CN114196643B - Process and method for purifying TPO - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses a process and a method for purifying TPO, wherein the process and the method disclosed by the invention are utilized, no label is required to be added for purifying TPO protein, the purification steps are greatly simplified, and the obtained TPO protein has good stability and cannot be precipitated in a preservation solution, so that the process and the method are beneficial to development and application of a subsequent kit. The invention also adds the step of screening and purifying TPO antibody, so that the purification process of the Ni column is stable, and the purified TPO protein has high purity.

Description

Process and method for purifying TPO

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a process and a method for purifying TPO.

Background

Thyroid Peroxidase (TPO) is a membrane-bound glycoprotein containing a heme prosthetic group, the whole molecule is divided into an intramembrane region, a transmembrane region and an extramembrane region, the cDNA is 3kb in full length, the presence of the transmembrane region makes the TPO difficult to secrete during expression, and a part with catalytic activity extends into the inside filled with a glial follicular cavity. TPO as an important enzyme in the synthesis process of thyroid hormone in organisms can participate in the processes of catalyzing the activation of iodine, the iodination of tyrosine residues on thyroglobulin and the coupling of iodinated tyrosine residues to finally generate thyroxine (T3 and T4). TPO is also the main antigen component of thyroid microsome, when the thyroid gland of organism is diseased and the biological structure of follicular cell is destroyed, TPO is missed to peripheral blood through the marginal part of thyroid gland follicular cell cavity, namely the top part of thyroid cell, thereby stimulating organism to regulate immune system, and the immune system generates TPO antibody (Anti-TPO) to respond stimulation.

The antiti-TPO antibody (Anti-TPO) is commonly found in people suffering from autoimmune thyroid diseases and is a thyroid autoantibody, the positive antiti-TPO is mainly found in patients with hashimoto thyroiditis, primary hypothyroidism, hyperthyroidism and the like, and the detection of the antiti-TPO antibody (Anti-TPO) in the thyroid function detection becomes one of the basic detection indexes of the thyroid function.

Therefore, for the detection of Anti-TPO with high specificity, the development of TPO with high expression and high purity is particularly important for clinical detection and experimental research, but at present, the TPO expressed in China is generally low in purity, the performance of the purified protein is poor, precipitation is easy to occur, and the problem of low recovery rate when the TPO protein is applied to a downstream reagent line greatly limits the development of TPO detection.

Disclosure of Invention

When purifying TPO, the purity of TPO is not high, which is found in the invention, mainly because the current commercial Ni column purification process is unstable and has low purity, and the purification of TPO using antibody must cross the isoelectric point, so that the purification yield is seriously influenced by the process precipitation. It is therefore necessary to find a way of elution that avoids crossing the isoelectric point, meeting the requirements and does not affect the TPO performance.

In order to solve the problem that protein is easily precipitated after purification, the present invention provides a process for purifying TPO, comprising,

(1) Screening and purifying TPO antibody;

(2) Mixing the TPO antibody purified in the step (1) with a filler according to a certain condition to prepare an affinity chromatography column;

(3) Adding a sample to be purified into the affinity chromatography column prepared in the step (2), eluting the affinity chromatography column by using eluent, and purifying TPO protein;

preferably, the eluent in step (3) contains 1-5M of a metal ion salt, and pH =6.5-8.0.

Preferably, the TPO antibody and the filler are mixed to form the TPO antibody: filler =10:1 and mixing.

Preferably, the concentration of the metal ion salt is 2-4M and the pH is 6.8-7.3.

Preferably, the metal ion salt is selected from NaCl, liCl or MgCl ₂ More preferably, the metal ion salt is selected from MgCl ₂ 。

The antibody purification usually adopts glycine with pH =2.7 for elution, but when the antibody raw material is applied to immune reaction, a neutral and alkaline buffer environment is needed, the isoelectric point of TPO is 6.13, when the pH of the eluted antibody solution with pH =2.7 is adjusted to be neutral or alkaline, the antibody solution passes through the isoelectric point of TPO, so that the TPO is precipitated, the purified TPO has poor stability, and the subsequent application is influenced.

