CN110964106A

CN110964106A - anti-sST 2 monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN110964106A
Application number: CN201911376360.0A
Authority: CN
Inventors: 王勇; 邱河
Original assignee: Suzhou Bofang Biotechnology Co Ltd
Current assignee: Suzhou Bofang Biotechnology Co Ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-07

Abstract

The invention discloses a preparation method of an anti-sST 2 monoclonal antibody, which comprises the steps of preparation of sST2 recombinant protein, animal immunization, preparation of hybridoma cells and preparation of a monoclonal antibody, wherein a specific sST2 gene segment is selected, the conditions of a prokaryotic expression system are optimized, the expression quantity of protein and the purity of recombinant protein are improved, an immune mouse can generate an antibody with higher titer, and the prepared hybridoma cell strain can durably and efficiently secrete the high-performance anti-sST 2 monoclonal antibody. The invention also discloses an anti-sST 2 monoclonal antibody, which is IgG1 subtype, and has the advantages of good antibody consistency, high titer, high immunocompetence, strong affinity, strong stability, high specificity and high sensitivity for detecting sST 2. The invention also discloses application of the sST2 monoclonal antibody in immunodetection of an sST2 product, and the detection specificity is strong, the sensitivity is high, and the accuracy is good.

Description

anti-sST 2 monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an anti-sST 2 monoclonal antibody and a preparation method and application thereof.

Background

Heart failure is a common outcome of many cardiovascular diseases, and its prevalence is increasing globally, and its pathophysiological mechanisms are not fully elucidated so far. Currently, the only beneficial biomarkers for the management of heart failure are the natriuretic peptides, including the B-type natriuretic peptide (BNP) and the amino terminal B-type natriuretic peptide (NT-proBNP). However, BNP is a single biomarker, only reflects one aspect of the pathophysiological mechanism of heart failure, and is affected by renal function, age, BMI and pulmonary hypertension caused by different reasons, so that more biomarkers are urgently needed in clinic to reflect different pathophysiological mechanisms, and more accurate and complete assessment is made on heart failure patients.

Among the various emerging biomarkers, sST2 reflects the strain of cardiomyocytes following biomechanical stimulation, is a marker of cardiomyocyte hypertrophy and fibrosis, and is not affected by age, renal dysfunction, and body mass index, and is considered to have high utility in heart failure and other cardiovascular diseases. Compared with the traditional heart failure marker, the sST2 has stronger death prediction capability, which has extremely important significance for the prognosis of heart failure. At present, the sST2 detection kit appears in foreign markets, but the price is high, the number of sST2 detection products in China is small, most of the sST2 detection products are scientific research reagents, and the clinical application reports are few. Therefore, the preparation of the anti-sST 2 monoclonal antibody with excellent performance and the development of the domestic sST2 quantitative detection kit with excellent performance have extremely important social significance for reducing medical cost and improving patient satisfaction.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a preparation method of an anti-sST 2 monoclonal antibody, select a specific sST2 gene segment, optimize the conditions of a prokaryotic expression system, improve the expression quantity of protein and the purity of recombinant protein, enable an immune mouse to produce an antibody with higher titer, and enable a prepared hybridoma cell strain to durably and efficiently secrete a high-performance anti-sST 2 monoclonal antibody.

The invention aims to provide an anti-sST 2 monoclonal antibody, which is prepared by the method, is IgG1 subtype, has good antibody consistency, high titer, high immunocompetence, strong affinity, strong stability, high specificity and high sensitivity for detecting sST 2.

The invention also aims to provide the application of the sST2 monoclonal antibody in immunodetection of an sST2 product, wherein the antibody has strong stability, strong detection specificity, high sensitivity and good accuracy.

