CN111303301B

CN111303301B - Preparation method of mycobacterium tuberculosis immunogen protein ESAT6

Info

Publication number: CN111303301B
Application number: CN202010193338.9A
Authority: CN
Inventors: 周亚凤; 申兆兴; 黎勇; 宋淑婷
Original assignee: Shanghai Gene Optimal Biotechnology Co ltd
Current assignee: Shanghai Gene Optimal Biotechnology Co ltd
Priority date: 2020-03-18
Filing date: 2020-03-18
Publication date: 2022-04-05
Anticipated expiration: 2040-03-18
Also published as: CN111303301A

Abstract

The invention discloses a preparation method of mycobacterium tuberculosis immunogen protein ESAT 6. The invention provides a CFP10-ESAT6 recombinant fusion protein, the amino acid sequence of which is shown in SEQ ID NO. 1. The recombinant fusion protein has high expression quantity, supernatant expression and good activity; purifying the recombinant fusion protein, and then carrying out enzyme digestion, nickel column separation and elution to prepare mycobacterium tuberculosis immunogen protein ESAT 6; the ESAT6 protein prepared by the method has high expression level, good antigenicity, good activity and high purity, solves the problems of poor antigenicity and poor solubility of the ESAT6 protein and the problems that most of prokaryotic expression is inclusion body, lays a foundation for the early diagnosis of tuberculosis and the development of novel vaccines, and has wide application prospect.

Description

Preparation method of mycobacterium tuberculosis immunogen protein ESAT6

Technical Field

The invention belongs to the technical field of biological medicines. More particularly relates to a preparation method of mycobacterium tuberculosis immunogen protein ESAT 6.

Background

Tuberculosis is an infectious disease caused by infection of Mycobacterium Tuberculosis (MTB), according to the report of the worldwide tuberculosis in 2019 issued by WHO, 1/4 people all over the world are infected with mycobacterium tuberculosis at present, about 1000 ten thousand new cases occur each year, and in HIV negative people, tuberculosis causes about 120 million deaths. China is one of 30 tuberculosis high-burden countries in the world, and according to the data of the fourth tuberculosis epidemiological survey in the country, the number of people infected by mycobacterium tuberculosis in the country exceeds 4 hundred million. Therefore, how to early detect and prevent tuberculosis increasingly attracts attention worldwide.

The ESAT6 protein is an early secretory protein of mycobacterium tuberculosis without signal peptide, which is secreted extracellularly, possibly in a signal peptide-independent manner. When the ESAT6 protein grows on a common culture medium, the amount of ESAT6 protein secreted by mycobacterium tuberculosis is very low, but when the mycobacterium tuberculosis grows in infected macrophages, the expression amount of ESAT6 protein is obviously improved, and the change is called macrophage induction effect. The ESAT6 protein has high-level transcription in the proliferation stage and the non-proliferation stage of the mycobacterium tuberculosis, so that the ESAT6 protein can effectively induce the immune response of the organism in both active tuberculosis patients and latent mycobacterium tuberculosis infected patients. The distribution specificity and good immunity of the ESAT6 protein determine that the ESAT6 protein can be used as a candidate antigen of a novel vaccine for tuberculosis prevention or used as a specific antigen for tuberculosis diagnosis. The existing research shows that the ESAT6 protein is superior to TST when used as antigen for detecting latent infection of mycobacterium tuberculosis, and the sensitivity and the specificity of the sample blood detection method of ESAT6 and CFP10 are superior to PPD skin test.

In the prior art, Wenzhang et al (2004) disclose the expression and purification of Mycobacterium-bound ESAT-6 protein in pET prokaryotic expression system, and successfully prepare the Mycobacterium-bound ESAT-6 protein. However, in the method for preparing the ESAT6 protein, the increase of fusion protein components still exists, and the antigenicity of the ESAT6 protein obtained is poor; or the obtained ESAT6 protein has good antigenicity, but most of the ESAT6 protein expressed by pronucleus exists in the precipitate in the form of inclusion body, and a few of the ESAT6 protein exists in the schizomycete supernatant in the form of soluble, so that the obtained ESAT6 protein has small molecular weight, and further, the problems of low expression quantity and low recovery rate of the soluble protein are caused.

