A sjhnf4 polypeptide antibody, preparation method and application

Abstract

The application discloses a SjHNF4 polypeptide antibody, a preparation method and application. The preparation method comprises the following steps: downloading a SjHNF4 protein sequence, screening a polypeptide sequence, adding a cysteine at an N terminal, then performing polypeptide synthesis and purification, coupling to obtain a recombinant polypeptide, mixing the coupled polypeptide with Freund's complete adjuvant to obtain a mixture, immunizing a mammal by using the mixture, taking heart blood of the mammal and separating serum to obtain a polypeptide antibody. The polypeptide antibody is detected by using an ELISA method, and then the polypeptide antibody is purified by using a polypeptide affinity chromatographic column. Finally, the binding affinity of the antibody and the antigen is detected by Western Blot and immunofluorescence experiments. The polypeptide antibody obtained by the application has high purity (not less than 90%) and high titer (not less than 512000), and has good purity and immunogenicity, can satisfy various experimental requirements and has commercial value.

Description

Technical Field

[0001] This invention relates to an SjHNF4 polypeptide antibody, its preparation method, and its application. It belongs to the field of molecular biology technology. Background Technology

[0002] Nuclear receptors (NRs) are key transcriptional regulators in metazoans, playing crucial regulatory roles in many vital life processes, including cell homeostasis, differentiation, proliferation, and development. A typical NR protein structure includes an N-terminal A / B domain, a C-terminal DNA-binding domain (DBD), a hinge (D-region), and a C-terminal ligand-binding domain (LBD). Schistosomiasis is a serious but neglected tropical disease that causes a series of pathological damages in humans. Schistosomiasis is mainly caused by three schistosomes: *Schistosoma japonicum*, *Schistosoma mansoni*, and *Schistosoma haematobium*. These schistosomes parasitize the host's veins, causing varying degrees of damage to the liver, intestines, and other parts of the body. Praziquantel is the only effective drug for treating schistosomiasis, but it has drawbacks such as side effects and drug resistance. Finding new drug targets is crucial, and nuclear receptors, as important target molecules in organisms, are increasingly being studied in schistosomiasis research.

[0003] Among them, the full-length cDNAs of four NRs in *Schistosoma japonicum* have been cloned and studied, including retinol X receptor 1 (SjRXR1), SjRXR2, thyroid hormone receptor (SjTHRβ), and peroxisome proliferator-activated receptor (PPAR-γ). Hepatocyte nuclear factor 4 (HNF4) belongs to the NR family and has been identified in the genomes of *Schistosoma mansoni* and *Schistosoma japonicum*. A 2020 study used single-cell sequencing analysis of *Schistosoma mansoni* to screen for HNF4, a target closely related to blood feeding, intestinal maintenance, and in vivo pathology. These studies show that HNF4 is crucial for various life activities of *Schistosoma japonicum*. However, there are currently no commercially available antibodies targeting *Schistosoma japonicum* HNF4, and recombinant protein antibodies have poor specificity, while monoclonal antibody preparation is time-consuming and difficult. Therefore, current research on HNF4 remains at the genetic level and has not yet characterized its specific biological functions. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a SjHNF4 polypeptide antibody, its preparation method, and its application. This antibody has good specificity and high purity and can be used for functional studies of the SjHNF4 protein level, which will help promote the progress of the nuclear receptor of Schistosoma japonicum.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] 1. A SjHNF4 polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1. The SjHNF4 protein sequence was downloaded from the NCBI database and analyzed using OptimumAntigen. TM The full-length protein sequence of SjHNF4 is analyzed online, and peptide sequences that meet the requirements are selected.

[0007] 2. A method for preparing an SjHNF4 polypeptide antibody, comprising first adding a cysteine ​​C residue to the N-terminus of the aforementioned SjHNF4 polypeptide to obtain a polypeptide as shown in SEQ ID NO.2, then cross-linking the polypeptide with a carrier protein to form a cross-linked polypeptide, then immunizing a mammal with the cross-linked polypeptide, collecting blood from the heart of the mammal and separating the serum, thereby obtaining the antibody.

[0008] As one of the preferred technical solutions, the titer of the polypeptide antibody is above 1:512000.