One of the purposes of the invention is realized by adopting the following technical scheme:

a preparation method of an anti-sST 2 monoclonal antibody comprises the following steps:

preparation of sST2 recombinant protein: inserting the sST2 gene fragment into pET-28A (+) plasmid to obtain PET-28A-sST2 expression vector, then transferring into escherichia coli, carrying out induced culture for 4-6h at 22-37 ℃ in LB liquid culture medium containing IPTG, extracting and purifying to obtain high-purity sST2 recombinant protein; the nucleotide sequence of the sST2 gene fragment is shown as SEQ ID NO.1

Animal immunization: after 45-55 μ g of the sST2 recombinant protein was fully emulsified with the same volume of freund's adjuvant, BALB/c mice were immunized by subcutaneous multiple injection at the back for 4 times, each immunization time interval being 14d, and the in vivo antibody titer was selected to be 1: (150000-;

preparation of hybridoma cells: injecting 95-105 mu g of sST2 recombinant protein without adjuvant into the abdominal cavity of the immunized mouse, extracting splenocyte suspension to perform cell fusion with mouse myeloma cells SP2/0, selectively culturing, and performing subcloning for 3 times to obtain hybridoma capable of stably secreting specific anti-sST 2 antibody;

preparation of monoclonal antibody: injecting the lipid-lowering alkane into the abdominal cavity of a BALB/c mouse aged 8-10 weeks, inoculating the hybridoma cells (0.8-1.2) multiplied by 10 after 7 days⁶And extracting ascites from one or more of the cells, and extracting and purifying to obtain the anti-sST 2 monoclonal antibody.

Preferably, the final concentration of the IPTG in the LB liquid medium is 0.4-0.6 mM; the temperature of the induction culture is 37 ℃, and the time of the induction culture is 6 h.

Further, the volume ratio of the mouse myeloma cell SP2/0 to the spleen cell suspension was 1: 5.

The purification method of the sST2 recombinant protein is NI-NTA affinity chromatography column purification.

The titer of the immunized mice was 1: 160000.

The culture medium adopted by the selective culture is a DMEM culture medium added with fetal calf serum, hypoxanthine, aminopterin and thymine.

The purification method of the anti-sST 2 monoclonal antibody is Protein G affinity chromatography.

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

an anti-sST 2 monoclonal antibody is prepared by the preparation method of the anti-sST 2 monoclonal antibody.

Further, the anti-sST 2 monoclonal antibody is of the IgG1 subtype.

an application of the anti-sST 2 monoclonal antibody in immunodetection of an sST2 product.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, a specific sST2 gene fragment is selected, so that the conditions of a prokaryotic expression system are optimized, and the expression quantity of the protein and the purity of the recombinant protein are improved; the sST2 recombinant protein is injected into a mouse 4 times subcutaneously at the back for immunization, so that the mouse can generate an antibody with higher titer in vivo, and the titer of the antibody is 1: 160000; the hybridoma is prepared from splenocytes of an immunized mouse with the in-vivo antibody titer of 1:160000, 2 hybridoma cell strains 5F2 and 1E4 which can stably secrete anti-sST 2 antibody are screened out through optimization, and the anti-sST 2 monoclonal antibody with high performance can be secreted durably and efficiently.

2. The anti-sST 2 monoclonal antibodies of the invention are IgG1 subtype antibodies, and have good consistency, high titer, high immunocompetence, strong affinity, strong stability, high specificity and high sensitivity for detecting sST 2. The kit can be independently used for immunodetection of sST2, and has strong detection specificity, high sensitivity and good accuracy; the kit can also be used for joint detection with other markers, so that the heart failure and other cardiovascular diseases can be detected more comprehensively, and the detection result is more accurate and reliable.

Drawings

FIG. 1 is a comparative sequence chart of the transfer of the PET-28A-sST2 expression vector into Escherichia coli in example 1;

FIG. 2 is an SDS-PAGE pattern of recombinant sST2 protein induced in example 2; 1. supernatant at 22 ℃; 2. precipitating at 22 ℃; 3. after 22 ℃ induction; 4. supernatant at 37 ℃; 5. precipitating at 37 ℃; 6. after induction at 37 ℃; 7. negative control; m, protein marker;