Therefore, the research and development of a preparation method of the ESAT6 protein with high expression level, good antigenicity, good activity and high purity is of great significance for the early diagnosis of tuberculosis by utilizing the ESAT6 protein and the development of novel tuberculosis vaccines.

Disclosure of Invention

The technical problem to be solved by the invention is to make up the defects of the existing preparation method of ESAT6 protein and provide a preparation method of mycobacterium tuberculosis immunogen protein ESAT 6.

The invention aims to provide a CFP10-ESAT6 recombinant fusion protein.

Another objective of the invention is to provide a CFP10-TEV-ESAT6 fusion gene encoding the recombinant fusion protein.

The invention also aims to provide application of the recombinant fusion protein or the fusion gene in preparing mycobacterium tuberculosis immunogen protein ESAT 6.

Still another objective of the invention is to provide a preparation method of mycobacterium tuberculosis immunogen protein ESAT 6.

The invention further aims to provide the immunogen protein ESAT6 prepared by the method.

The above purpose of the invention is realized by the following technical scheme:

the invention firstly provides a CFP10-ESAT6 recombinant fusion protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO. 1.

Preferably, the preparation method of the CFP10-ESAT6 recombinant fusion protein comprises the following steps: amplifying CFP10 gene and ESAT6 gene, connecting with plasmid pET-28a, constructing pET-28a-CFP10-ESAT6 recombinant plasmid, and inducing and expressing in BL21(DE3) escherichia coli to obtain the CFP10-ESAT6 recombinant fusion protein.

The invention also provides a CFP10-TEV-ESAT6 fusion gene for coding the recombinant fusion protein, and the nucleotide sequence of the fusion gene is shown as SEQ ID NO. 2.

The application of the recombinant fusion protein or the fusion gene in the preparation of mycobacterium tuberculosis immunogen protein ESAT6 also belongs to the protection scope of the invention.

The invention also provides a preparation method of the mycobacterium tuberculosis immunogen protein ESAT6, after the recombinant fusion protein is purified, the immunogen protein ESAT6 is obtained by enzyme digestion, nickel column separation and elution.

Preferably, the method comprises the steps of:

s1, purifying the recombinant fusion protein by nickel column affinity chromatography, chromatographic desalting and anion exchange chromatography in sequence;

s2, carrying out enzyme digestion on the recombinant fusion protein purified in the step S1 by using protease TEV, separating by using a nickel column, and collecting flow-through;

s3, treating the fluid obtained in the step S2 with 1-3% triton, and eluting to obtain the immunogen protein ESAT 6.

Preferably, the washing buffer used in the nickel column affinity chromatography of step S1 comprises 10-50 mM Tris-HCl and 50-80 mM imidazole.

More preferably, the washing buffer used for the nickel column affinity chromatography of step S1 comprises 20mM Tris-HCl and 60mM imidazole.

Preferably, the elution buffer used in the nickel column affinity chromatography of step S1 comprises 10-50 mM Tris-HCl and 250-300 mM imidazole.

More preferably, the elution buffer used for the nickel column affinity chromatography of step S1 comprises 20mM Tris-HCl and 300mM imidazole.

Preferably, the elution buffer used in the anion exchange chromatography of step S1 comprises 10-50 mM Tris-HCl and 0-0.5M NaCl.

More preferably, the elution buffer used in the anion exchange chromatography described in step S1 comprises 10mM Tris-HCl and 0.5M NaCl.

Preferably, the elution of step S3 is: eluting with the first and second equilibration buffers, and eluting with elution buffer.

Preferably, the first balance buffer solution comprises 10-50 mM Tris-HCl and 1% -3% triton.

More preferably, the first equilibration buffer comprises 20mM Tris-HCl and 2% triton.

Preferably, the second equilibration buffer is 10-50 mM Tris-HCl.

More preferably, the second equilibration buffer is 20mM Tris-HCl.

