CN102344490B - Anti-mouse Stk40 polyclonal antibody and its preparation - Google Patents

Abstract

The present application relates to a polyclonal antibody against mouse Stk40 and its preparation. Specifically, the application relates to an antigen or its fusion protein for preparing an anti-mouse Stk40 polyclonal antibody, its coding sequence, a composition for immunization containing the antigen or its fusion protein, preparation of an anti-mouse Stk40 polyclonal antibody The method for the polyclonal antibody of Stk40, the anti-mouse Stk40 polyclonal antibody prepared by the method and the use thereof.

Description

Anti-mouse Stk40 polyclonal antibody and its preparation

technical field

The present application relates to a polyclonal antibody against mouse Stk40 and its preparation.

Background technique

Stk40 is a protein with a serine/threonine kinase domain, which is highly conserved in evolution, especially with more than 94% homology between mammals (see the table below). The expression level of Stk40 in undifferentiated mouse ES cells is very low, but its RNA (Fig. 1B left) and protein level (Fig. 1B right) both increase with the differentiation of mouse ES cells and embryoid bodies (embryoid bodies, EBs) formed and gradually increased. Most importantly, when Stk40 was overexpressed in mouse ES cells, the mouse ES cells showed the morphology of differentiated cells (Fig. 1C) and the expression of extraembryonic endoderm cell marker genes (Fig. 1D).

It has been reported in the literature that Stk40 is a downstream gene directly negatively regulated by Oct4. When mouse ES cells overexpressing Stk40 and labeled with H2B-GFP fusion protein were injected into mouse 3.5-day-old (embryoday 3.5, E3.5) blastocysts for in vivo chimeric embryo experiments, it was found that most of these cells Distributed to the ExEn region of the embryo (Figure 2). This is consistent with the localization of cells with down-regulated Oct4 expression in chimeric embryo experiments, and further functionally supports the upstream and downstream regulatory relationship between Oct4 and Stk40.

Stk40, as a new target gene repressed by Oct4, plays an important role in the differentiation of ES cells into extraembryonic endoderm cells. By trying a variety of signal transduction pathway inhibitors, it was found that Stk40 caused ES cells to differentiate towards ExEn by activating the Ras-Erk/MAPK pathway. Further through the strategies of affinity chromatography and mass spectrometry, a calcium-binding protein Rcn2 that can directly interact with Stk40 was found, and a recent study reported that Rcn2 was highly expressed in ExEn cells. Interestingly, overexpression of Rcn2 in mouse ES cells can also cause a similar differentiation phenotype to that of Stk40 overexpression, and Rcn2 is required for Stk40-induced differentiation in the ExEn direction. In addition, literature has proved that Rcn2 can form a protein complex with an important scaffold protein Ksr1 in the Ras-Erk/MAPK signaling pathway, suggesting that Rcn2 may participate in the regulation of the Ras-Erk/MAPK signaling pathway by activating Ksr1 (Figure 3).

In addition, the Stk40 gene is expressed in multiple important organs of mice, and this gene is highly conserved with its homologous gene STK40 in humans, suggesting that it may have important links with human diseases and health.

Anti-mouse Stk40 polyclonal antibody provides convenience for the study of the function and mechanism of Stk40 in mouse embryonic development and in various systems (such as the immune system), and for the study of molecular mechanisms involved in the occurrence and development of human diseases offers the possibility. Although there are some commercial anti-Stk40 antibodies on the market, the effects of these antibodies cannot meet the requirements of Western blotting applications. To this end, the present invention provides a new anti-mouse Stk40 antibody, which can meet the requirements of Western blotting applications, thereby solving the problems in this field.

Contents of the invention

The first aspect of the present application provides an isolated polypeptide, the amino acid sequence of which is composed of amino acid fragments with a length of 60-85 amino acids between amino acid residues 340-430 of SEQ ID NO:1.

In a specific embodiment, the amino acid of the polypeptide consists of an amino acid fragment with a length of 63-75 amino acids between positions 345-425 of SEQ ID NO:1.