FIG. 3 is an SDS-PAGE picture of the recombinant protein of example 3sST 2; m, protein marker; 1. before induction; 2. after induction; 3. precipitating; 4. washing the supernatant with 2mol/L urea; 5. washing the supernatant with 4mol/L urea; 6. washing supernatant by 8mol/L urea;

FIG. 4 is a graph of titers of antibodies against the recombinant sST2 protein in sera of the immunized mice in example 4;

FIG. 5 is an SDS-PAGE electrophoresis of the sST2 monoclonal antibody of example 6;

FIG. 6 is a titer detection graph of the sST2 monoclonal antibody of example 7;

FIG. 7 shows the subtype identification of the sST2 monoclonal antibody of example 8.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

preparation of sST2 recombinant protein: inserting the sST2 gene fragment into pET-28A (+) plasmid to obtain PET-28A-sST2 expression vector, transferring into Escherichia coli, performing induced culture at 22-37 ℃ for 4-6h in LB liquid culture medium containing 0.4-0.6mM IPTG, extracting, and purifying to obtain high-purity sST2 recombinant protein; the nucleotide sequence of the sST2 gene fragment is shown as SEQ ID NO.1

Animal immunization: after fully emulsifying 45-55 μ g of sST2 recombinant protein and the same volume of Freund's adjuvant, injecting the mixture to BALB/c mice back by subcutaneous multi-point injection for 4 times, wherein the time interval of each immunization is 14d, and screening the antibody titer in vivo to be 1: (150000-;

preparation of hybridoma cells: injecting 95-105 mu g of sST2 recombinant protein without adjuvant into the abdominal cavity of an immunized mouse, extracting splenocyte suspension to perform cell fusion with mouse myeloma cells SP2/0, selectively culturing, and performing subcloning for 3 times to obtain hybridoma capable of stably secreting specific anti-sST 2 antibody;

preparation of monoclonal antibody: injecting the lipid-lowering alkane into the abdominal cavity of a BALB/c mouse aged 8-10 weeks, inoculating hybridoma cells (0.8-1.2) multiplied by 10 after 7 days⁶And extracting ascites from one or more of the cells, and extracting and purifying to obtain the anti-sST 2 monoclonal antibody.

As a further preferred embodiment, the volume ratio of mouse myeloma cell SP2/0 to spleen cell suspension is 1: 5.

As a further preferred scheme, the purification method of the sST2 recombinant protein is NI-NTA affinity chromatography column purification; the purification method of the anti-sST 2 monoclonal antibody is Protein G affinity chromatography.

The anti-sST 2 monoclonal antibody is prepared by the preparation method of the anti-sST 2 monoclonal antibody, and the anti-sST 2 monoclonal antibody is an IgG1 subtype.

Example 1

(1) Construction of recombinant plasmid

1) The basic information of the human sST2 gene was found from NCBI, and the human sST2 gene selected in this example had the nucleotide sequence shown in SEQ ID NO.1 and encoded the protein shown in SEQ ID NO. 2. The human sST2 gene (SEQ ID NO:1) can be obtained by artificial synthesis or purchase;

2) according to the conventional recombinant plasmid double-enzyme digestion construction method in the field, the human sST2 gene in 1) is subjected to double enzyme digestion and then is connected to a plasmid PET-28A (+) to obtain a PET-28A-sST2 expression vector;

3) escherichia coli BL21 competent cells were prepared by chemical induction, and the PET-28A-sST2 expression vector obtained in 2) was transformed into Escherichia coli BL21 competent cells, which were then applied to LB solid medium containing 50mg/ml Kan (kanamycin) and cultured overnight at 37 ℃. Selecting a faint yellow colony (escherichia coli containing a PET-28A-sST2 expression vector) in the plate, and culturing in an LB liquid culture medium containing Kan for 4-6h to obtain the clone bacteria. After the completion, the bacterial liquid is divided into two parts which are respectively used for preparing strains and identifying and analyzing. The cloned bacteria are positive cloned bacteria through colony PCR identification, and the PET-28A-sST2 expression vector is successfully transferred into escherichia coli BL 21;

4) sequencing and identifying: the clones of 3) were randomly selected and sequenced, and the alignment of the sequence of sST2 with the sequencing results is shown in FIG. 1. The result shows that the sequencing result is completely consistent with the sequence of the sST2 gene inquired in NCBI, namely the PET-28A-sST2 expression vector is successfully transferred into escherichia coli.

