CN114196643A

CN114196643A - Process and method for purifying TPO

Info

Publication number: CN114196643A
Application number: CN202111596915.XA
Authority: CN
Inventors: 冯奇; 林晏因; 李乾; 付鹏德; 蒋雪; 姬成东
Original assignee: Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-18
Anticipated expiration: 2041-12-24
Also published as: CN114196643B

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 TPO is difficult to secrete during expression due to the existence of the transmembrane region, and a part with catalytic activity extends into the interior filled with a colloid follicular cavity. TPO, 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, 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 damaged, 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 the organism to regulate the immune system, and the immune system generates TPO antibody (Anti-TPO) to deal with 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), and eluting the affinity chromatography column by using an eluent to purify the TPO protein;

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

Preferably, the TPO antibody and the filler are mixed into the TPO antibody according to certain conditions: 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 MgCl₂More preferably, the metal ion salt is selected from MgCl₂。

The antibody purification usually adopts glycine with pH 2.7 for elution, but 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 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 recombinant TPO gene contains a tag or does not contain a tag.

The eluent in the step (6) contains 1-5M of metal ion salt, and the pH value is 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 main one, and among them, 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, histidine tag is not added, and the purification effect of protein is better than that of protein added with histidine tag.

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

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

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

(4) screening and purifying TPO antibody;

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 into the TPO antibody according to certain conditions: filler 10: 1.

Preferably, the metal ion salt is selected from NaCl, LiCl or MgCl₂More preferably, the metal ion salt is selected from MgCl₂

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 the present invention;

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

FIG. 4: performing a linear experiment;

FIG. 5: and (3) clinical relevance experiments.

Detailed Description

Examples

Example 1 Virus preparation

Transformation of DH10bac

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

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

3. standing on ice for 2-5 min;

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

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

(II) extraction of rod-shaped plasmids

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

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

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 flask (supplemented with anti/anti) medium and incubated at 28 ℃ for about 72 h.

2. After the confluence of Sf9 cells in the T25 culture bottle reaches 90% -100%, the old culture medium is removed, 15ml of fresh culture medium is added, and the culture medium is inoculated into a new T25 culture bottle in a third-to-third ratio, namely a 4ml/T25 culture bottle (the culture medium is not added with anti/anti any more). And (3) standing and culturing for 24 hours in a drying incubator at 28 ℃.

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

3. After 24h of transfection, the cell state is observed, the complex particles can be seen, and whether the pollution exists or not can be detected, so that the timely response can be realized.

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 passage virus, short-term storage at 4 ℃ (three months of validity), long-term storage at 20 ℃ (one year of validity). The remaining cells can be analyzed by WB to determine whether TPO is expressed. After confirming the expression, the TPO-P1 virus was amplified.

5. The seeding density is 1.5x10⁶(viability > 95%) of Sf9 cells 30ml in 125ml shake flasks, P0 virus was added to Sf9 cells at 28 ℃ in an MOI of 0.05 calculated according to the titer of 1E6, and cultured at rest. 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 defined as TPO-P1 virus, and after the subsequent titer determination, the supernatant can be directly used for large-scale amplification of TPO-P2 virus.

6. The seeding density is 1.5x10⁶(viability > 95%) SF9 cells (1000 ml) were grown in 2.8L flasks at 28 ℃ in which P1 virus had an MOI of 0.05, calculated to titer 1E7, and added to Sf9 cells in static culture. 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 was filtered through a 0.22um filter, defined as TPO-P2 passage virus, for subsequent dropsAfter the degree determination, the method 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, and performing titer determination by using TPO standard plasmid as a standard substance and TPO-P2 virus DNA as a sample after extraction, wherein the virus titer is more than 1E 8.

(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; calculating the required cell suspension volume according to the number of living cells, transferring the cell suspension to a 50ml sterile centrifuge tube, centrifuging for 5min at 800rpm/min in a centrifuge, collecting the cells, decanting most of the old medium, leaving 5-10ml of old medium/tube, resuspending the cells, pouring into a 2.8L shake flask, adding 45ml of 200mM glutamine (9mM) to 1lh.five cell fresh medium, pouring the 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, sucking out about 1ml by a pipette, adding into a sterile 1.5ml and 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, wherein the cell activity is more than 98%.

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 to a 50ml sterile centrifuge tube, centrifuging for 5min at 1500rpm/min in a centrifuge, collecting the cells, re-suspending the cells with fresh Sf9 cell culture medium, pouring into a 2.8L shake flask with a final volume of 1L.