In a specific embodiment, the amino acids of the polypeptide consist of amino acids 349-420 of SEQ ID NO: 1.

The second aspect of the present application provides an isolated polypeptide, which consists of an amino acid fragment with a length of 60-85 amino acids between amino acid residues 340-430 of SEQ ID NO: 1 and a polypeptide for facilitating purification.

In a specific embodiment, the polypeptide for promoting purification is GST or His.

In a specific embodiment, the amino acid fragment is composed of an amino acid fragment with a length of 63-75 amino acids between positions 345-425 of SEQ ID NO:1.

In another specific embodiment, the amino acid fragment consists of amino acids 349-420 of SEQ ID NO:1.

In a specific embodiment, the sequence of the polypeptide is shown in SEQ ID NO:5.

Another aspect of the present application provides a composition for immunization, which composition contains the isolated polypeptide described in the present application.

In a specific embodiment, the isolated polypeptide consists of an amino acid fragment with a length of 60-85 amino acids between amino acid residues 340-430 of SEQ ID NO:1.

In another specific embodiment, the polypeptide consists of an amino acid fragment with a length of 60-85 amino acids between amino acid residues 340-430 of SEQ ID NO: 1 and a polypeptide for promoting purification.

In a specific embodiment, the amino acid fragment is composed of an amino acid fragment with a length of 63-75 amino acids between positions 345-425 of SEQ ID NO:1.

In another specific embodiment, the amino acid fragment consists of amino acids 349-420 of SEQ ID NO:1.

In another specific embodiment, the isolated polypeptide is shown in SEQ ID NO:5.

Another aspect of the present application provides a method for preparing a polyclonal antibody against mouse Stk40, which is characterized in that the method comprises using the isolated polypeptide of the present application or a composition thereof to immunize a mammal.

In a specific embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 4 or 5.

In a specific embodiment, the method includes:

(1) Constructing an antigen expression vector containing the polypeptide;

(2) using the expression vector to transform Escherichia coli;

(3) purifying the polypeptide;

(4) immunizing a mammal with the polypeptide or a composition comprising the polypeptide; and

(5) purifying the antibody from the serum of the mammal, thereby preparing the antibody.

Another aspect of the present application relates to the anti-mouse Stk40 polyclonal antibody prepared by the method of any one of claims 6-8.

The present application also relates to the use of the isolated polypeptide or its composition in the preparation of polyclonal antibodies against mouse Stk40.

The present application also relates to an isolated polynucleotide sequence encoding the isolated polypeptide described herein.

Description of drawings

Figure 1A shows a schematic diagram of the structure of Stk40; Figure 1B shows that the expression of Stk40 in undifferentiated mouse ES cells is very low, but its RNA (Fig. 1B left) and protein levels (Fig. Differentiation and formation of embryoid bodies gradually increased; Figure 1C shows that when Stk40 is overexpressed in mouse ES cells, mouse ES cells will appear as differentiated cells; Figure 1D shows the expression of extraembryonic endoderm cell marker genes .

Figure 2 shows that when mouse ES cells overexpressing Stk40 and labeled with H2B-GFP fusion protein were injected into mouse 3.5-day-old blastocysts for in vivo chimeric embryo experiments, it was found that most of these cells were distributed to the ExEn of the embryo area.

Figure 3 shows a schematic diagram of the mechanism of action of Stk40.

Figure 4 shows the results of WB detection of the anti-mouse Stk40 polyclonal antibody of the present application.

Figure 5 shows the results of WB detection of the antibody (Stk40-C98) prepared from the C-terminal 98 amino acids (C98) of Stk40 as the antigenic fragment.

Figure 6 shows the antigenicity index map of mouse Stk40. It can be seen from the figure that the Stk40 amino acid sequence (SEQ ID NO: 1) amino acid residues 349-375 and SEQ ID NO: 1 amino acid residues 386-424 have strong immunogenicity.

