CN116769056A - Fusion protein, purification preparation method and application - Google Patents
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention belongs to the field of immunology, and particularly discloses a fusion protein, a purification preparation method and application thereof, wherein the fusion protein is an adjuvant of BCG vaccine, the fusion protein comprises a CD19 single-chain antibody and a fusion protein of full-length ESAT-6 protein, and the CD19 single-chain antibody is connected with the full-length ESAT-6 protein. The invention provides a fusion protein which can avoid cross reaction caused by shared antigen in subunit vaccine and enhance immune response in a targeted amplification B cell activation path manner, thereby improving the immunity of BCG, and a purification preparation method and application thereof.
Description
Technical Field
The invention belongs to the field of immunology, relates to a fusion protein, a purification preparation method and application thereof, and in particular relates to a fusion protein formed by combining a CD19 single chain antibody variable region (CD 19 scFv) and an antigen ESAT-6 for triggering immune response against pathogenic mycobacterium tuberculosis (Mycobacterial tuberculosis, M.tb), and a preparation method and application thereof.
Background
At present, tuberculosis is still the leading infectious cause of death in the world, and new preventive strategies are urgently needed to relieve the burden of the disease. At present, bacillus Calmette-Guerin (BCG) is widely applied to clinical vaccine for preventing mycobacterium tuberculosis, has better effect of preventing tuberculosis of children, but has the defect of insufficient protective effect on adults [1] . Many candidate vaccines are designed almost as enhancers or modifiers of BCG, having a shared antigen component with BCG, known as subunit vaccines, the presence of "cross-reactions" caused by the shared antigen component makes it still difficult for most new candidate vaccines to effectively boost the protective efficacy of BCG [2] 。
ESAT-6 as a strong antigen eliciting specific anti-m.tb immune response does not share antigenic components with BCG [3] Is widely used in tuberculosis vaccine research. Research proves that macrophage TLR2 recognition toxicity M.tb immunogenic molecule ESAT-6 can activate intracellular PI3K-Akt pathway [4] . ESAT-6 can also modulate autophagy by SOD-2, thereby inducing intracellular survival of mycobacterium bovis BCG [5] . When BCG is combined with ESAT-6, it is possible to not only activate the TLR2-PI3K-Akt pathway, but also improve BCG survival in vivo, thus enhancing the immune memory response of vaccinated individuals against pathogenic m.tb.
There has also been growing evidence in recent years that B cells, which are the major participants in humoral immune responses, play an important role in regulating T cell function, such as B cell TLR2 signaling leading to NF- κb activation, triggering interleukin IL-6 production, and thus promoting CD4 + Differentiation of T cells into helper T cells 17 (Th 17) [6] While Th17 cells mediate inflammation and neutrophil recruitment to help clear infected M.tb [7] The method comprises the steps of carrying out a first treatment on the surface of the Follicular helper T cells (T) constituting the vast majority of lymphoid follicles, including the inducible bronchi-associated lymphoid tissue iBALT fh ) Is amplified from memory T cells in a B cell assisted manner [8] The method comprises the steps of carrying out a first treatment on the surface of the B fineThe interaction between cells and T cells is critical for the normal development of granulomatous protective responses to tuberculosis [9] 。
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides a fusion protein which can avoid cross reaction caused by shared antigens in subunit vaccine and enhance immune response in a targeted amplification B cell activation pathway manner, thereby improving the immune efficacy of BCG, and a purification preparation method and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a fusion protein, characterized in that: the fusion protein is an adjuvant of BCG vaccine, the fusion protein comprises a CD19 single-chain antibody and a fusion protein of full-length ESAT-6 protein, and the CD19 single-chain antibody is connected with the full-length ESAT-6 protein.
Preferably, the fusion mode of the fusion protein adopted by the invention is CD19scFv-ESAT-6; the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
A nucleotide for encoding a fusion protein as hereinbefore described, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.