[0009] As one of the preferred technical solutions, the carrier protein is keyhole hemocyanin (KLH), and the thiol group of the polypeptide and the amino group of KLH are cross-linked with MBS cross-linking agent to form a cross-linked polypeptide.

[0010] As one of the preferred technical solutions, the cross-linked peptide is mixed with Freund's complete adjuvant to obtain a mixture, and the mixture is injected subcutaneously into mammals to achieve immunization.

[0011] As one of the preferred technical solutions, immunization includes three times: booster immunizations are performed on days 14 and 35 after the initial immunization, and blood is collected from the heart of the mammal seven days after the third immunization.

[0012] As one of the preferred technical solutions, the mammal is selected from any one of the following: New Zealand white rabbit, Japanese white rabbit, and mouse.

[0013] As one of the preferred technical solutions, the obtained polypeptide antibody is purified using an affinity chromatography column.

[0014] 3. An SjHNF4 polypeptide antibody obtained using the above preparation method.

[0015] 4. Application of the above-mentioned SjHNF4 polypeptide antibody in the preparation of a schistosomiasis detection kit.

[0016] As one of the preferred technical solutions, the kit is used to detect the SjHNF4 protein level in different developmental stages of Schistosoma japonicum.

[0017] 5. Application of the above-mentioned SjHNF4 polypeptide antibody in detecting the interaction between SjHNF4 and protein or DNA.

[0018] The beneficial effects of this invention are:

[0019] This invention downloads the SjHNF4 protein sequence from the NCBI database and uses OptimumAntigen. TM The full-length SjHNF4 protein sequence was analyzed online, and suitable peptide sequences were screened. A cysteine ​​residue was added to the N-terminus to facilitate cross-linking. Following peptide synthesis and purification, the synthesized peptide was conjugated using MBS-activated KLH to obtain a recombinant peptide. The conjugated peptide was then mixed with Freund's complete adjuvant to obtain a mixture. This mixture was subcutaneously injected into two New Zealand white rabbits for two booster immunizations. Finally, heart blood was collected from the New Zealand white rabbits, and serum was separated to obtain peptide antibodies. The antibody titer was detected using ELISA, and the peptide antibodies were then purified using peptide affinity chromatography. Finally, the binding affinity of the antibodies to endogenous antigens was detected by Western blotting and immunofluorescence assays.

[0020] The polypeptide antigens used in this invention are screened based on criteria such as immunogenicity, hydrophilicity, flexibility, and ease of synthesis. This screening method is applicable to proteins from other species. The cross-linked polypeptides and their mixing ratio with adjuvants used in this invention can be implemented according to conventional methods in the art. Animals used for immunization include New Zealand white rabbits, Japanese white rabbits, and mice. The animal immunization includes three immunizations: booster immunizations on days 14 and 35 after the initial immunization, with the immune response level detected by indirect ELISA seven days after each immunization. Finally, the polypeptide antibodies are purified using an affinity chromatography column coated with the antigen. The polypeptide polyclonal antibodies obtained using the method provided in this invention have a purity of not less than 90% and a titer of not less than 512,000, exhibiting good purity and immunogenicity, and can meet various experimental requirements.

[0021] The specific advantages are as follows:

[0022] (1) The antibody preparation process of this invention solves the shortcomings of traditional recombinant protein polyclonal antibodies, such as difficulty in obtaining and purifying recombinant proteins, low antibody titer, and poor specificity;

[0023] (2) Using the online website OptimumAntigen TM The screened peptide sequences are effective, easy to synthesize, and low in cost, making them ideal for mass production of antigens for preparing peptide antibodies.

[0024] (3) The entire process of this invention is much shorter than the traditional preparation cycle of polyclonal and monoclonal antibodies. The entire process from antigen screening to antibody purification takes only 45 days, which improves research efficiency.

[0025] (4) The polypeptide antibody obtained by the preparation method of the present invention was identified by SDS-PAGE for purity and detected by ELISA for titer. It not only has high purity (>90%), but also high titer (>512000), which fully meets the experimental requirements.

