effect of the cancer vaccine with MUC1 as a target in a mouse (FIG. 13).

3.2 MBP (43.12KD) combined with CpG2006 can improve Th1 type immune response in mice

Cytokines produced by Th cells are involved in inducing a switch from IgM to other Ab isotypes. Th1 cell secretes IFN-gamma, the cytokine can promote the antibody to be converted into IgG2, Th2 cell secretes IL-4, IL-4 can promote the antibody to be converted into IgG 1. Thus, IgG2c/IgG1 indirectly reflected the predominant Th response pattern. In the experiment, the titer and subtype of the specific antibody of MUC1 in the serum of the mouse are detected by ELISA, and an NS group is used as a control group. Immunization with MUC1+ CpG2006 and MUC1+ MBP + CpG2006 all induced anti-MUC 1-specific IgG, IgG1 and IgG2c antibodies compared to the control group (fig. 14A). From the results we found that IgG2c/IgG1 increased compared to the control group, indicating that MBP and CpG2006 as vaccine adjuvants induced immune responses in mice were inclined to Th1 type (fig. 14B). We further analyzed representative Th1 (IFN-. gamma.) and Th2(IL-4) cytokines in mouse sera. Spleen of mice was obtained after vaccination, spleen lymphocytes were stimulated with MUC1, and the cultured supernatant was collected and assayed by ELISA, and the results are shown in FIGS. 14C and D. Compared with a control group, the immunization of MUC1+ CpG2006 and MUC1+ MBP + CpG2006 both up-regulate IFN-gamma secretion in splenic lymphocytes of mice and down-regulate IL-4 secretion, and the similar support that the immune response induced by the MUC1+ MBP + CpG2006 vaccine in mice is inclined to a Th1 type. The level of IFN-gamma in the MUC1+ MBP + CpG2006 group is higher than that in the MUC1+ MBP and MUC1+ CpG2006 groups, which indicates that the MBP and the CpG2006 synergistically promote the secretion of Th1 cytokine (IFN-gamma).

3.3 MBP (43.12KD) combined with CpG2006 can improve CTL killing activity in mice immunized by the vaccine

In this study, we used RTCA technology to dynamically monitor CTL killing activity of splenic lymphocytes from vaccine immunized mice. As shown in fig. 15, three vaccine groups showed CTL killing activity compared to the NC group, and the killing rate was proportional to the efficacy-to-target ratio. CTL killing activity: MUC1+ MBP + CpG2006 > MUC1+ CpG2006 > MUC1+ MBP (fig. 15), suggesting that MBP and CpG2006 synergistically enhance the CTL killing activity of the vaccine when used as a vaccine adjuvant.

4. Conclusion MBP (43.12KD) and CpG2006 are combined to be used as an adjuvant of MUC1 cancer vaccine, a B16 melanoma tumor-bearing mouse model which over-expresses MUC1 is constructed after the cancer vaccine is prophylactically inoculated, the in vivo immune activity of the mouse is detected, and the tumor inhibition effect of the vaccine is observed. The results of the experiment support that MBP (43.12KD) and CpG2006 are combined as a vaccine adjuvant to enable the immune response in mice to be prone to Th1 type, and meanwhile, the CTL killing activity induced by the vaccine is enhanced, so that the tumor inhibition effect of the MUC1 cancer vaccine is synergistically improved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> kang Jiu Biotechnology (Changchun) Ltd

<120> maltose-binding protein, maltose-binding protein expression vector, recombinant engineering bacteria and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 402

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agttagtgtt gagatgatgc tttgacaaaa ggtaatccat ctgttcagaa attctacaat 60