On the other hand, in order to solve the problem that the purity of TPO is not high, the present invention provides a method for producing high purity TPO, comprising,

(1) Transferring the recombinant TPO gene into bacterial cells to prepare plasmids;

(2) Transfecting the plasmid prepared in the step (1) into Sf9 cells to prepare TPO virus;

(3) Introducing TPO virus into Sf9 cells for expression to obtain TPO protein;

(4) Screening and purifying TPO antibody;

(5) Mixing the purified TPO antibody with filler under certain conditions to prepare an affinity chromatography column;

(6) Adding the TPO protein in the step (3) into the affinity chromatographic column prepared in the step (5), and eluting the affinity chromatographic column by using an eluent to purify the TPO protein;

the recombinant TPO gene may or may not contain a tag.

The eluent in the step (6) contains 1-5M of metal ion salt, and the pH =6.5-8.0.

Preferably, the recombinant gene TPO does not contain a tag.

In the prior art, the protein is separated by utilizing the principle that some amino acids on the surface of the protein, such as histidine, tryptophan, cysteine and the like, can generate special interaction with metal ions. These include dative bonding, electrostatic adsorption, covalent bonding, among which dative bonding is the dominant one, and among these, 6-histidine Tag (His-Tag) is most widely used. The method is generally characterized in that the His-Tag and the recombinant protein are fused to purify the protein, and the recombinant protein with the His-Tag can be adsorbed by a Ni column by utilizing the characteristic that histidine is chelated with divalent metal ions, so that the protein can be purified by metal chelating affinity chromatography conveniently, and the purification rate of the protein is increased.

The invention creatively discovers that when Sf9 cells are used as host cells to purify TPO protein, no histidine tag is added, and the protein purification effect is better than that of the protein added with histidine tag.

Preferably, the TPO antibody and the filler are mixed into the TPO antibody according to certain conditions: filler =10:1 and mixing.

Preferably, the eluent in the step (6) contains 1-5M of metal ion salt, and pH =6.5-8.0.

Preferably, the concentration of the metal ion salt is 2-4m, and the ph =6.8-7.3.

Preferably, the metal ion salt is selected from NaCl, liCl or MgCl ₂ More preferably, the metal ion salt is selected from MgCl ₂ 。

In another aspect, the present invention provides a process for producing high purity TPO comprising,

(1) Transferring the recombinant TPO gene into bacterial cells to prepare plasmids;

(2) Transfecting the plasmid prepared in the step (1) into Sf9 cells to prepare TPO virus;

(3) Introducing TPO virus into H.five cell for expression to obtain TPO protein;

(4) Screening and purifying TPO antibody;

(5) Mixing the purified TPO antibody with a filler according to a certain condition to prepare an affinity chromatographic column;

(6) Adding the TPO protein in the step (3) into the affinity chromatographic column prepared in the step (5), and eluting the affinity chromatographic column by using an eluent to purify the TPO protein;

the eluent in the step (6) contains 1-5M of metal ion salt, and the pH =6.5-8.0.

Preferably, the recombinant TPO gene does not contain any tag.

The invention changes host cells, and finds that the effect of adding and not adding labels is larger, the purification effect without adding labels is better, and the purification effect can be influenced even by adding labels by using H.five cells.

Expression purification with h.five cells required two cells compared to expression directly with Sf9 cells.

Preferably, the TPO antibody and the filler are mixed to form the TPO antibody: filler =10:1.

preferably, the concentration of the metal ion salt is 2-4m, and the ph =6.8-7.3.

Preferably, the metal ion salt is selected from NaCl, liCl or MgCl ₂ More preferably, the metal ion salt is selected from MgCl ₂

The recombinant TPO gene sequence is shown in SEQ NO. 1.