2. And (3) virus infection:

a. virus was added to host cells (h.five or Sf9 cells) at the required virus density x volume x MOI/virus titer

b. According to the calculation result, after the virus was mixed by gentle inversion, TPO-P2 generation virus was added according to MOI 2, gently mixed, and then placed in a dry incubator at 28 ℃ and 120rpm/min for suspension culture with shaking. 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 carried out at pH 8.

(I) purification by conventional method

The method comprises the following specific steps:

50mg of TPO antibody was dialyzed against PBS (pH7.2) at 4 ℃ for 12 hours, and the dialyzate was filtered through 0.45uM filter 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 filling was washed with a further 50ml of PBS (pH 7.2).

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

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

5. The filler was collected and washed 5 times with 10ml portions of pH 2.7 glycine eluent and PBS (pH7.2) alternately. Defined as purified for use.

6. The column was equilibrated by adding 15mL of equilibration buffer (pH7.2 PBS).

7.1L of the supernatant was initially loaded at a flow rate of 5ml/min and after completion of the run 50ml of PBS (pH7.2) was equilibrated.

8. 10mL of elution buffer (pH 2.7, 0.1M glycine) was added to elute, and the eluate was collected.

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

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 result showed that the eluted product precipitated in the preservation buffer at pH 8.0 and 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 column was equilibrated by adding 15mL of equilibration buffer (pH7.2 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 (pH7.2) was equilibrated.

4. 10mL of elution buffer (3M magnesium chloride, pH 7.0) was added for elution, and the eluate was collected.

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

Sf9 cells were selected for 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: five did not contain his-tag, and it can be seen that the recombinant protein containing his-tag was not as effective in purification as it did not contain his-tag.

The result shows that by using the purification method of the invention, H.five and Sf9 cells are selected as host cells for expression and purification, no label is needed 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 the concentration of the eluent is in the range of 1-5M, and the purified TPO with pH 6.5-8 has high purity, good stability and no precipitation, wherein the metal ion salt MgCl is₂And 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 determination reagent comprises R1, R2 and a magnetic bead solution

Said R1 includes: biotin labeled TPO antigen, concentration used: 1ug/ml.

Said R2 includes: 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 R1 and R2 working solutions, 4ml of magnetic bead solutions, 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 GmbH), and anti-TPO items (10 ul samples, R160 ul, R2125 ul and 30ul magnetic bead solution) 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 when the TPO antigen purified according to the present invention is applied to chemiluminescence detection, the sensitivity of the detection is 3IU/ml or less 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

1. A process for purifying TPO comprising,

(1) screening and purifying TPO antibody;

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

2. The process of claim 1, wherein the metal ion salt is present at a concentration of 2 to 4M and a pH of 6.8 to 7.3.

3. The process of claim 2, wherein the metal ion salt is selected from NaCl, LiCl, or MgCl₂More preferably, the metal ion salt is selected from MgCl₂And mixing the TPO antibody and the filler according to certain conditions to obtain the TPO antibody: filler 10: 1.

4. a process for producing high purity TPO comprising,

(3) introducing TPO virus into Sf9 cell for expression to obtain TPO protein;

(4) screening and purifying TPO antibody;

the recombinant TPO gene contains a tag or does not contain a tag,

5. The method of claim 4, wherein said recombinant gene TPO is tag-free.

6. A process according to claim 4 or claim 5, wherein the metal ion salt is present at a concentration of 2 to 4M and a pH of 6.8 to 7.3.

7. The method of claim 6, wherein the metal ion salt is selected from NaCl, LiCl, or MgCl₂(ii) a More preferably, the metal ion salt is selected from MgCl₂And mixing the TPO antibody and the filler according to certain conditions to obtain the TPO antibody: filler 10: 1 and mixing.

8. A process for producing high purity TPO comprising,

(4) screening and purifying TPO antibody;

9. The method of claim 8, wherein said recombinant TPO gene does not contain any tag.

10. The method of claim 9, wherein the metal ion salt is eluted in step (6)The concentration of (A) is 2-4M, and the pH value is 6.8-7.3; more preferably, the metal ion salt is selected from NaCl, LiCl or MgCl₂And mixing the TPO antibody and the filler according to certain conditions to obtain the TPO antibody: filler 10: 1.