Adding glycerol into the bacterial liquid which is identified by PCR and sequenced correctly, then adding glycerol and storing at-80 ℃ to obtain the glycerol strain.

Example 2

sST2 recombinant protein Induction Condition screening

The glycerol strain obtained in example 1 is taken and placed in LB liquid culture medium for culture overnight, the glycerol strain is shaken at 22 ℃ and 220rmp until OD is 0.6, IPTG (isopropyl thio- β -D-galactoside) is added until the final concentration is 0.5mM, induction expression is continued for 6h, the thalli are collected to prepare an electrophoresis sample, the expression condition of SST2 protein is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the result is shown in a lane 3 in figure 2, the collected thalli are crushed and centrifuged, supernatant (a lane 1 in figure 2) and sediment (a lane 2 in figure 2) are reserved, and the expression condition of SST2 protein is detected by SDS-PAGE, and the result is shown in a lane 2-3 in figure 2.

The glycerol strain is taken to be cultured in LB liquid culture medium overnight, shaken at 37 ℃ and 220rmp until OD is 0.6, IPTG is added to the final concentration of 0.5mM, and the induction expression is continued for 6 h. Collecting the thallus, preparing an electrophoresis sample, and detecting the expression of the sST2 recombinant protein by SDS-PAGE, wherein the result is shown in lane 6 of FIG. 2; the collected cells were disrupted and centrifuged, and the supernatant (lane 4 in FIG. 2) and the precipitate (lane 5 in FIG. 2) were retained, and SDS-PAGE was performed to detect the expression of sST2 recombinant protein, the results of which are shown in lanes 4-5 in FIG. 2.

Mu.l of the suspension was used as a negative control, and the suspension was shaken at 37 ℃ and 220rmp to OD 0.6, and then the cells were harvested to prepare an electrophoresis sample, and SDS-PAGE was performed to detect the expression of sST2 recombinant protein, as shown in lane 7 of FIG. 2.

As can be seen in FIG. 2, there was a clear protein band at about 29kD after IPTG induction, which was absent from the negative control. The optimal temperature for induced expression of the sST2 protein is 37 ℃, a target band is clearer in precipitation at 37 ℃, and the protein expression amount is high.

Example 3

Mass induction and purification of sST2 recombinant proteins

1) Mass induction of sST2 recombinant protein

The glycerol strain is taken to be cultured in LB liquid culture medium overnight, shaken at 37 ℃ and 220rmp until OD is 0.6, IPTG is added to the final concentration of 0.5mM, and the induction expression is continued for 6 h. Electrophoresis samples were prepared from the cells before induction (before IPTG addition) and after induction, and SDS-PAGE was used to detect the expression of sST2 recombinant protein, as shown in FIG. 3, lane 1 (before induction) and lane 2 (after induction). The induced bacteria were taken, disrupted, centrifuged, and the precipitate was retained to prepare an electrophoretic sample, and the expression of the recombinant protein of sST2 was detected by SDS-PAGE, and the result is shown in lane 3 of FIG. 3.

And then washing the precipitated electrophoresis sample by respectively adopting washing liquids containing urea with different concentrations, wherein the washing liquids comprise: 50mmol/L Tris, 0.2mol/L NaCl and Urea, wherein the Urea concentration is 2mol/L, 4mol/L and 8mol/L respectively, and SDS-PAGE detection is carried out on the washed supernatant, and the result is shown in lanes 4-6 of FIG. 3.

As can be seen in FIG. 3, a distinct protein band at about 29kD was observed in the 2M urea wash supernatant.

2) sST2 recombinant protein purification

And (3) purifying the 2M urea washing supernatant obtained in the example 3 after the mass induction according to the specification of an NI-NTA affinity chromatographic column to obtain the sST2 recombinant protein with high purity.