A method for preparing a fusion protein based on the above, characterized in that: the preparation method comprises the following steps:
1) Designing the structure of the fusion protein as described above;
2) Constructing a recombinant expression vector pET-28a-CD19scFv-ESAT-6;
3) Converting the recombinant expression vector pET-28a-CD19scFv-ESAT-6 obtained in the step 2) into a protein expression strain BL-21, and performing expansion culture on the protein expression strain BL-21;
4) Prokaryotic expression of the fusion protein CD19scFv-ESAT-6.
Preferably, the specific implementation manner of the step 1) adopted by the invention is as follows: the nucleotide sequence of the early secretion protein antigen ESAT-6 is searched in NCBI database, tool molecule CD19scFv and ESAT-6 molecule are assembled together, and corresponding connecting sequence is added through analysis of bioinformatics software, so that the fusion protein with scientific and reasonable structure is designed.
Preferably, the specific implementation manner of the step 2) adopted by the invention is as follows: and respectively synthesizing a CD19scFv gene and an ESAT-6 gene by utilizing a molecular cloning technology, integrating the CD19scFv gene and the ESAT-6 gene into the CD19scFv-ESAT-6 gene by a mode of overlapping PCR, and finally cloning the CD19scFv-ESAT-6 gene into a pET28a vector to construct a recombinant expression vector pET-28a-CD19scFv-ESAT-6.
Preferably, the specific implementation manner of the step 4) adopted by the invention is as follows: the fusion protein is induced to be expressed in inclusion bodies by IPTG, and is dissolved by urea and then dialyzed by Beyoold His-tag purification Resin nickel ion affinity chromatography and dialysis bags, so that the fusion protein CD19scFv-ESAT-6 is obtained.
Use of a fusion protein as hereinbefore described as an adjuvant to a BCG vaccine or for the prevention of tuberculosis.
Preferably, the fusion protein used in the present invention is present in an amount not less than 8. Mu.g in an amount effective to elicit a significant immune response.
The fusion protein adjuvant adopts a prokaryotic expression system, is used for improving the immunopotency of BCG after expression and purification, and has the following advantages compared with the prior candidate vaccine:
the fusion protein provided by the invention comprises a CD19 single-chain antibody and a fusion protein of full-length ESAT-6 protein, wherein the fusion mode is CD19scFv-ESAT-6, the preparation process is simple, and the cost is low. According to the potential effect of B cells in the development process of tuberculosis and the immunological function of ESAT-6, the invention takes the activity of enhanced B cells as an entry point, forms fusion protein from CD19scFv and ESAT-6 which can target and activate the B cells, successfully constructs the CD19scFv-ESAT-6 through molecular cloning and other technologies, and obtains purified protein CD19scFv-ESAT-6 through prokaryotic expression, protein purification and other technologies, so as to be used as an immunopotentiator of BCG, and improve the immunopotentiator of vaccine. In the BCG immunization process, the CD19scFv in the fusion protein has the capability of targeted activation of B cells, and ESAT-6 is promoted to activate B cell TLR2 signals independently, so that the efficacy of protective immune response is remarkably improved. ESAT-6 in the fusion protein does not share an antigen component with BCG, can supplement immune effects caused by BCG, and avoids "cross reactions" caused between antigen components. ESAT-6 in the fusion protein may improve survival of BCG in vivo, helping to enhance vaccine efficacy of BCG. Compared with the existing candidate vaccine, the fusion protein provided herein has the advantages of simple preparation process, low cost and good effect.
Drawings
FIG. 1 shows the results of double restriction enzyme assay of the fusion protein expression plasmid successfully constructed by amplifying the genes of the CD19scFv and ESAT-6 fragments in example 1.
FIG. 2 shows the result of SDS-PAGE electrophoresis of the fusion protein of example 2.