Detailed ways

In this application, the terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, polymers, etc. are included within this definition. Full length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Additionally, for the purposes of the present invention, "polypeptide" is meant to include modifications of the native sequence, such as deletions, additions and substitutions (often conservative in nature), so long as the protein maintains the desired activity. These modifications can be engineered by site-directed mutagenesis, or can be accidental, for example by mutation of the protein-producing host, or errors due to PCR amplification.

The present application includes analogs of polypeptides which preferably include substitutions which are conservative in nature, ie, substitutions which occur within a class of amino acids with respect to their side chains. Specifically, amino acids are generally divided into four categories: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) nonpolar Sex - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polarity - glycine, Asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to predict that substitution of isoleucine or valine alone for leucine, for glutamic acid for aspartic acid, for serine for threonine, or for similarly conserved amino acids with structurally related amino acids Amino acids, such substitutions will not have a significant effect on biological activity. For example, a polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, e.g., 1-8, 1-5, 1-3 conservative or non-conservative amino acid substitutions, as long as The desired function of the molecule remains intact. Those skilled in the art can readily determine the regions of a molecule of interest that are tolerated to be altered by combining the Hopp/Woods and Kyte-Doolittle plots well known in the art. The present application also includes 1 or several (eg 1, 2, 3, 4, 5 or 6 or more) amino acid deletions or insertions in the polypeptides of the present application.

In this application, the term "fragment" refers to a polypeptide consisting of only a part of the complete full-length polypeptide sequence and structure. Therefore, the "amino acid fragment with a length of 63-75 amino acids between positions 345-425 of SEQ ID NO: 1" described herein refers to a part of the sequence of amino acids 345-425 of SEQ ID NO: 1 (ie, this amino acid A "fragment" of a sequence), which is 63-75 amino acids in length.

In this application, when referring to a polypeptide or polynucleotide, "isolated" means that said molecule is separated and separated from the whole organism in which it is found naturally occurring, or substantially free from other biomacromolecules of the same type .

In the present application, "contains", "comprising" and similar terms include "consisting of" and "consisting of".

As used herein, an "expression cassette," "expression construct," or "expression vector" refers to an assembly capable of directing the expression of a sequence or gene of interest. The expression cassette includes the aforementioned control elements, such as a promoter operably linked to the sequence or gene of interest (to direct its transcription), and often also includes a polyadenylation sequence. Expression vectors are constructed such that the specific coding sequence is located within the vector with appropriate regulatory sequences, the coding sequence relative to the control sequences being positioned and oriented such that the coding sequence is transcribed under the "control" of the control sequence (i.e., incorporated into the control sequence). RNA polymerase of the DNA molecule transcribes the coding sequence). Modifications to the sequence encoding the molecule of interest may be required to accomplish this. For example, in some cases it may be necessary to modify the sequence so that it is ligated to a control sequence in the proper orientation, ie, to maintain the reading frame. Control and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and appropriate restriction sites.

"Transformation" herein refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method of insertion: eg, transformation by direct uptake, transfection, infection, and the like. Specific transfection methods are described further below. The exogenous polynucleotide can be maintained as an unintegrated vector, such as an episome, or can be integrated into the host genome.

A "host cell" is a cell that has been transformed, or is capable of being transformed by an exogenous DNA sequence.

The first aspect of the present application provides an isolated polypeptide, the amino acid sequence of which is composed of amino acid fragments with a length of 60-85 amino acids between amino acid residues 340-430 of SEQ ID NO:1. SEQ ID NO: 1 shows the amino acid sequence of mouse Stk40 protein.

The length of the amino acid fragment is 63-80 amino acids, 63-75 amino acids, 70-75 amino acids, 72-75 amino acids. A preferred amino acid fragment is shown in SEQ ID NO:4.

In addition, it is preferred that the isolated polypeptide is one of the 340-430 amino acid residues of SEQ ID NO: 1 containing the amino acid sequence shown in SEQ ID NO: 4, and its length is between 72-78 amino acids. amino acid fragments.

In other preferred embodiments, the amino acid fragment preferably contains amino acid residues 349-375 of SEQ ID NO: 1. In other preferred embodiments, the amino acid fragment preferably contains amino acid residues 386-423 of SEQ ID NO: 1.