Preferably, the elution buffer comprises 10-50 mM Tris-HCl and 250-300 mM imidazole.

More preferably, the elution buffer comprises 20mM Tris-HCl and 300mM imidazole.

Preferably, the pH value of Tris-HCl is 7.5-8.5.

More preferably, the Tris-HCl has a pH of 8 in the present invention.

In addition, the immunogen protein ESAT6 prepared by the method also falls into the protection scope of the invention.

The invention has the following beneficial effects:

the invention provides a preparation method of mycobacterium tuberculosis immunogen protein ESAT 6. The invention utilizes a prokaryotic expression system to obtain CFP10-ESAT6 recombinant fusion protein with high secretory expression quantity and good activity, and then CFP10-ESAT6 recombinant fusion protein and CFP10 protein are separated by purification, enzyme digestion and a nickel column to obtain protein ESAT 6; the CFP10-ESAT6 recombinant fusion protein has high expression quantity, supernatant expression and good activity, so that the expression and purification of the ESAT6 protein are more efficient;

the ESAT6 protein prepared by the method has high expression quantity, good antigenicity, high purity (up to more than 95 percent) and good activity, and the ESAT6 protein 3mg can be prepared from 1L of target engineering bacterium fermentation liquor, so that the problems of poor antigenicity, poor solubility, inclusion body expression and the like of the ESAT6 protein are solved, a foundation is laid for the early diagnosis of tuberculosis and the development of novel vaccines, and the method has wide application prospects.

Drawings

FIG. 1 is a schematic structural diagram of a CFP10-ESAT6 recombinant fusion protein; wherein CFP10 represents the objective gene CFP10, ESAT6 represents the objective gene ESAT6, TEV represents protease TEV, and HIS represents HIS tag.

FIG. 2 is a nickel column affinity chromatography electrophoretogram of CFP10-ESAT6 recombinant fusion protein; wherein 1 is before the Ni column, 2 is the Ni column flow-through, 3 is 60mM imidazole wash, and 4 is 300mM imidazole elution.

FIG. 3 is a DEAE anion exchange chromatography electrophoretogram of CFP10-ESAT6 recombinant fusion protein; wherein 1 is DEAE flow through, 2 is eluted with 10% 0.5M NaCl, 3 is eluted before the peak with 25% 0.5M NaCl, 4 is eluted with 25% 0.5M NaCl, and 5 is eluted with 100% 0.5M NaCl.

FIG. 4 is the first Ni column electrophoresis of CFP10-ESAT6 recombinant fusion protein after digestion with protease TEV; wherein, after 1 is protease TEV for enzyme digestion of CFP10-ESAT6 recombinant fusion protein, 2 is Ni column flow-through, 3 is Ni column leaching, and 4 is 300mM imidazole elution.

FIG. 5 is a second Ni column electrophoresis of CFP10-ESAT6 recombinant fusion protein after digestion with protease TEV; wherein, 1 is Ni column and 2% Triton flow-through, and 2 is 300mM imidazole elution.

Figure 6 is a mass diagram of the immunogenic protein ESAT 6.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 construction and characterization of pET-28a-CFP10-ESAT6 recombinant plasmid

1. Primer design, synthesis and amplification of target genes CFP10 and ESAT6

The method comprises the steps of using a mycobacterium tuberculosis H37Rv genome as a template, amplifying a CFP10 gene by using a primer CFP10-F/R (the nucleotide sequences of the primer CFP10-F/R are respectively shown as SEQ ID No.3 and SEQ ID No. 4), amplifying an ESAT6 gene by using a primer ESAT6-F/R (the nucleotide sequences of the primer ESAT6-F/R are respectively shown as SEQ ID No.5 and SEQ ID No. 6), separating a target DNA fragment by nucleic acid gel electrophoresis of an amplification product, recovering the target DNA fragment by using an Omega glue back kit, respectively using NcoI/BamHI and BamHI/XhoI restriction endonucleases to cut the recovered DNA, and recovering the cut DNA by using the Omega Cycle-pure recovery kit for later use.