FIG. 3 shows the proportion of fusion protein-bound C57BL/6J mouse spleen cells and the proportion of B cells in fusion protein-bound spleen cells in example 3;
FIG. 4 shows the ratio of the peripheral blood cells of the C57BL/6J mice bound by the fusion proteins at different concentrations in example 3,
FIG. 5 shows the intravenous injection of BCG (1.0X10) into the tail of C57BL/6J mice in example 4 and example 5 6 After CFU) and fusion protein (fig. A, B is dose gradient, fig. C is 8 mg), splenocytes were taken at 15/30 days for in vitro culture and incubated with inactivated m.tb H37Rv (fig. A, B)/inactivated BCG/ESAT-6 (inactivated m.tb H37Rv/BCG CFU: cell number = 10:1,0.1g/mL ESAT-6) was stimulated for 24h, and the proportion of spleen Th1 cells was determined by flow-through.
Detailed Description
The invention is further illustrated below in connection with specific examples, without limiting the scope of the invention. Unless otherwise indicated, all examples were carried out under conventional conditions or according to the instructions of the manufacturer, and all reagents used in the examples were those conventional in the art, and all methods used were conventional.
The amino acid sequences of the CD19scFv used in the following examples are set forth in SEQ ID NOs: 3 is shown in the figure; the amino acid sequence of ESAT-6 is shown as SEQ ID NO: 4. The nucleic acid sequence of the CD19scFv is shown in SEQ ID NO:5 is shown in the figure; ESAT-6 has a nucleic acid sequence shown in SEQ ID NO: shown at 6.
Before the following experiments were carried out, the structure of the fusion protein CD19scFv-ESAT-6 molecule was designed, specifically:
the nucleotide sequence of the early secretion protein antigen ESAT-6 is searched in NCBI database, the tool molecule CD19scFv (single-chain fragment variable, CD19 single-chain antibody variable region) established in the early work and ESAT-6 molecule are assembled together, and the corresponding connecting sequence is added through analysis of bioinformatics software, so that the fusion protein with a scientific and reasonable structure is designed.
Example 1: construction of CD19scFv-ESAT-6 fusion protein expression plasmid and engineering bacterium
1. Primers are designed and synthesized for the gene sequences of CD19scFv (shown as SEQ ID NO: 5) and ESAT-6 (shown as SEQ ID NO: 6), the existing plasmids pET-28a-CD19scFv-IL-10R in the laboratory are used as templates, bamHI enzyme cutting sites are respectively added at the upstream of CD19scFv (single-chain fragment variable, CD19 single-chain antibody variable region) through gene amplification, and HindIII enzyme cutting sites are added at the downstream of ESAT-6; a complementary repetitive nucleotide sequence (shown as SEQ ID NO: 9) was added downstream of the CD19scFv and upstream of the ESAT-6 as a junction fragment of overlap PCR (Overlap PCR). The conditions for gene amplification in example 1 were those given in the description of the gold plate Mix reagent (TSE 101, product number: TSE101, new industry, beginnings, and qing, and the reaction system and conditions were as follows:
| gold medal Mix (Green) | 45μL |
| 10μM Primer A | 2μL |
| 10μM Primer B | 2μL |
| Template DNA | as required |
| 50μL Reaction |
Pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s; annealing Tm + -5 ℃ for 10-25s; extending at 72℃for 5-15s/kb; finally, the extension is carried out for 1 to 5 minutes at the temperature of 72 ℃.2. Designing primers for the designed CD19scFv-ESAT-6 sequence (shown as SEQ ID NO: 2) and synthesizing (the primer sequence is shown as SEQ ID NO: 10), and carrying out overlapping PCR amplification on the CD19scFv with the enzyme digestion site and the connecting sequence obtained by the amplification (the amplification conditions are the corresponding amplification conditions given by the instruction of the gold plate Mix reagent), and the ESAT-6 fragment (shown as figure 1A and figure 1B) after cutting gel recovery, wherein the two fragments are placed in the same system as a template for amplification by a common complementary sequence, namely, the two fragments are amplified and integrated into the CD19scFv-ESAT-6 gene (shown as figure 1C).