Advantageous effects

(1) The invention creatively discovers that the purified TPO protein does not need to be added with a label, thereby greatly simplifying the purification steps.

(2) The TPO protein obtained by using the high-concentration metal ion salt eluent TPO disclosed by the invention has good stability, cannot be precipitated in a preservation solution, and is beneficial to the development and application of a subsequent kit.

(3) The step of screening and purifying TPO antibody is added, so that the purification process of the Ni column is stable, and the purified TPO protein has high purity.

Drawings

FIG. 1: gel map of purified TPO by conventional method

FIG. 2: gel profiles of purified TPO of this invention;

FIG. 3: comparing gel maps for different host cell purifications;

FIG. 4: performing a linear experiment;

FIG. 5: and (4) performing clinical relevance experiments.

Detailed Description

Examples

Example 1 Virus preparation

Transformation of DH10bac

1. 100ul DH10bac competent cells were taken and placed on ice for 10min until completely thawed. Adding 50-200ng (about 1 ul) plasmid, mixing, and standing on ice for 30min;

heat shock is carried out for 45s at the temperature of 2.42 ℃;

3. standing on ice for 2-5min;

4. adding 700ul LB culture medium, 37 ℃,225rpm,4h;

5. taking 100ul of the X-gal plate, and carrying out inverted culture at 37 ℃ overnight;

6. taking white colonies on an x-gal plate for streaking, and carrying out inverted culture at 37 ℃ for overnight;

7. the colony was observed to remain white overnight, which was confirmed to be successful in transformation, and was again verified by colony PCR.

(II) extraction of rod-shaped plasmids

1. Selecting white clone in 2ml LB culture medium (50 ug/ml kanamycin, 7ug/ml gentamicin, 10ug/ml tetracycline, X-gal 100ug/ml, IPTG 40 ug/ml), and culturing at 37 deg.C with shaking table at 250-300rpm for more than 24 hr;

2. the rod-shaped plasmids were extracted using PureLink HiPupure Plasmid DNA Purification Kits.

3. Dissolving plasmid with water after extraction, and determining the concentration of the plasmid to be more than 500ng/ul for backup

(III) Virus preparation and amplification

1. Sf9 cells were recovered by liquid nitrogen extraction and cultured in T25 flasks (medium supplemented with anti/anti) and incubated at 28 ℃ for about 72 hours at rest.

2. And (3) removing the old culture medium after the confluence of Sf9 cells in the T25 culture bottle reaches 90-100%, adding 15ml of fresh culture medium, inoculating the culture medium into a new T25 culture bottle in a third-to-third ratio, and adding 4ml of anti/anti in the culture medium. And (3) standing and culturing for 24 hours in a drying incubator at 28 ℃.

2. Preparation of transfection mixture: adding 2ug of rod-shaped plasmid DNA into a 200ul Sf900II culture medium, adding 10ug of transfection reagent into the 200ul Sf900II culture medium, slightly and uniformly mixing the prepared rod-shaped plasmid DNA working solution and the transfection reagent working solution, incubating at room temperature for 20min, adding into 4ml of SF9 cell sap, and standing in a 28 ℃ culture box for culture.

3. After 24h of transfection, the cell state is observed, and the complex particles can be seen to detect whether the cells are polluted or not so as to respond in time.

4. After 72h of transfection, the cells were obviously diseased and became round and large. After 3-5 days, the supernatant was collected and centrifuged at 1500rpm/min at room temperature. The supernatant was filtered through a 0.22um filter and defined as TPO-P0 generation virus, stored at 4 ℃ for a short period (three months of validity) and at-20 ℃ for a long period (one year of validity). The residual cells can be analyzed by WB to detect the expression of TPO. After the expression was confirmed, the TPO-P1 virus was further amplified.