[0026] (5) Western blotting and immunofluorescence experiments have demonstrated that the polypeptide polyclonal antibody prepared in this invention can recognize SjHNF4 at different developmental stages in Schistosoma japonicum and can be used to detect the SjHNF4 protein level. It also meets the experimental requirements of immunoprecipitation (IP) and chromatin immunoprecipitation (CHIP), which is beneficial for the study of SjHNF4 interacting proteins and the analysis of promoter motifs that bind to downstream genes. This has greatly promoted the study of nuclear receptors in Schistosoma japonicum and laid the foundation for finding reliable drug targets for Schistosoma japonicum from nuclear receptors. Attached Figure Description

[0027] Figure 1 To use OptimumAntigen TM Online websites analyze the antigenicity, surface accessibility, and hydrophilicity of the full-length SjHNF4 protein sequence.

[0028] Figure 2 The HPLC purity test results for the synthesized polypeptide show a purity of 90.1%.

[0029] Figure 3 The SDS-PAGE purity test results for the SjHNF4 peptide antibody are as follows: purity is 97%; Lane M: Marker; Lane 1: SjHNF4 peptide antibody; Lane 2: Rabbit IgG.

[0030] Figure 4 The results are from the ELISA titer assay of the SjHNF4 polypeptide antibody.

[0031] Figure 5 The results of Western blotting detection of SjHNF4 in Schistosoma japonicum.

[0032] Figure 6 The results of immunofluorescence of SjHNF4 at different developmental stages of Schistosoma japonicum are shown. Among them, AD represents the control group of female, male, cercariae, and eggs of Schistosoma japonicum, respectively, and EH represents the experimental group of female, male, cercariae, and eggs of Schistosoma japonicum. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0034] Example 1: Protein sequence analysis of HNF4 from Schistosoma japonicum ( Figure 1 )

[0035] The protein sequence of SjHNF4 was obtained from GenBank (accession number: TNN13336.1), and the amino acid sequence is shown in SEQ ID NO.3, consisting of a total of 712 amino acids. ProtParam tool was used to analyze the sequence.

[0036] (https: / / web.expasy.org / protparam / ) The molecular weight of SjHNF4 is calculated to be approximately 80964.55 Daltons, and the isoelectric point is 6.28.

[0037] Example 2: Design and Synthesis of HNF4 Peptide from Schistosoma japonicum

[0038] via OptimumAntigen TM Online website design tools were used to predict the antigenic epitopes of SjHNF4 and to perform comprehensive analysis on the listed epitopes based on multiple aspects, including antigenicity, hydrophilicity, hydrophobicity, surface probability, transmembrane activity, homology, flexible region, helical region, sheet region, signal peptide, and modifications. Considering factors such as the difficulty of peptide synthesis, solubility, and heterologity with the host, a peptide with the amino acid sequence YNDHNREYSKSNTH (SEQ ID NO.1) was ultimately selected as the antigen (284aa-297aa) (Table 1). Adding a cysteine ​​C residue to the N-terminus of the above peptide facilitates cross-linking with the carrier protein and improves the immunogenicity of the peptide. Therefore, the final synthesized peptide with the sequence CYNDHNREYSKSNTH (e.g., SEQ ID NO.2) has a total length of 15aa and a theoretical molecular weight of 1867.92. The peptide was synthesized using a solid-phase peptide synthesis (SPPS) method commonly used in the art, then purified using a thiol-containing antigen-specific affinity column, and finally identified as having a purity of 90.1% by high-performance liquid chromatography and mass spectrometry. Figure 2 The actual molecular mass is 1867.92, which is consistent with the theoretical molecular mass.

[0039] Table 1

[0040]

[0041] Example 3: Crosslinking of peptides with carrier proteins

[0042] Antigens were prepared by conjugating KLH (Nanjing Weiwo Biotechnology Co., Ltd.) carrier protein with MBS (Dalian Meilun Biotechnology Co., Ltd.), a commonly used cross-linking agent in this field. KLH protein was dissolved in 10 mmol / L PBS (pH 7.4) to a final concentration of 10 mg / mL, and then dialyzed overnight at 4°C. MBS was dissolved in DMF to a final concentration of 10 mg / mL. KLH and MBS were then mixed at a 10:1 (w / w) ratio and incubated at room temperature for 30 min to activate KLH. The mixture was then slowly added to a Sephadex G-25 chromatography column, eluted with cross-linking buffer, and the activated KLH was collected in an EP tube. Finally, 2 mg of peptide was dissolved in 10 mmol / L PBS (pH 7.4) and mixed with activated KLH at a 1:1 (w / w) ratio. The mixture was incubated at room temperature for 3 h, dialyzed overnight at 4°C, and the peptide-KLH cross-linked mixture was lyophilized and stored at -20°C.