2. Construction of pET-28a-CFP10-ESAT6 recombinant plasmid

The E.coli DH5 a strain containing pET-28a plasmid was cultured on LB solid plate, plasmid was extracted using Omega plasmid extraction kit, the obtained pET-28a plasmid was digested with NcoI/XhoI restriction enzyme and subjected to nucleic acid gel electrophoresis, and the linearized plasmid pET-28a was recovered using Omega gel recovery kit. The recovered DNA fragment and linearized plasmid pET-28a were digested, reacted with T4 DNA ligase at 16 ℃ overnight, the ligation product transformed E.coli DH 5. alpha. competent cells, and plated on LB solid plate (containing 40. mu.g/mL kanamycin sulfate), which was incubated at 37 ℃ in a constant temperature incubator overnight. And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, the plasmid was extracted using an Omega plasmid extraction kit to obtain a pET-28a-CFP10-ESAT6 recombinant plasmid.

Example 2 expression and purification of CFP10-ESAT6 recombinant fusion protein

1. Transformation and validation of Escherichia coli BL21(DE3)

BL21(DE3) competent cells and 5 μ L of pET-28a-CFP10-ESAT6 recombinant plasmid obtained in example 1 are taken to be mixed, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, ice bath is taken out for 2min, 900 μ L SOC culture medium is added and placed in a 37 ℃ shaking table, recovery is carried out for 1h at 200rpm, centrifugation is carried out for 1min at 5000rpm, supernatant is removed after 100 μ L is remained, thalli are blown and uniformly mixed lightly, then the mixture is coated on an LB solid plate (containing 40 μ g/mL kanamycin sulfate), the mixture is placed in a 37 ℃ incubator, after 12h of culture, single colony growing on the plate is picked and inoculated with LB liquid culture medium, shaking table is carried out at 37 ℃ for 200rpm overnight culture, the bacterial liquid is subjected to sequencing verification by engine company, and then the induction expression of CFP10-ESAT6 recombinant fusion protein is carried out.

2. Inducible expression of CFP10-ESAT6 recombinant fusion protein

In the preparation process of the CFP10-ESAT6 recombinant fusion protein, the terminator of CFP10 is removed, two fragments are connected by enzyme cutting site BamHI, TEV protease is arranged between the two proteins, 6 XHIS label is arranged at the C end of ESAT6 protein, and the amino terminal sequence of the enzyme cutting site is ENLYFQG.

Then, the bacterial liquid of CFP10-ESAT6 recombinant fusion protein is taken to prepare 1L of bacterial liquid for fermentation through two-stage amplification culture, shaking culture is carried out at 37 ℃ and 200rpm for 4-5 hours (OD value is 0.6-0.8), IPTG with final concentration of 0.1mM is added, induction expression overnight culture is carried out at 16 ℃ and 220rpm, 8000rpm is carried out after fermentation is finished, and the thalli are collected through centrifugation for 10 min.

The structural schematic diagram of the CFP10-ESAT6 recombinant fusion protein is shown in FIG. 1.

The amino acid sequence of the CFP10-ESAT6 recombinant fusion protein is shown in SEQ ID NO. 1. The nucleotide sequence of the CFP10-TEV-ESAT6 fusion gene for coding the recombinant fusion protein is shown as SEQ ID NO. 2.

3. Disruption of cells

According to the weight of the collected thallus, adding balance liquid with 10 times volume (for example, adding 100mL balance liquid into 10g thallus), re-suspending in a homogenizer at 3000rpm, centrifuging at 8000rpm for 30min, removing supernatant, collecting precipitate, re-suspending, and homogenizing under high pressure to break thallus. After the thalli are broken, the thalli are centrifuged at 8000rpm for 30min, and supernatant is left to remove precipitates.