5. The inoculation density is 1.5x10 ⁶ (viability > 95%) of Sf9 cells 30ml in 125ml shake flasks, P0 virus was added to Sf9 cells at 28 ℃ in a MOI =0.05 calculated according to the titer of 1E 6. Cytopathic effects were observed for 72h, and after 3-4 days, the supernatant was collected and centrifuged at 1500rpm/min at room temperature. The supernatant is filtered by a 0.22um filter and is defined as TPO-P1 virus, and after the subsequent titer determination, the supernatant can be directly used for large-scale amplification of the TPO-P2 virus.

6. The inoculation density is 1.5x10 ⁶ (viability > 95%) SF9 cells in a total of 1000ml in 2.8L flasks, P1 virus was added to Sf9 cells at 28 ℃ in MOI =0.05 calculated according to the titer of 1E 7. Cytopathic effect was observed for 72h, and after 3-4 days, the supernatant was collected and centrifuged at 1500rpm/min at room temperature. The supernatant is filtered by a 0.22um filter and is defined as TPO-P2 virus, and after the subsequent titer determination, the supernatant can be directly used for large-scale infection expression of TPO protein.

7. And (3) performing titer determination by using a fluorescent quantitative PCR method, taking TPO standard plasmid as a standard substance, extracting TPO-P2 virus DNA and then taking the extracted TPO-P2 virus DNA as a sample to perform titer determination, wherein the virus titer is more than 1E8 and can be used.

(IV) fermentation expression Process

First, shake flask fermentation

1. Five cell suspension preparation:

a. shaking the cell suspension, sucking out about 1ml by a pipette, adding into a sterile 1.5ml EP tube, sucking out 10ul of cell suspension, mixing with 10ul of trypan blue, sucking 10ul to a cell counting plate, automatically counting in a cell counter, and observing the cell state under a microscope.

b. The transfection density of the cells is 3E6, and the volume is 1L; calculate the required cell suspension volume based on the number of viable cells, transfer the cell suspension to a 50ml sterile centrifuge tube, centrifuge at 800rpm/min for 5min, collect the cells, decant most of the old medium, leave 5-10ml of old medium/tube, resuspend the cells, pour into a 2.8L shake flask, add 45ml 200mM glutamine (9 mM) to 1lh. Five fresh cell medium, pour medium containing 9mM glutamine into a 2.8L shake flask to a final volume of 1L.

2. Sf9 cell suspension preparation:

a. shaking the cell suspension evenly, sucking out about 1ml by a pipette, adding the cell suspension into a sterile 1.5ml and EP tube, sucking out 10ul of cell suspension, mixing the cell suspension with 10ul of trypan blue evenly, sucking 10ul of cell suspension to a cell counting plate, placing the cell counting plate into a cell counter for automatic counting, and observing the cell state under a microscope, wherein the cell activity is more than 98 percent.

b. The transfection density of the cells is 3E6, and the volume is 1L; calculating the required cell suspension volume according to the number of living cells, transferring the cell suspension into a 50ml sterile centrifuge tube, placing the tube in a centrifuge for 5min at 1500rpm/min, collecting the cells, resuspending the cells with fresh Sf9 cell culture medium, pouring into a 2.8L shake flask, and obtaining a final volume of 1L.

2. And (3) virus infection:

a. addition of virus to host cells (h.five cells or Sf9 cells) required virus = cell density x volume x MOI/virus titer

b. According to the calculation result, after the virus is mixed by gentle inversion, TPO-P2 generation virus is added according to MOI =2, mixed by gentle mixing, and placed in a dry incubator at 28, DEG C and 120rpm/min for shake suspension culture. The fermentation process was continued for 72h, the cell dynamic parameter changes were monitored every 24h, and samples were collected after 3 days.

3. Pouring the fermentation liquor into a 250ml centrifuge bottle, balancing, placing in a centrifuge at 3000rpm/min, centrifuging at 4 deg.C for 10min, collecting supernatant, and measuring to confirm final volume. PMSF was added to the supernatant at a final concentration of 1mM/L and the broth was stored at 4 ℃ for purification.