[0043] Example 4: Preparation of immunizing and antiserum in experimental animals

[0044] Two 4-month-old New Zealand White rabbits (GenScript Biotechnology Co., Ltd.) were selected as immunization animals. Four days before immunization, 2-5 mL of blood was collected from their marginal ear veins, and the serum was separated and stored at -80℃ as negative control serum for subsequent experiments. 500 mg of the previously prepared peptide-KLH cross-linked mixture was dissolved in 500 μl of PBS (pH 7.4) and thoroughly mixed with Freund's complete adjuvant at a 1:1 (v / v) ratio. The mixture was then vortexed to emulsify thoroughly and used as the immunogen for subsequent immunization experiments. On day 1, the initial immunization was performed, with the immunogen injected subcutaneously into each of the two New Zealand White rabbits at a dose of 200 μg. 500 mg of the previously prepared peptide-KLH cross-linked mixture was dissolved in 500 μl of PBS (pH 7.4) and thoroughly mixed with an equal volume of Freund's incomplete adjuvant. The mixture was then vortexed to emulsify thoroughly and used as the immunogen for booster immunizations. Two booster immunizations were performed on days 14 and 35, with the same dosage and procedure as the initial immunization. Seven days after each immunization, the serum titer of the immunized animals was measured using an indirect ELISA method to determine the level of the immune response. Seven days after the third immunization, blood was collected from the heart of New Zealand white rabbits, and polyclonal antibodies were isolated from the serum and their titers were determined.

[0045] Example 5: Purification of polyclonal antibodies using antigen affinity chromatography column

[0046] Transfer 1 mL of activated antigen affinity resin to a chromatography column. After the resin precipitates, equilibrate it with 10 column volumes of PBS (pH 7.4). Add the separated antiserum to the column and continuously monitor the absorbance at A280. Wash the column with PBS (pH 7.4) until the absorbance at A280 stabilizes. After column equilibration, add elution buffer to the column. Based on the absorbance at A280, collect the antibody-containing eluent into a tube and neutralize the antibody eluent with neutralization buffer. Add 20 column volumes of PBS (pH 7.4) to the column to equilibrate the antigen affinity resin. Dialyze the antibody eluent to PBS (pH 7.4) overnight at 4°C. Finally, add sodium azide buffer to the purified antibody solution to a final concentration of 0.02% and store at 4°C. Figure 3 )

[0047] Example 6: Indirect ELISA determination of antibody titer

[0048] The peptide-KLH cross-linker was diluted to 4 μg / mL using coating buffer and incubated overnight at 4°C for coating the ELISA plate. After thorough washing with 10 mmol / L PBST (pH 7.4), 5% skim milk powder was added, and the plate was blocked at 25°C for 2 h, followed by thorough washing with PBST. The SjHNF4 polyclonal antibody was diluted with PBS (pH 7.4) to different concentrations (1:1000, 1:2000, 1:4000, etc., and 100 μL of each solution was added to the ELISA plate. Pre-immunization serum was used as a negative control. The plate was incubated at room temperature for 2 h, and after thorough washing with PBST, 100 μL of horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added to each well. The plate was incubated at 37°C for 1 h, followed by washing with PBST. 100 μL of TMB chromogenic solution was added to each well, and the reaction was terminated with 2 mol / L HCl. The OD450 absorbance was then measured. Serum titer was calculated when the ratio of SjHNF4 antibody OD450 to negative OD450 was ≥2.1. The SjHNF4 polyclonal antibody titer detected by this invention is greater than 1:512000. Figure 4 )

[0049] Example 7: Western Blot Analysis of SjHNF4 Protein in Schistosoma japonicum