3. And (3) respectively carrying out BamHI and HindIII double digestion on the amplified CD19scFv-ESAT-6 gene and the plasmid pET-28a, connecting the digested target gene and the digested plasmid overnight (16 ℃) under the action of T4 DNA ligase to obtain a recombinant plasmid, transferring the recombinant plasmid into escherichia coli DH5 alpha according to a conventional method, preparing a large amount of recombinant plasmid, and carrying out enzyme digestion identification (shown in figure 1D) on the plasmid successfully, and then, converting the extracted recombinant plasmid into a protein expression strain BL-21 (DE 3), wherein the strain is used for expression preparation of fusion protein.
Example 2: preparation of CD19scFv-ESAT-6 fusion proteins
1. Protein expression: inoculating a strain (transformed protein expression strain BL-21 (DE 3)) into 5mL LB medium containing kanamycin resistance, culturing at 37deg.C (rotation speed of shaking table 200 rpm) for 8 hr, adding the bacterial liquid into 2YT medium containing kanamycin resistance, culturing at 37deg.C (rotation speed of shaking table 200 rpm) for about 3 hr to bacterial liquid OD 600 When the content is more than 0.6, isopropyl thiogalactose is addedThe glycoside IPTG (final concentration of 0.5 mM/L) was then induced overnight at 37 ℃.
2. And (3) thallus crushing: and (3) centrifuging at 8000rpm for 10min to collect thalli, re-suspending and washing 600ml of Lysis Buffer (1×), centrifuging, re-suspending thalli with 100ml of Lysis Buffer (1×), crushing thalli by a high crushing bacteria meter at 800bar under the crushing pressure of 5000rpm for 20min after crushing, and collecting inclusion body sediment.
3. Protein purification: after the inclusion body pellet was dissolved with 8M urea (pH 8.0, 1X Lysis Buffer), beyoold His-tag purification Resin (Shanghai Biyun Biotechnology Co., ltd., cat# P2233) was added and incubated overnight in a chromatography cabinet with rotation (rotation speed of the rotary incubator 20 rpm). The following day after centrifugation at 1500rpm for 5min the incubated beads were collected, washed off the beads with 8M urea at pH6.3 (pH adjusted to different pH values by hydrochloric acid) and 8M urea at pH4.3 (pH adjusted to different pH values by hydrochloric acid) in the affinity column, and finally the fusion protein was eluted with 8M urea (pH 8.0) containing 250mM imidazole.
4. Dialysis renaturation: the eluted protein solution was packed into a dialysis bag, and the dialysis bag was placed in 300mL of PBS dialysate (pH 7.4) containing 0.1mM DTT, 1mM reduced glutathione, 0.2mM oxidized glutathione and 5% glycerol, dialyzed 3 times at 4℃for 24 hours (one dialysate change), and then placed in PBS solution (pH 7.4) for 2 times for 24 hours (one dialysate change), and the protein solution system was changed to 1 XPBS.
5. Ultrafiltration concentration: the protein solution was concentrated by ultrafiltration using a 10K ultrafiltration tube, the concentrated protein solution was collected and filtered for sterilization, and finally the prepared fusion protein was verified by SDS-PAGE electrophoresis (as shown in FIG. 2).
Example 3: fusion protein can target and bind B cells and has stronger binding capacity
Spleen cells and peripheral blood of a C57BL/6J mouse are taken, cell suspension is prepared after red breaking treatment, fusion protein (CD 19scFv-ESAT-6 fusion protein) prepared in example 2 is added, the mixture is evenly mixed, incubation is carried out for 30min at 4 ℃, PBS (phosphate buffered saline) is used for washing 3 times, and then flow type antibody is added for dyeing according to groups, and flow type analysis results show that:
1.CD19his in spleen cells of scFv-ESAT-6 fusion proteins + The highest proportion of cells, indicating that the fusion protein binds to spleen cells more strongly than either the CD19scFv protein or ESAT-6 protein alone (fig. 3A (top) is representative flow image and fig. 3A (bottom) is statistical).