The isolated polypeptide may have one or several amino acid mutations, including 1, 2, 3, 4, 5, 6 or more amino acid insertions, deletions, and mutations, as long as these mutations do not alter the immunogenicity of the isolated polypeptide Just sex.

The present application also includes fusion proteins of the isolated polypeptide, wherein the isolated polypeptide is fused to a polypeptide to facilitate purification. The purification-facilitating polypeptides include GST and His. GST is a tag protein that binds to glutathione. Therefore, after the polypeptide of the present application is fused with it, the GST on the fusion protein can be bound to the glutathione solid-phased on the carrier, and then the fusion protein can be purified by using the principle of glutathione exchange and elution . The sequence of His tagged protein is 6 consecutive histidines. Usually, the expression vector of His tagged protein is PET-30a (the vector is purchased from Novagen). It should be understood that the polypeptides used for fusion with the immunogenic polypeptides of the present application, such as GST and His, etc., should not weaken or affect the immunogenicity of the immunogenic polypeptides of the present application.

The present application also provides coding sequences of the polypeptides of the present application, including fusion proteins, and expression vectors containing the coding sequences.

The present application also provides a composition for immunizing mammals, which contains the polypeptide or fusion protein of the present application, a pharmaceutically acceptable carrier and/or adjuvant.

The term "adjuvant" refers to any substance that assists or modulates the action of a drug, including but not limited to an immunological adjuvant, which enhances the immune response to an antigen. Carriers usually include

"Pharmaceutically acceptable" means that the carrier can be administered to an individual together with the polypeptides and fusion proteins of the present application without causing undesirable biological reactions in the individual or harmful interactions with any other components contained in the composition . Usable carriers include water, saline, glycerin, polyethylene glycol, hyaluronic acid, ethanol and the like.

The amount of polypeptide or fusion protein in the composition depends on the actual situation, which is within the knowledge of those skilled in the art. For example, the content of the polypeptide or fusion protein in the composition varies from 0.001-50% by weight, 0.001-20% by weight, 0.001-10% by weight, and 0.001-1% by weight.

The present application provides a method for preparing a polyclonal antibody against mouse Stk40, the method comprising immunizing mammals with the polypeptide described in the present application or its fusion protein.

More specifically, the preparation method of the present invention includes the steps of constructing an antigen expression vector containing the polypeptide, transforming, inducing expression, and purifying to obtain the antigen, immunizing, and purifying the antibody.

The antigens of the present invention can be prepared by methods well known in the art. For example, an expression vector for the antigen can be constructed, and then a host cell can be transformed with the expression vector so that the host cell can express the antigen, and the antigen can be isolated and purified from the host cell culture.

There is no special limitation on the choice of expression vector, as long as it can express the antigen of the present invention in host cells. Generally, an appropriate expression vector can be selected according to the polypeptide actually used to facilitate purification. For example, when using GST fusions, the PGEX-4T-1 vector can be used as an option. When His is selected for fusion, PET-30a vector can be used. The selection of polypeptides to facilitate purification and the selection of their corresponding expression vectors are within the purview of the art.

The host cell is usually E. coli. Preferably, different bacterial species are selected according to different protein expression vectors, and cultured in different mediums. In a preferred embodiment, the PGEX-4T-1 vector expressing GST fusion protein corresponds to BL21 strain and YTA medium, and the PET-30a vector expressing His fusion protein corresponds to BL21-DE3 strain and LB medium.

There is no special limitation on the cultivation of Escherichia coli, and existing conventional cultivation methods can be used.

The purified antigen can be immunized into a mammal. Immunization can be carried out by conventional methods. In a specific embodiment, two adult male rabbits are injected with the antigen, and the immunization is carried out twice, with an interval of about 1-2 months between the two times, and a total of 5 mg of the antigen is used. After the second immunization, the antibody titer in the rabbit serum was tested by ELISA. If it was greater than 2.5 million, the rabbit was bled, and the serum was collected for antibody purification.