4. Purification of CFP10-ESAT6 recombinant fusion protein

The purification of the CFP10-ESAT6 recombinant fusion protein sequentially comprises 3 steps of nickel column affinity chromatography, chromatographic desalting and anion exchange chromatography, and specifically comprises the following steps:

(1) nickel column affinity chromatography: after Ni is hung on the chromatographic column, 10CV of the solution is treated by an equilibration buffer solution A at the flow rate of 90cm/h until the pH value of the effluent is the same as that of the equilibration solution. Loading the crushed thalli at a flow rate of 60cm/h, washing a sample with a washing buffer solution B at a flow rate of 90cm/h, and then eluting with an elution buffer solution C at a flow rate of 90cm/h to obtain CFP10-ESAT6 recombinant fusion protein;

the formula of the equilibrium buffer A is as follows: pH 8.0, 20mM Tris-HCl +300mM NaCl +20mM imidazole;

the formulation of wash buffer B was: pH 8.0, 20mM Tris-HCl +60mM imidazole;

the formulation of elution buffer C was: pH 8.0, 20mM Tris-HCl +300mM imidazole.

The electrophoresis chart of the CFP10-ESAT6 recombinant fusion protein Ni column is shown in FIG. 2, and it can be seen that after the sample passes through the Ni column, the pure CFP10-ESAT6 recombinant fusion protein can be obtained by washing and eluting with imidazole (lanes 3 and 4);

samples

3 and 4 were then collected for further purification.

(2) Chromatographic desalting: the column was treated with 10mM TE (5CV), treated with 0.5M NaOH (0.5CV), and then equilibrated with 10mM TE until the effluent pH was the same as that of the equilibration solution, at a flow rate of 120 cm/h. The CFP10-ESAT6 recombinant fusion protein eluted by the Ni column elution buffer C was loaded at a flow rate of 120cm/h, and the sample collection was started at a volume of 60mL when the UV was increased.

(3) Anion exchange chromatography: the column was treated with 10CV of equilibration buffer D, treated with 5CV of 0.5M NaOH, and equilibrated with equilibration buffer D until the pH of the effluent was the same as that of the equilibration solution, and the flow rate was 90 cm/h. After desalting G25, the sample was loaded at a flow rate of 60cm/h and eluted with elution buffer E at a flow rate of 90 cm/h; and (3) an elution mode: isocratic elution (isocratic elution with 10%, 25% and 100% elution buffer E, respectively).

The formula of the equilibrium buffer D is as follows: 10mM Tris-HCl, pH 8.0;

the formulation of elution buffer E was: pH 8.0, 10mM Tris-HCl +0.5M NaCl.

The DEAE anion exchange chromatophoresis chart of the CFP10-ESAT6 recombinant fusion protein is shown in FIG. 3, and it can be seen that both DEAE flow-through, 10% elution buffer E and 25% elution buffer E (lanes 1-4) can better purify the CFP10-ESAT6 recombinant fusion protein, while the purification effect of 100% elution buffer E is not good (lane 5); samples 1-4 were then collected for further experiments.

EXAMPLE 3 obtaining of the immunogenic protein ESAT6

1. Enzyme digestion of CFP10-ESAT6 recombinant fusion protein

The CFP10-ESAT6 recombinant fusion protein purified in example 2 was digested by dialysis with protease TEV at 4 ℃ overnight, and the fusion protein eluted from DEAE column with 25% elution buffer E was digested by dialysis with a macromolecular dialysis bag at pH 8.0 and 20mM Tris-HCl for 3-4 hours at 4 ℃ overnight.

2. Purification of ESAT6 immunogenic protein

(1) Removal of CFP10-ESAT6 recombinant fusion protein

The principle is as follows: removing the CFP10-ESAT6 recombinant fusion protein which is not cut after enzyme digestion, separating the CFP10-ESAT6 recombinant fusion protein, the cut CFP10 protein and the cut ESAT6 protein by using a Ni column, wherein the CFP10-ESAT6 recombinant fusion protein is combined on the Ni column due to the HIS label, the CFP10 protein and the ESAT6 protein interact, and the two proteins exist in the flow-through during washing, so that the flow-through is collected, and the target protein (the CFP10 protein and the ESAT6 protein) is obtained.