Spleen cells (His) to which CD19scFv-ESAT-6 fusion proteins bind + cells) showed the highest proportion of B cells, indicating that the fusion protein was more targeted to B cells than CD19scFv protein or ESAT-6 protein alone (fig. 3B, fig. 3B (top) representative flow image, fig. 3B (bottom) statistical). The results in fig. 3A and 3B are shown as mean ± standard deviation (n=6). Statistical differences in the figures: * P (P)<0.05，**P<0.01; ANOVA analysis and Neuman-Keuls test.
3. In peripheral blood cells, when the concentration of CD19scFv-ESAT-6 fusion protein was higher, the bound cells (His + cells), i.e., the proportion of fusion protein bound to B cells, was positively correlated with concentration (fig. 4A-C), indicating that fusion proteins were targeted to bind B cells in peripheral blood. Fig. 4A and 4B are representative streaming images, and fig. 4C is a statistical chart. Results are shown as mean ± standard deviation (n=3). Statistical differences in fig. 4: * P<0.01，****P<0.0001; ANOVA analysis and Neuman-Keuls test.
Example 4:8 μg is an effective dose of the fusion protein to elicit a significant immune response in mice
The fusion protein (CD 19scFv-ESAT-6 fusion protein) prepared in example 2 was injected in gradient doses into the tail vein of each group of C57BL/6J mice and the same amount (1.0X10) 6 CFU), the state of mice after immunization was observed, and spleen cells were sampled at 15 days and 30 days, respectively, cultured in vitro and stimulated with inactivated m.tb H37Rv, and immune effects were determined by flow. Wherein, FIG. 5A, FIG. 5B and FIG. 5C show the proportion of effector Th1 cells in the spleen of mice after 15 or 30 days of combined immunization of mice with BCG at different concentrations (1 ug,4ug,8 ug) of fusion protein (CD 19scFv-ESAT-6 fusion protein), respectively, and as can be seen from the data, the proportion of Th1 shows an increasing general trend with increasing concentration, and the data of 4ug below 1ug in FIG. 5B may be caused by individual differences of mice, but notIt was concluded that 8ug was the most effective dose and that the fusion proteins produced more Th1 effector cells when used in combination with BCG (1 ug,4ug,8 ug) than when BCG alone (PBS group, BCG injected alone), i.e., had a stronger protective immune effect against tuberculosis. Fig. 5A, 5B and 5C are all statistical diagrams. Results are shown as mean ± standard deviation (n=3). Statistical differences in the figures: * P (P)<0.05，**P<0.01，***P<0.001，****P<0.0001; ANOVA analysis and Neuman-Keuls test. The results show that immunized mice injected with 8 μg of fusion protein showed stronger Th1 response at 15 days (fig. 5A) and 30 days (fig. 5B), respectively, than other dose groups, indicating that 8 μg is an effective dose of fusion protein to elicit a significant immune response in mice (i.e., the maximum dose used without death of the mice). In fig. 5A and 5B, the CD19scFv-ESAT-6 fusion protein set (used as an adjuvant in combination with BCG) was able to generate a stronger Th1 immunoprotection response upon re-stimulation of Rv (virulent mycobacterium tuberculosis) compared to PBS set (immunized BCG only), both in experiments with splenocytes re-stimulated after Rv culture.