Conventional methods in the art can be used to collect serum and purify antibodies.

This application also includes the Stk40 polyclonal antibody prepared by the above method, the composition containing the antibody, and the polyclonal antibody or its composition in the study of Stk40 in the mouse embryonic development process and in various systems (such as the immune system) functions in and uses in mechanisms.

Hereinafter, the present invention will be described in detail by means of specific examples. The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, known to those skilled in the art. These techniques are fully explained in the literature. See, e.g., Fundamental Virology, Second Edition, Volumes I and II (eds. B.N. Fields and D.M. Knipe); Handbook of Experimental Immunology, Volumes I-IV ( D.M. Weir and C.C. Blackwell, eds., Blackwell Scientific Publications); T.E. Creighton, Proteins: Structures and Molecular properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry ( Worth Publishers, Inc. latest edition); Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition, 1989; Methods in Engymology (S. Colowick and Ed. N. Kaplan, Academic Press, Inc.).

It should be understood that the present application is not limited to these specific embodiments. Those skilled in the art can make appropriate changes to the present invention without departing from the spirit and scope of the present application. Unless otherwise indicated, the reagents used and their dosage units are conventional in the art.

Example 1: Construction of antigen expression vector

The Stk40-C72 antigen encodes a total of 72 amino acids at positions 349-420 of the C-terminus of Stk40, and its sequence is shown in SEQ ID NO:4.

The construction process of Stk40-C72 antigen expression vector is as follows:

1. Design cloning primers introducing restriction enzyme sites:

C72-F (BamH I): 5'TTG GGA TCC ATG CCG GAC ATT GAT GAC 3' (SEQ ID NO: 2)

C72-R (Xho I): 5'TGT CTC GAG ACT ACA TTG GCT GTG CGT 3' (SEQID NO: 3)

2. Use the Flag-LYK4/ppyCAGIP plasmid (Lingjie Li ^* , Lei Sun ^* , etc., Stk40 Links the Pluripotency Factor Oct4 to the Erk/MAPK Pathway and Controls Extraembryonic Endoderm Differentiation PNAS.2010 Jan; 107(4): 1402-1407) as Template, use C72-F (BamH I) and C72-R (Xho I) primers to perform PCR to obtain a 210bp target fragment; [Note: LYK4 is Stk40]

3. Digest the PCR product with BamH I and Xho I, and perform gel recovery. At the same time, digest the pGEX-4T-1 plasmid (purchased from Amersham Biosciences) with BamH I and Xho I, and run the gel to recover the 5Kb vector fragment;

4. Ligate the insert fragment and the vector fragment, overnight at 4°C;

5. Transform the ligation product into Escherichia coli DH5α;

6. Bacteria were picked, identified by enzyme digestion, and the correct LYK4-C72/pGEX-4T-1 clone was sequenced.

Example 2: Antigen Purification

1. Transform the LYK4-C72/pGEX-4T-1 plasmid prepared in Example 1 into BL21 competent;

2. Pick the bacteria into 5ml YTA (peptone 16g/L; yeast extract 10g/L; sodium chloride 5g/L) and shake overnight;

3. Inoculate 2L YTA at 1:30 and shake for 3 hours; induce with 0.2mM (GST) or 1mM (His) IPTG at 30°C for 3 hours;

4. Collect bacteria by centrifugation at 10000g for 3 minutes, divide into 4 tubes, resuspend with 15ml PBS (GST) respectively, add PMSF (final concentration 1mM) at 1:100;

5. Ultrasound, intensity: 10-12; time: 8-10min; interval: 2s;

6. After ultrasonication, add 1% TritonX-100, centrifuge at 10000g for 10min, and take the supernatant;

7. Handle GST beads during centrifugation: wash 3 times, 10 times the volume of PBS, 500g, 5min; (storage: 80%)

Mix the supernatant of the centrifuged bacterial liquid with the treated GST beads (5-10mg protein per ml), and rotate at 4°C for 2-4h;

8. Washing: Wash 3 times with 10 times the volume of PBS, rotate at RT for 5-10min each time, and centrifuge;

9. Elution; elution buffer: 0.1M Tris-cl, 0.2M Nacl, 61.5ng Glutathine, 0.1% TritonX-100; rotate at 4°C for 30min-1h, centrifuge; elution 2-3 times;

10. Gel running, Coomassie Brilliant Blue staining and quantification.

This embodiment obtains the fusion protein of SEQ ID NO: 4 and GST, its sequence is shown in SEQ ID NO: 5.