After Ni is hung on the chromatographic column, 10CV of the effluent is treated with the equilibration buffer solution F at the flow rate of 90cm/h until the pH value of the effluent is the same as that of the equilibration solution. And (3) carrying out chromatography treatment on the protein after enzyme digestion, wherein the sampling flow rate is 60cm/h, impurities are eluted at the same degree by using the elution buffer solution C, the elution flow rate is 90cm/h, and the target protein in flow-through is collected to obtain the CFP10 protein and the ESAT6 protein.

The formula of the equilibration buffer F is as follows: Tris-HCl, pH 8.0, 20 mM;

The first Ni column electrophoresis after the CFP10-ESAT6 recombinant fusion protein is digested by protease TEV is shown in FIG. 4, and it can be seen that after the CFP10-ESAT6 recombinant fusion protein passes through a Ni column, the flow-through is collected, and the uncut CFP10-ESAT6 recombinant fusion protein can be better removed (lanes 2 and 3);

samples

2 and 3 were then collected for further experiments.

(2) Obtaining of the immunogenic protein ESAT6 (removal of CFP10 protein)

The principle is as follows: treating the collected flow-through (CFP10 protein, ESAT6 protein) with 2% triton to separate CFP10 protein and ESAT6 protein, separating the two proteins by passing through a nickel column because the ESAT6 protein has a HIS label, and eluting to obtain the immunogen protein ESAT 6.

After Ni is hung on the chromatographic column, 10CV of the effluent is treated with an equilibration buffer G at a flow rate of 90cm/h until the pH value of the effluent is the same as that of the equilibration solution. To the upstream Ni-column flow-through and eluted sample was added 2% triton to separate the interacting CFP10 protein and ESAT6-6His, and the Ni-column bound ESAT6-6His was used to flow through the CFP10 protein. After elution with equilibration buffer G, the residual triton was eluted with equilibration buffer F, followed by elution with ESAT6-6His with elution buffer C. The flow rate of the sample is 60cm/h, and the flow rate of the elution is 90cm/h, so that the immunogen protein ESAT6 can be obtained.

The formula of the equilibrium buffer G is as follows: pH 8.0, 20mM Tris-HCl + 2% Triton;

The second Ni column electrophoresis after CFP10-ESAT6 recombinant fusion protein is digested by protease TEV is shown in FIG. 5, and it can be seen that 2% triton is added into the collected mixed protein of CFP10 protein and ESAT6 protein, so that the two proteins can interact less effectively, and after passing through a nickel column, CFP10 protein can be removed by elution, and finally ESAT6 protein can be obtained.

The quality diagram of the immunogen protein ESAT6 is shown in FIG. 6, and it can be seen that the purity of the obtained ESAT6 protein is high, and the purity of the obtained ESAT6 protein can reach more than 95% by analyzing the purity of the ESAT6 protein through gel imaging system image analysis software.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. A preparation method of mycobacterium tuberculosis immunogen protein ESAT6 is characterized by comprising the following steps:

s1, sequentially purifying the CFP10-ESAT6 recombinant fusion protein with the amino acid sequence shown as SEQ ID NO.1 through nickel column affinity chromatography, chromatographic desalting and anion exchange chromatography;

2. The method of claim 1, wherein the washing buffer used in the nickel column affinity chromatography of step S1 comprises 10 to 50mM Tris-HCl and 50 to 80mM imidazole; the elution buffer used in the nickel column affinity chromatography of step S1 includes 10-50 mM Tris-HCl and 250-300 mM imidazole.

3. The method of claim 1, wherein the elution buffer used in the anion exchange chromatography of step S1 comprises 10-50 mM Tris-HCl and 0-0.5M NaCl.

4. The method of claim 1, wherein the elution of step S3 is: eluting with the first and second equilibration buffers, and eluting with elution buffer.

5. The method of claim 4, wherein the first equilibration buffer comprises 10-50 mM TrisHCl and 1-3% Triton; the second equilibrium buffer solution is 10-50 mM Tris-HCl; the elution buffer comprises 10-50 mM Tris-HCl and 250-300 mM imidazole.