ggttgctgtc tcatcagcat attcacacat gaatgttgtt tcagatccct ttagttccag 120

aactattacg ttgatattgc tgattaagtc cctgggtctt aagtgaaagt ttttgctttg 180

agctaaattt agcacttcct ccagaggttt gagttcttct tctagacact gaagatgttt 240

cagttctgtg gccttcttgg gcatgtaaaa cttaaatgtg agcatcctgg tgagtttggg 300

attcttgtaa ttattaattc cattcaaaat catctgtaaa tccagcagta aatgctccag 360

ttgtagctgt gttttctttg tagaacttga agtaggtgcc at 402

<210> 2

<211> 7046

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 60

gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 120

gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 180

cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 240

aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 300

acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 360

tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 420

ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 480

ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 540

cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 600

tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 660

cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 720

cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 780

atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 840

tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 900

acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 960

gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1020

agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1080

cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1140

cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag 1200

gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1260

tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1320

tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1380

ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1440

attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1500

gcacttcacc aacaaggacc atagattatg aaaactcgaa gaaggtaaac tggtaatctg 1560

gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga 1620

taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt 1680

tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta 1740

cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta 1800

tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt 1860

tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga 1920

agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa 1980

cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa 2040

gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc 2100

gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta 2160

ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg 2220

ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt 2280

caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag 2340

tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct 2400

ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga 2460

gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat 2520

gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc 2580

cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc 2640

gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggaagga tttcagaatt 2700

cagttagtgt tgagatgatg ctttgacaaa aggtaatcca tctgttcaga aattctacaa 2760

tggttgctgt ctcatcagca tattcacaca tgaatgttgt ttcagatccc tttagttcca 2820

gaactattac gttgatattg ctgattaagt ccctgggtct taagtgaaag tttttgcttt 2880

gagctaaatt tagcacttcc tccagaggtt tgagttcttc ttctagacac tgaagatgtt 2940

tcagttctgt ggccttcttg ggcatgtaaa acttaaatgt gagcatcctg gtgagtttgg 3000

gattcttgta attattaatt ccattcaaaa tcatctgtaa atccagcagt aaatgctcca 3060

gttgtagctg tgttttcttt gtagaacttg aagtaggtgc catggatcct ctagagtcga 3120

cctgcaggca agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 3180

cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 3240

agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcagct 3300

tggctgtttt ggcggatgag ataagatttt cagcctgata cagattaaat cagaacgcag 3360

aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc 3420

catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc 3480

gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 3540

ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag 3600

cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa 3660

ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac 3720

aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 3780

ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 3840

gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 3900

ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 3960

gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt cccaatgatg 4020

agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 4080

caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 4140

gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 4200

agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 4260

gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 4320

aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 4380

ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 4440

tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 4500

tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 4560

gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 4620

atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 4680

ctgtcagacc aagtttactc atatatactt tagattgatt taccccggtt gataatcaga 4740

aaagccccaa aaacaggaag attgtataag caaatattta aattgtaaac gttaatattt 4800

tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa 4860

tcggcaaaat cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag 4920

tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg 4980

tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga 5040

ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg 5100

gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg 5160

cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc 5220

cgctacaggg cgcgtaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5280

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 5340

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 5400

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 5460

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 5520

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 5580

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 5640

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 5700

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 5760

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 5820

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 5880

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 5940

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6000

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6060

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 6120

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 6180

ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 6240

gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 6300

cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 6360

gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 6420

cgtggtcgtg cagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 6480

tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 6540

cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac 6600

cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 6660

tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 6720

ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 6780

agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 6840

gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 6900

tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 6960

ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg 7020

gccaggaccc aacgctgccc gaaatt 7046

<210> 3

<211> 392

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys

1 5 10 15

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

20 25 30

Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe

35 40 45

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

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu

100 105 110

Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys

115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly

130 135 140

Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

145 150 155 160

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

165 170 175

Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly

180 185 190

Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp

195 200 205

Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala

210 215 220

Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys

225 230 235 240

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

245 250 255

Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro

260 265 270

Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp

275 280 285

Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala

290 295 300

Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala

305 310 315 320

Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

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

355 360 365

Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile

370 375 380

Glu Gly Arg Ile Ser Glu Phe Ser

385 390

Claims (9)

1. A maltose-binding protein expression vector, which uses pMAL-C2 as the original vector, and inserts a gene containing a termination code after malE gene.

2. The maltose binding protein expression vector of claim 1, wherein the gene containing the termination codon is inserted between EcoRI and BamHI sites on pMAL-C2.

3. A recombinant engineered bacterium comprising the maltose binding protein expression vector of claim 1 or 2.

4. Use of the maltose binding protein expression vector of claim 1 or 2 or the recombinant bacterium of claim 3 for preparing maltose binding protein.

5. A maltose-binding protein prepared based on the maltose-binding protein expression vector of claim 1 or 2 or the recombinant engineered bacteria of claim 3.

6. An immunopotentiator comprising CPG-ODN and the maltose binding protein of claim 5.

7. Use of the immunopotentiator according to claim 6 for the preparation of a medicament for immunopotentiation.

8. Use according to claim 7, wherein the immunopotentiator is used as a vaccine adjuvant.

9. The use of claim 7, wherein the maltose binding protein and CPG-ODN enhance vaccine immunity by promoting lymphocyte proliferation, inducing lymphocyte proliferation and differentiation to Th1 cells, increasing DC ratio, increasing cDC1 ratio, increasing cDC2 ratio and up-regulating DC maturation.

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