[0050] Total protein from Schistosoma was extracted using RIPA lysis buffer and sonication, and the total protein concentration was determined using a BCA protein quantification kit. A 5% stacking gel and a 10% separating gel were prepared. The total Schistosoma protein was boiled at 95°C for 10 min to ensure complete denaturation. Then, 40 μg of total Schistosoma protein was separated by SDS-PAGE electrophoresis at a constant voltage of 80 V. When the sample just emerged from the stacking gel, the voltage was adjusted to 120 V, and electrophoresis was stopped when the bromophenol blue indicator reached the bottom. The protein product was transferred to a PVDF membrane using a wet transfer method and kept at a constant current of 200 mA for 90 min. The PVDF membrane was then placed in a 5% skim milk blocking buffer and blocked on a shaker at room temperature for 3 h. The SjHNF4 polyclonal antibody obtained in Example 6 was used as the primary antibody, and pre-immunization serum was used as the negative control primary antibody. After dilution at a volume ratio of 1:1000, the antibody was incubated overnight at 4°C with a blocked PVDF membrane. The hybridized PVDF membrane was washed four times with 1×TBST (pH 7.4) on a shaker for 10 min each time. The membrane was then incubated with diluted HRP-labeled goat anti-rabbit secondary antibody (1:5000) at 37°C for 1 h, followed by washing five times with 1×TBST. After incubation in ECL chemiluminescence solution for 2 min, the membrane was developed and photographed using a chemiluminescence analyzer. The experimental results of this example show that the expression level of SjHNF4 is higher in female Schistosoma japonicum, while the expression level of SjHNF4 in males is too low to be detected by Western blotting. Figure 5 )

[0051] Example 8: Immunofluorescence analysis of SjHNF4 protein in Schistosoma japonicum

[0052] Samples of *Schistosoma japonicum* at four developmental stages—eggs, cercariae, females, and males—were fixed with ice-cold acetone and incubated at 4°C for at least 24 h. After washing with PBS (pH 7.4), each sample was treated with 1% glycine aqueous solution and then infiltrated with 0.3% Triton X-100 aqueous solution for 30 min. Following PBS (pH 7.4) washing, the samples were blocked with 10% goat serum for 2 h. Different samples were then incubated with diluted SjHNF4 polyclonal antibody (1:100 v / v) at 4°C for 18 h. The primary antibody was discarded, and the samples were thoroughly washed with PBS (pH 7.4), followed by incubation with Alexa Fluor 488 (1:500 v / v) fluorescent secondary antibody at 37°C for 1 h in the dark. Secondary antibody was discarded, and samples were thoroughly washed with PBS. Then, samples were fixed onto adhesive slides, and 50 μl of anti-fluorescence quencher (containing DAPI) was added. After incubation for 5 min, fluorescence was captured using a fluorescence microscope to observe the fluorescence of different samples of female and male *Schistosoma japonicum*, cercariae, and eggs. The experimental results in this example show that SjHNF4 is expressed in different developmental stages of *Schistosoma japonicum*: the female reproductive system, capsule, etc.; the male capsule; the cercarial head; and the eggs. Figure 6 )

[0053] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A SjHNF4 polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing an SjHNF4 polypeptide antibody, characterized in that, First, add a cysteine ​​C residue to the N-terminus of the polypeptide described in claim 1 to obtain the polypeptide shown in SEQ ID NO.

2. Then, crosslink the polypeptide with a carrier protein to form a crosslinked polypeptide. Next, use the crosslinked polypeptide to immunize a mammal, collect blood from the heart of the mammal, and separate the serum to obtain the polypeptide. The carrier protein is keyhole hemocyanin, and the thiol group of the polypeptide and the amino group of KLH are cross-linked with MBS cross-linking agent to form a cross-linked polypeptide; Cross-linked peptides were mixed with Freund's complete adjuvant to obtain a mixture, which was then injected subcutaneously into mammals to induce immunization. Immunization consists of three doses: booster immunizations are given on days 14 and 35 after the initial immunization, and blood is drawn from the heart of the mammal seven days after the third immunization. The mammal is selected from any one of the following: New Zealand white rabbit, Japanese white rabbit, and mouse.

3. The preparation method according to claim 2, characterized in that, The titer of the polypeptide antibody is above 1:512000.

4. An SjHNF4 polypeptide antibody obtained using the preparation method described in claim 2 or 3.

5. The use of the SjHNF4 polypeptide antibody as described in claim 4 in the preparation of a schistosomiasis detection kit.

6. The use of the SjHNF4 polypeptide antibody as described in claim 4 in detecting the interaction between SjHNF4 and protein or DNA.

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