EXAMPLE 2 TPO purification

Purified TPO needs to be stored in a buffer for subsequent immunoreaction, which occurs under conditions of pH around neutral, and this example was performed with pH =8.

(I) purification by conventional method

The method comprises the following specific steps:

1.50 mg of TPO antibody was dialyzed against PBS (pH 7.2) at 4 ℃ for 12h, and the dialyzate was filtered through 0.45uM membrane to remove the precipitate

2. 5ml of NHS plug (GE) were taken and the plug was washed with 50ml of 1Mm HCl pre-cooled beforehand. The filler was washed with another 50ml PBS (pH = 7.2).

3. 5ml of the filler and 50mg of the antibody were mixed and the mixture was left at 12h at 50rpm at 4 ℃.

4. Recovering the filler according to the proportion of 1: blocking solution (0.5M ethanolamine pH = 8.0) was added at 2 ratio and left at room temperature for 3h,50rpm.

5. The packing was harvested and washed 5 times with 10ml each time with pH =2.7 glycine eluent and PBS (pH = 7.2) in alternation. Defined as purified for use.

6. The purification column was equilibrated by adding 15mL of equilibration buffer (pH =7.2 PBS).

7.1L of supernatant was initially loaded at a flow rate of 5ml/min, and after completion PBS (pH = 7.2) was equilibrated by 50ml.

8. Elution was performed by adding 10mL of elution buffer (pH =2.7,0.1m glycine), and the eluate was collected.

9. The collected eluate was dropped into a storage buffer (1M Tris, pH = 8.0) and stored, and a precipitate was found.

10. The purity of the stored TPO antigen was confirmed by SDS-PAGE.

The results are shown in FIG. 1, lane 1: supernatant, lane 2: flow through, lane 3: elution fraction 1, lane 4: elution fraction 2, lane 5: elution fraction 3, lane 6: precipitate after elution, lane 7: marker, the results showed that the eluted product precipitated in the storage buffer at pH =8.0 and that the precipitated product was the protein of interest.

(II) purification by the method of the invention

The method comprises the following specific steps:

(1) Screening for TPO antibodies

(a) Externally purchased TPO (purchased from Hytest company) is used for carrying out mouse immune screening, 35 effective antibodies are obtained, and three antibodies with different reactivities of high, medium and low are preliminarily selected from the 35 effective antibodies according to the ELISA reactivities, and are named as #17, # 31 and # 11.

(b) Coupled purification validation

In summary, the antibodies #17, # 31 and # 11 can be used for preparing the purification column, i.e. the antibodies: filler =10:1 preparing an affinity chromatography column.

1. Purifying by using the self-made purification column

2. The purification column was equilibrated by adding 15mL of equilibration buffer (pH =7.2 PBS).

3.1L of fermentation supernatant was initially loaded at a flow rate of 5ml/min, and after completion of the run, 50ml of PBS (pH 7.2) was equilibrated.

4. Elution was performed by adding 10mL of elution buffer (3M magnesium chloride, pH = 7.0), and the eluate was collected.

5. Confirming no precipitation of the eluate, and verifying purity by SDS-PAGE

Sf9 cells were selected in this example and did not contain his-tag.

The total yield of #17, # 31 and # 11 after purification is 96.8%,67.8% and 25.2%, respectively. Therefore, selecting #17 for subsequent experiments, the TPO protein purified from #17 was stored in the storage buffer without any precipitation, and the purity was verified by gel, as shown in FIG. 2, lane 1: supernatant, lane 2: flow through, lane 3: marker, lane 4: and storing the eluent after the elution.

The result shows that the antibody eluted and stored by the method has good stability, does not precipitate, and is beneficial to the requirement of subsequent immune experiments.

Example 3 Effect of host cells and tags on purification

The host cells were h.five and Sf9 cells, with and without his-tag, and the purification results are shown in fig. 3, lane 1: sf9 with his-tag, lane 2: five with his-tag, lane 3: marker, lane 4: sf9 cells do not contain his-tag, lane 5: the five protein does not contain his-tag, and the recombinant protein containing the his-tag is shown to have a purification effect not as good as that of the recombinant protein without the his-tag.