Example 3: Immunization of Rabbits

The work of immunizing rabbits was done by Shanghai Zhongke Yingmu Biotechnology Co., Ltd. The general procedure is as follows: the GST fusion protein antigen prepared in Example 2 was injected into two adult male rabbits respectively, and the immunization was carried out twice, with an interval of about 1-2 months between the two times, prepared with a total of 5 mg of Example 2 GST fusion protein antigen. After the second immunization, the antibody titer in the rabbit serum was tested by ELISA. If it was greater than 2.5 million, the rabbit was bled, and the serum was collected for antibody purification.

Example 4: Antibody Purification

1) Get the rabbit serum of the rabbit immunized in Example 3: the rabbit is immunized 3 times (about 2 months) with the GST fusion protein (dissolved in PBS) prepared in Example 2, and blood is taken (70-90ml/only) , exudate serum overnight at 4°C;

2) The antigen used to purify the antibody was passed through a desalting column PD-10 (GE Healthcare Life Sciences), and the elution buffer was replaced with NaPi buffer (100mM NaH ₂ PO ₄ /Na ₂ HPO ₄ (pH 8.2); 500mM NaCl ; 0.2 mM EDTA,; 0.1% NP-40; 0.5 mM PMSF);

3) Centrifuge at 15000g for 10min, take serum, aliquot 9-10ml per tube, store at -80°C for later use;

4) Purify the antibody by combining CNBr-activated sepharose 4B beads (toxic) (GE Healthcare LifeSciences) with the antigen (the GST fusion protein prepared in Example 2). Beads have to be activated first. According to experience, 0.6g beads can purify 9-10ml serum. Weigh 0.6g beads into a centrifuge tube filled with pre-cooled 1mM HCl, mix until completely dissolved;

5) Prepare a large suction device, a large Erlenmeyer flask with a suction port, an enamel funnel (with holes inside), and two pieces of 3M filter paper that match the funnel. Pour 50ml of the liquid containing the beads into the funnel, add 450ml of pre-cooled 1mM HCl, and at the same time pump slowly, pay attention not to let the beads dry out during the suction process. The whole suction process takes about 15 minutes;

6) Suction again with 500ml NaPi buffer (continuous suction, complete in about 40 seconds). When about 5ml of bf remains (be careful not to drain it), pull out the enamel funnel, collect the beads with a 5ml gun, and add 5ml of NaPi buffer to collect the remaining beads. Steps 5 and 6 should be completed within 2 minutes as soon as possible.

7) Rotate the 50ml centrifuge tube at room temperature for 1-3 hours (beads and antigen binding), or overnight at 4°C. After binding, centrifuge at 500g at 4°C for 5 minutes, measure the OD280 of the supernatant, and compare the readings before and after binding to determine the binding efficiency. (Calculation method: total amount of protein=OD×dilution factor×protein volume). The precipitation volume of 0.6g beads is about 2ml. Mix the beads with 5 times the precipitation volume (10ml) of NaPi buffer, then centrifuge and discard the supernatant.

8) Add 100mM Tris-HCl PH8.0 to the precipitation, 10 times the precipitation volume (20ml), mix well, and rotate at room temperature for 2 hours or overnight at 4°C to block the redundant protein binding sites on the beads;

9) Pour 20ml of the liquid containing beads into two previously prepared glass wool columns (10ml syringe, about 1ml of glass wool), plug the stopper and let it stand for 3 minutes to allow the beads to settle, then remove the stopper to allow the liquid to Drop freely (connect with 500ml plastic bottle, put foam plastic in the middle).