Example 4

Animal immunization experiment

Using the sST2 recombinant protein purified in example 3 as an immunogen, 50. mu.g/mouse of sST2 recombinant protein was thoroughly emulsified with equal volume of Freund's adjuvant, and the mice were immunized by multiple injections subcutaneously in BALB/c for 4 times, with the interval of each immunization being two weeks. After one week of completion of 4 immunizations, two of the mice were randomly selected and designated as sST2-1# and sST2-2#, and blood was collected and serum was isolated for indirect ELISA to detect the titer of anti-sST 2 recombinant protein antibodies in the mice, and the results are shown in FIG. 4. And selecting an immune mouse with high immune titer to take the spleen, and preparing a hybridoma cell experiment.

As can be seen from FIG. 4, the OD value at 1:160000 exceeded 2.1 times the OD value of the negative control (blank experiment, mouse serum of the non-immune sST2 recombinant protein), while at 1:320000, the OD value was 2.1 times less than the OD value of the negative control, which is a negative result. Namely, the antibody titer of the mice immunized with the sST2 recombinant protein 4 times after the purification of the example 3 reaches 1: 160000.

Example 5

Cell fusion and screening of hybridoma cells

Mice were injected intraperitoneally 3d prior to cell fusion with 100 μ g of sST2 recombinant protein without adjuvant to boost immunity. 3d, separating the spleen of the mouse in an aseptic environment, preparing cell suspension, mixing the SP2/0 myeloma cell which is serum-free, clean and good in state and the mouse spleen cell suspension in a ratio of 1:5, and fusing at 37 ℃ under the action of PEG 1450. Then, D μ lbecco's modified eagle (DMEM) medium was added to terminate the fusion, and after centrifugation, 20% fetal bovine serum-hypoxanthine-aminopterin-thymine-DMEM (FBS-HAT-DMEM) medium was added for selective culture, and seeded in a 96-well plate and placed in an incubator for culture. At fusion 11d, culture wells with hybridoma cell growth were subjected to supernatant detection and supernatant antibody titer was measured by indirect ELISA. Positive wells were cloned by limiting dilution to 50-60 cells/mL. After 3 times of subcloning, two hybridoma cell strains which can stably secrete specific anti-SST 2 antibodies are obtained and are respectively named as 5F2 and 1E 4. The hybridoma cell lines 5F2 and 1E4 were expanded and cultured, respectively, and stored in liquid nitrogen. Hybridoma cell lines were resuscitated in a 37 ℃ water bath prior to use. Centrifuging after completely thawing, removing supernatant, transferring into culture flask, and placing at 37 deg.C and 5% CO₂And (5) performing incubator static culture and observing growth conditions. Selected logarithmic growthAnd (3) centrifuging the hybridoma cells in the stage to remove the culture medium, adding a proper amount of prepared cryopreservation liquid, and subpackaging the cells into cryopreservation tubes. Cloning and proliferating, and transferring the cells to liquid nitrogen for freezing after gradient cooling. The hybridoma cells of this example were used once recovered.

Example 6

Preparation of monoclonal antibodies

Injecting the lipid-lowering alkane into the abdominal cavity of a BALB/c mouse aged 8-10 weeks, inoculating the recovered hybridoma cells 5F2 and 1E4 of the example 4 respectively after one week, wherein the inoculation amount is 1 multiplied by 10⁶A/only. When the abdomen of the mouse is obviously enlarged, aseptically extracting ascites after the mouse is sacrificed, and collecting the middle-layer ascites after centrifugation. And (3) precipitating the crude monoclonal antibody by using a saturated ammonium sulfate solution, purifying the monoclonal antibody according to the protocol of Protein G affinity chromatography, and collecting the purified anti-sST 2 monoclonal antibody. The purity of the sST2 monoclonal antibody was confirmed by SDS-PAGE electrophoresis, and the results are shown in FIG. 5. Two clear bands of 55kD and 25kD can be seen in both of the two hybridoma cell lines in FIG. 5, wherein the heavy chain of the anti-sST 2 recombinant protein antibody is located at 55kD, and the light chain is located at 25 kD.