Example 5: fusion protein (CD 19scFv-ESAT-6 fusion protein) for combined immunization against BCG to elicit specific immune response against virulent Mycobacterium tuberculosis Mycobacterial tuberculosis H Rv
A fusion protein (CD 19scFv-ESAT-6 prepared in example 2, the same applies hereinafter) was injected intravenously at a dose of 8. Mu.g in C57BL/6J mice with 1.0X10 6 CFU BCG, splenocytes were taken at 30 days, stimulated with inactivated m.tb H37Rv, inactivated BCG and ESAT-6 (inactivated m.tb H37Rv/BCG CFU: cell number = 10:1,0.1 μg/mL ESAT-6) respectively, and after 24H incubation, flow analysis showed that BCG specific Th1 response was significantly weaker than m.tb H37Rv and ESAT-6 specific Th1 response (fig. 5C), three groups in fig. 5C were the ratio of specific Th1 produced by splenocytes against Rv, BCG, ESAT-6 antigen re-stimulation (ESAT-6 is an exoprotein of virulent tuberculosis strain Rv) respectively 30 days after combined injection with BCG, the result showed a lower ratio of Th1 against BCG, suggesting that the primary effect of fusion protein in combination with BCG was not to cause cross-reactivity against BCG, possibly favoring survival of BCG, and showed that strain Rv was viable in vivoThe Th1 response was very strong, indicating that the fusion protein when used in combination with BCG was able to elicit protective effects against virulent strain Rv. The immune response generated by the combination of the fusion protein and BCG is effective against M.tb H37Rv virulent strains.
The references referred to in the background of the invention are:
[1]LANGE C，AABY P，BEHR M A，et al.100years of Mycobacterium bovis bacille Calmette-Guérin[J].The Lancet Infectious Diseases，2022，22(1)：e2-e12.
[2]WOODWORTH J S，CLEMMENSEN H S，BATTEY H，et al.A Mycobacterium tuberculosis-specific subunit vaccine that provides synergistic immunity upon co-administration with Bacillus Calmette-Guerin[J].Nat Commun，2021，12(1)：6658.
[3]GANGULY N，SIDDIQUI I，SHARMA P.Role of M.tuberculosis RD-1region encoded secretory proteins in protective response and virulence[J].Tuberculosis(Edinburgh，Scotland)，2008，88(6)：510-7.
[4]PATHAK S K，BASU S，BASU K K，et al.Direct extracellular interaction between the early secreted antigen ESAT-6of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages[J].Nat Immunol，2007，8(6)：610-8.
[5]YABAJI S M，DHAMIJA E，MISHRA A K，et al.ESAT-6regulates autophagous response through SOD-2and as a result induces intracellular survival of Mycobacterium bovis BCG[J].Biochimica et biophysica acta Proteins and proteomics，2020，1868(10)：140470.
[6]FERREIRA-GOMES M，WICH M，et al.B Cell Recognition of Candida albicans Hyphae via TLR 2Promotes IgG1 and IL-6Secretion for T(H)17Differentiation[J].Frontiers in immunology，2021，12：698849.
[7]SCRIBA T J，NETEA M G，GINSBERG A M.Key recent advances in TB vaccine development and understanding of protective immune responses against Mycobacterium tuberculosis[J].Seminars in immunology，2020，50：101431.
[8]FAIRFAX K C，EVERTS B，AMIEL E，et al.IL-4-secreting secondary T follicular helper(Tfh)cells arise from memory T cells，not persisting Tfh cells，through a B cell-dependent mechanism[J].Journal of immunology(Baltimore，Md：1950)，2015，194(7)：2999-3010.
[9]KAUSHAL D，FOREMAN T W，GAUTAM U S，et al.Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis[J].Nat Commun，2015，6：8533.
Claims (9)
1. a fusion protein, characterized in that: the fusion protein is an adjuvant of BCG vaccine, the fusion protein comprises a CD19 single-chain antibody and a fusion protein of full-length ESAT-6 protein, and the CD19 single-chain antibody is connected with the full-length ESAT-6 protein.
2. The fusion protein of claim 1, wherein: the fusion mode of the fusion protein is CD19scFv-ESAT-6; the amino acid sequence of the fusion protein is shown as SEQ ID NO: 1.
3. A nucleotide for encoding the fusion protein of claim 1 or 2, characterized in that: the nucleotide sequence is shown as SEQ ID NO: 2.