10) Wash the beads with the following five buffer solutions, each volume is 10 times the bead precipitation volume, about 20ml:

a) 100mM Tris-HCl, pH 8.0, containing 0.5M NaCl

b) 10mM Tris-HCl, pH 8.0, containing 0.5M NaCl

c) 200mM glycine, pH 2.5, containing 0.5M NaCl

d) 0.5M NaCl

e) 200mM triethylamine, pH11.5, containing 0.5M NaCl

f) 0.5M NaCl

11) Rinse the beads again with about 20ml of NaPi buffer of 10 times the precipitation volume.

12) Add 1ml of 1M NaPi, pH8.2, to every 9ml of serum and mix well. Inactivate complement at 56°C for 30 minutes, centrifuge at 12,500 g for 10 minutes, take the supernatant and add it to the column for filtration, and return the filtered serum to the column for another 2-3 times.

13) Wash the beads with about 100ml of 10mM Na ₂ HPO ₄ /NaH ₂ PO ₄ , pH8.2, and 0.5M NaCl, and drop the buffer into the column through a 50ml syringe and dropper;

14) Prepare two 15ml centrifuge tubes, add 1ml 1M Na ₂ HPO ₄ /NaH2PO ₄ , pH8.2 to each of them, add 5ml 100mM glycine, pH2.5, and 0.5M NaCl to the column, collect 1M NaPibuffer, pH8 .2 Mix well in a 15ml centrifuge tube;

15) Repeat step 13;

16) Prepare two 15ml centrifuge tubes, add 1ml 1M Na ₂ HPO ₄ /NaH2PO ₄ , pH8.2, add 5ml 100mM triethylamine, pH11.5, with 0.5M NaCl to the column, collect the buffer containing 1M NaPi Solution, mix in a 15ml centrifuge tube with pH 8.2. Combine the antibodies filtered twice (about 12ml);

17) Replace the Protein A column with a connector (only one) that connects to the syringe. Fill the syringe with 10ml of PBS, unplug the column, and rinse slowly with PBS (1ml/min). The liquid will drip out and no air bubbles can enter. (Protein A column can be reused to purify the same antibody, stored in 20% ethanol);

18) Pass 12ml antibody through the column, you can pass it twice. Use the corresponding bf to zero, measure the OD280 of the leaked liquid to check the binding efficiency, and ensure that the read value of the effluent is 0;

19) Wash the column again with 10ml PBS;

20) Elute the antibody with 3ml 0.1M citric acid pH 3.6, collect the eluate in 6 ep tubes (500ul/tube), add 50ul 1M Tris-HCl pH 9.0 to each tube, and mix well. Use the corresponding bf to zero, measure the OD280 of each tube, and determine the concentration. Take 2-6 tubes of antibody with higher concentration, a total of 2.5ml and mix well;

21) Pass through the desalting column PD-10, and replace the elution buffer with PBS (2.5ml in, 3.5ml out). The final OD280 and total amount of antibodies were determined. Aliquot 500 per tube and store at -80°C. The appropriate antibody concentration is 1ug/ul. If the antibody concentration is too low, BSA should be added to prevent degradation. If it is too high, NP-40 should be added to increase the solubility; the total amount of antibody (IgG) = 0.75*OD280*volume;

22) The following WB (Western Blotting) detection antibody:

(a) Collect the embryoid body (EB) samples formed by the differentiation of wild-type (WT) and Stk40-deleted (KO) mouse embryonic stem cells for 9 days, wash twice with pre-cooled 1×PBS, and add an appropriate volume of Co -IP lysis buffer (50mM Tris-Cl, pH 7.5, 150mM NaCl, 2mM EDTA, 10% (v/v) glycerol, 0.5% NP-40, 1mM NaF, 1mM PMSF) for lysis.

(b) Shake at 4°C for 15 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and take the supernatant.

(c) After BCA quantification, take 20 μg of total protein, add 1/3 volume of 4×SDS loading buffer (200mM Tris-Cl, pH 7.4, 8% SDS, 20% β-mercaptoethanol, 40% glycerol, 0.04% bromophenol blue) in boiling water for 5 minutes.