The result shows that by using the purification method of the invention, H.five and Sf9 cells are selected as hosts for expression and purification, no label is required to be added, the operation is simplified, and the purification effect is better.

Example 4: effect of eluent on purification

H.five cells were selected as host cells, and no his-tag was contained.

The affinity column was #17 mixed with packing.

The eluent was changed in composition and purity as follows:

TABLE 1 eluent purification effect

It can be seen that TPO purified from the eluent with the concentration range of 1-5M and the pH =6.5-8 has high purity, good stability and no precipitation, wherein the metal ion salt MgCl ₂ And the purification effect is not influenced by replacement of LiCl and NaCl.

EXAMPLE 5 TPO Performance validation

In order to verify the effect of applying the purified TPO antigen to an immunoassay, the present embodiment applies the antigen to a kit, and currently, the detection of an antibody mainly includes immunoturbidimetry, immunochromatography, chemiluminescence, and the like, wherein the performance requirement of chemiluminescence on the antibody is the most strict, so chemiluminescence is selected for the determination in the present embodiment. Chemiluminescent kits are commercially available.

This example selects the TPO antigen purified in example 2 for testing.

The assay reagent comprises R1, R2 and magnetic bead solution

The R1 comprises: biotin labeled TPO antigen, concentration used: 1ug/ml.

The R2 comprises: alkaline phosphatase labeled protein A used at a concentration of 0.1ug/ml

The specific operation steps are as follows:

1. preparation of the experiment: 5ml of each of the R1 and R2 working solutions, 4ml of a magnetic bead solution, a calibrator and 24 clinical samples. (supplementary detection step)

2. The components are respectively placed in a reagent kit, the reagent kit is placed in a full-automatic chemiluminescence immunoassay analyzer EXI1800 (from Zhongyuan Virgi Biotechnology Co., ltd.), and anti-TPO items (10 ul samples, 60ul samples, 125ul samples and 30ul magnetic bead solutions) are selected for experiment.

(1) Linear experiment

The results of the experiment are shown in table 2 and fig. 4:

TABLE 2 Linear experiment

As shown in FIG. 4, it can be seen from FIG. 4 that the TPO antigen purified by the present invention can be applied to chemiluminescence detection, and the detection sensitivity is less than or equal to 3IU/ml, and the linear range is 5-600IU/ml, indicating that the TPO antigen is excellent.

(2) Clinical validation

The TPO preparation kit produced by the invention and the commercially available kit are subjected to correlation analysis.

The result is shown in fig. 5, the correlation coefficient is 0.99, which indicates that the product correlation is good and is suitable for clinical detection.

Claims (3)

1. A process for producing high purity TPO comprising,

(1) Transferring the recombinant TPO gene into bacterial cells to prepare plasmids;

(2) Transfecting the plasmid prepared in the step (1) into Sf9 cells to prepare TPO virus;

(3) Introducing TPO virus into H.five cell for expression to obtain TPO protein;

(4) Screening and purifying TPO antibody;

(5) Mixing the purified TPO antibody with a filler according to a certain condition to prepare an affinity chromatographic column;

(6) Adding the TPO protein in the step (3) into the affinity chromatographic column prepared in the step (5), and eluting the affinity chromatographic column by using an eluent to purify the TPO protein;

the eluent in step (6) comprises 1-5M of a metal ion salt, pH = 6.5-8.0;

the recombinant TPO gene does not contain any tag;

the above-mentionedThe metal ion salt is selected from NaCl, liCl or MgCl ₂ 。

2. The method according to claim 1, wherein the metal ion salt concentration of the eluent in the step (6) is 2-4M, and the pH is 6.8-7.3.

3. The method of claim 1 or 2, wherein said TPO antibody is mixed with a filler under conditions such that the TPO antibody: filler =10:1.

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