4. A method of preparing a fusion protein according to claim 2, wherein: the preparation method comprises the following steps:
1) Designing the structure of the fusion protein according to claim 2;
2) Constructing a recombinant expression vector pET-28a-CD19scFv-ESAT-6;
3) Converting the recombinant expression vector pET-28a-CD19scFv-ESAT-6 obtained in the step 2) into a protein expression strain BL-21, and performing expansion culture on the protein expression strain BL-21;
4) Prokaryotic expression of the fusion protein CD19scFv-ESAT-6.
5. The method of manufacturing according to claim 4, wherein: the specific implementation mode of the step 1) is as follows: the nucleotide sequence of the early secretion protein antigen ESAT-6 is searched in NCBI database, tool molecule CD19scFv and ESAT-6 molecule are assembled together, and corresponding connecting sequence is added through analysis of bioinformatics software, so that the fusion protein with scientific and reasonable structure is designed.
6. The method of manufacturing according to claim 5, wherein: the specific implementation manner of the step 2) is as follows: and respectively synthesizing a CD19scFv gene and an ESAT-6 gene by utilizing a molecular cloning technology, integrating the CD19scFv gene and the ESAT-6 gene into the CD19scFv-ESAT-6 gene by a mode of overlapping PCR, and finally cloning the CD19scFv-ESAT-6 gene into a pET28a vector to construct a recombinant expression vector pET-28a-CD19scFv-ESAT-6.
7. The method of manufacturing according to claim 6, wherein: the specific implementation manner of the step 4) is as follows: the fusion protein is induced to be expressed in inclusion bodies by IPTG, and is dissolved by urea and then dialyzed by Beyoold His-tag purification Resin nickel ion affinity chromatography and dialysis bags, so that the fusion protein CD19scFv-ESAT-6 is obtained.
8. Use of the fusion protein according to claim 1 or 2 as an adjuvant to BCG vaccine or for the prevention of tuberculosis.
9. The use according to claim 8, characterized in that: the fusion protein is effective in inducing an apparent immune response at a dose of not less than 8 μg.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210859A (en) * | 2011-05-31 | 2011-10-12 | 昆明理工大学 | Mycobacterium tuberculosis ESAT-6-F2 fusion protein subunit vaccine and preparation method thereof |
| CN109306012A (en) * | 2017-07-26 | 2019-02-05 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor and application thereof of targeted mouse CD19 |
| CN110684116A (en) * | 2019-08-23 | 2020-01-14 | 北京恩元华生物科技有限公司 | Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof |
| WO2020249756A1 (en) * | 2019-06-14 | 2020-12-17 | Statens Serum Institut | Fusion proteins for tuberculosis vaccines |
-
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- 2023-07-07 CN CN202310832438.5A patent/CN116769056A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210859A (en) * | 2011-05-31 | 2011-10-12 | 昆明理工大学 | Mycobacterium tuberculosis ESAT-6-F2 fusion protein subunit vaccine and preparation method thereof |
| CN109306012A (en) * | 2017-07-26 | 2019-02-05 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor and application thereof of targeted mouse CD19 |
| WO2020249756A1 (en) * | 2019-06-14 | 2020-12-17 | Statens Serum Institut | Fusion proteins for tuberculosis vaccines |
| CN110684116A (en) * | 2019-08-23 | 2020-01-14 | 北京恩元华生物科技有限公司 | Mycobacterium tuberculosis EEC fusion protein, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SHEN, HB等: "Novel recombinant BCG coexpressing Ag85B, ESAT-6 and mouse TNF-α induces significantly enhanced cellular immune and antibody responses in C57BL/6 mice", 《MICROBIOLOGY AND IMMUNOLOGY》, vol. 54, no. 8, 31 August 2010 (2010-08-31), pages 435 - 441 * |
| 孟闯等: "结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性", 《畜牧兽医学报》, vol. 45, no. 05, 31 May 2014 (2014-05-31), pages 789 - 794 * |
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