(d) SDS-PAGE electrophoresis separation. The electrophoresis buffer formula is: Tris 0.25M, glycine 1.92M, SDS 1%.

(e) Transfer the protein in the gel to the nitrocellulose membrane by electrotransfer (200mA, 4 hours). The transfer solution formula is 0.025M Tris, 0.192M Glycine and 20% methanol.

(f) Block with TBS-T buffer (0.02M Tris-Cl, pH7.6, 0.137M NaCl, 0.1% Tween-20) containing 5% skimmed milk powder or 5% BSA for 1 hour at room temperature, discard the blocking solution .

(g) Add a certain amount of primary antibody against mouse Stk40 protein (Stk40-C72 and Stk40-C98 polyclonal antibody) properly diluted in TBS-T buffer containing 5% skimmed milk powder, overnight at 4°C. (h) Discard the primary antibody, wash with TBS-T buffer 3 times, 5 minutes each time.

(i) Dilute a certain amount of anti-rabbit horseradish peroxidase-conjugated secondary antibody (Ab2 shown in Figure 4) in TBS-T buffer containing 5% skimmed milk powder, and incubate at room temperature for 1-2 hours ; Discard the secondary antibody, wash with TBS-T buffer 3 times, 5 minutes each time.

(j) Add Pierce chemiluminescent substrate dropwise on the film, then expose it with X-ray film, and then develop and fix it automatically by a processor.

Figure 4 shows the Stk40-C72 polyclonal antibody was used to detect the protein expression of Stk40 in embryoid bodies (EBs) formed by the differentiation of wild-type (WT) and Stk40-deficient (KO) mouse embryonic stem cells for 9 days by Western blotting. The results showed that the obvious expression of Stk40 (~50kDa band) could be detected in the wild-type EB samples, but the corresponding band could not be detected in the Stk40-deleted EB samples, indicating that the polyclonal antibody can specifically Recognizes mouse Stk40 protein.

Example 5: Detection of antibody activity

Perform WB detection according to step (22) of Example 4, except that Stk40-C98 antibody (LingjieLi ^* , Lei Sun ^* , etc., Stk40 Links the Pluripotency Factor Oct4 to the Erk/MAPK Pathway and Controls Extraembryonic Endoderm Differentiation PNAS.2010 Jan; 107(4):1402-1407) to replace the Stk40-C72 polyclonal antibody of the present application for detection.

The results are shown in Figure 5. Figure 5 shows that although the antibody (Stk40-C98) prepared by using the C-terminal 98 amino acids (C98) of Stk40 as the antigen segment can recognize the Stk40 protein, there are many non-specific bands (miscellaneous bands), and some miscellaneous bands are off The bands of interest are very close together and can only be seen when the proteins are well separated during the gel run.

Although the present application has been described in terms of specific embodiments, it should be understood that appropriate changes and modifications can be made to the present application without departing from the spirit of the present application. These are all within the scope of protection defined by the claims of the present application.

Claims (6)

1. an isolated polypeptide, its sequence is as shown in SEQ ID NO:4.

2. an isolated polypeptide, its sequence is as shown in SEQ ID NO:5.

3. for an immune composition, it is characterized in that, said composition contains the isolated polypeptide described in any one in claim 1-2.

4. a method of preparing the polyclonal antibody of anti-mouse Stk40, is characterized in that, described method comprises that right to use requires the composition immunity Mammals described in any one in the isolated polypeptide described in any one in 1-2 or claim 3.

5. method as claimed in claim 4, is characterized in that, described method comprises:

(1) build the antigen presentation carrier that contains described polypeptide;

(2) use this expression vector to transform intestinal bacteria;

(3) polypeptide described in purifying;

(4) the composition immunity Mammals of using described polypeptide or containing described polypeptide; With

(5) antibody purification from described mammalian blood serum, thus make described antibody.

6. a separated polynucleotide sequence, is characterized in that, the polypeptide in this polynucleotide sequence coding claim 1-2 described in any one.

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