Example 7

The indirect ELISA kit is used for detecting the titer of the sST2 monoclonal antibody in the ascites in example 6, and the specific steps are as follows:

A. coating: the sST2 recombinant protein purified in example 3 was coated on a microplate at 1. mu.g/mL, 100. mu.l/well and incubated overnight at 4 ℃.

B. And (3) sealing: the ELISA plates were washed with PBST, 200. mu.l/well of 5% nonfat dry milk was added, and blocked at 37 ℃ for 2h to occupy the sites in the solid phase that were not coated with the recombinant antigen, reducing non-specific reactions.

C. Reaction: after washing, 100. mu.l/well of the diluted concentrations of the monoclonal antibody of example 6 (200, 100, 50, 25, 12.5, 6.25, 3.125, 0ng/ml, respectively) was added and incubated at 37 ℃ for 1 h.

D. Adding a secondary antibody: after washing the plate, the appropriate dilution of horseradish peroxidase-labeled goat anti-mouse antibody was added and incubated at 37 ℃ for 1 h.

E. Color development: 100. mu.l/well of TMB chromogenic substrate was added and the reaction was carried out for 10 min.

F. And (4) terminating: the reaction was terminated by adding 50. mu.l of a 2mol/L sulfuric acid stop solution.

G. Reading: the OD value of each well is read by a microplate reader at 450 nm.

The detection result is shown in FIG. 6, and the result shows that the sST2 monoclonal antibody titer in the mouse ascites is 6.25ng/mL, and the antibody immunization effect is good.

Example 8

Subtype identification of monoclonal antibodies

The subtype of the monoclonal antibody of example 6 was detected using a commercially available mouse monoclonal antibody subtype detection reagent strip, and 70. mu.l of cell culture supernatant was applied to the application area of the reagent strip according to the instructions for use, and after 10min, the antibody subtype was determined by comparison with a reference card. The results are shown in FIG. 7, and it can be intuitively understood that the monoclonal antibodies secreted by the 2 hybridoma cells are all IgG1 types.

The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of the present invention claimed in the present invention.

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Gln Gly Ser Arg Tyr Arg Ala His Lys Ser Phe Leu Val Ile Asp Asn

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Val Met Thr Glu Asp Ala Gly Asp Tyr Thr Cys Lys Phe Ile His Asn

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Claims

1. A preparation method of an anti-sST 2 monoclonal antibody is characterized by comprising the following steps:

2. The method for preparing an anti-sST 2 monoclonal antibody according to claim 1, wherein the IPTG is present in the LB liquid medium at a final concentration of 0.4-0.6 mM; the temperature of the induction culture is 37 ℃, and the time of the induction culture is 6 h.

3. The method of claim 1, wherein the volume ratio of the mouse myeloma cell SP2/0 to the spleen cell suspension is 1: 5.

4. The method for preparing the anti-sST 2 monoclonal antibody according to claim 1, wherein the purification method of the sST2 recombinant protein is NI-NTA affinity chromatography column purification.

5. The method of claim 1, wherein the titer of the immunized mouse is 1: 160000.

6. The method of claim 1, wherein the selective culture medium is DMEM medium supplemented with fetal bovine serum, hypoxanthine, aminopterin, and thymidine.

7. The method for producing the anti-sST 2 monoclonal antibody according to claim 1, wherein the method for purifying the anti-sST 2 monoclonal antibody is Protein G affinity chromatography.

8. An anti-sST 2 monoclonal antibody produced by the method for producing an anti-sST 2 monoclonal antibody according to any one of claims 1 to 7.

9. The anti-sST 2 monoclonal antibody of claim 8, wherein the anti-sST 2 monoclonal antibody is of the IgG1 subtype.

10. The use of the anti-sST 2 monoclonal antibody of claim 9 in the immunoassay of a sST2 product.