A rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, its preparation method and application

Abstract

The present invention discloses a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, its preparation method and application. The amino acid sequence of the NPHP4 protein is shown in SEQ ID NO.1. Preparation method: Analysis and design of the antigen epitope of Chlamydomonas reinhardtii NPHP4 protein, and selection of the 859-1058aa amino acid fragment to construct a prokaryotic cell recombinant expression vector; transferring the recombinant expression vector into prokaryotic expression cells to obtain a recombinant expression strain and inducing the expression of the antigen protein; using the purified antigen protein as an immunogen to immunize rabbits to obtain a rabbit polyclonal antiserum against the NPHP4 protein, and further purifying it; the purified polyclonal antibody can be used to verify the labeling of the ciliary transition zone of Chlamydomonas reinhardtii by the IF method, proving that it can be used for specific fluorescence labeling of the ciliary transition zone; it can also be used for qualitative and quantitative analysis of the Chlamydomonas reinhardtii NPHP4 protein by the immunoblotting WB method.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, a preparation method thereof, and applications thereof. Background Art

[0002] Chlamydomonas reinhardtii is the most commonly used model organism in laboratory research. This single-celled eukaryotic green alga is known as the "photosynthetic yeast" because of its simple culture conditions, short growth cycle, fast growth rate, and high photosynthetic efficiency. So far, genetic transformation of the nuclear genome, chloroplast genome, and mitochondrial genome of Chlamydomonas reinhardtii has been achieved.

[0003] Cilia are highly conserved organelles with multiple functions in cell biology. The complex ultrastructure of cilia has co-evolved with their multiple roles, including motility and sensing functions. Despite their diverse functions, there remains a generally conserved structural core, including the microtubule-based axoneme, with nine-fold axial symmetry. The structure and symmetry of the axoneme are determined by the mother centriole or basal body from which it emanates. After the basal body is correctly anchored to the plasma membrane, the developing cilium must establish a selective barrier to sequester molecules crucial for its construction, as it lacks an autonomous translation mechanism. This region connecting the basal body and the nascent cilium is called the transition zone (TZ). Since the TZ was first observed by electron microscopy (EM) in the 1950s, a great deal of knowledge has been accumulated to elucidate the structure and composition of this key ciliary region. Notably, the importance of the TZ in scientific research has become increasingly prominent due to the increasing number of ciliary-related diseases (ciliopathies) caused by mutations in TZ proteins. Chlamydomonas reinhardtii is one of the main model organisms for studying the structure, function, and potential molecular mechanisms of eukaryotic cilia, and is widely used in the fields of cell and molecular biology, and as a research model for ciliary diseases.

[0004] The NPHP4 gene encodes nephrocystin-4 (NPHP4), which is localized in the ciliary transition zone (TZ). NPHP4 is an important component of the selective gating structure that functions in the transition zone to control the movement of soluble and membrane-associated proteins between the cilium and the cytoplasmic compartment. The various phenotypic consequences of NPHP4 mutations in humans and other organisms are likely due to protein mislocalization caused by transition zone defects. To address the scientific problem of studying the important ciliary functions of NPHP4, it is particularly important to seek an antibody that can specifically recognize Chlamydomonas reinhardtii NPHP4 protein and a preparation method thereof. Summary of the Invention

[0005] The present invention provides a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, its preparation method and application. The polyclonal antibody can specifically recognize Chlamydomonas reinhardtii NPHP4 protein and can be used for studying the localization marker of the ciliary transition zone. In the application in the field of biological research, the NPHP4 protein in Chlamydomonas reinhardtii cells can be qualitatively and quantitatively analyzed by Western blot (WB) method, and the NPHP4 protein can also be labeled by immunofluorescence (IF) method to explore the localization and mutual relationship of each protein in the transition zone, thereby laying an application foundation for the research on the ciliary phenotype changes and related regulatory mechanisms such as ciliary gated transport using Chlamydomonas reinhardtii as a model organism.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, the polyclonal antibody can specifically recognize the NPHP4 protein in the ciliary transition zone of Chlamydomonas reinhardtii; the amino acid sequence of the NPHP4 protein is as shown in SEQ ID NO.1 in the sequence listing.

[0007] The present invention also provides a preparation method of the above rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, including the following steps:

[0008] (1) Using bioinformatics analysis to obtain the amino acid sequence suitable as an antigen fragment in the NPHP4 protein, selecting the cDNA fragment of NPHP4 located at 859-1058aa as the immunogenic antigen polypeptide, and performing whole gene synthesis according to the codon preference of Escherichia coli; the cDNA fragment of NPHP4 located at 859-1058aa is as shown in SEQ ID NO.2 in the sequence listing, and the corresponding amino acid sequence is as shown in SEQ ID NO.3 in the sequence listing;

[0009] (2) Using the NPHP4 antigen cDNA fragment synthesized in step (1) and the prokaryotic cell recombinant expression vector pSUMO to construct a NPHP4 prokaryotic expression vector;

[0010] (3) Transforming the NPHP4 prokaryotic expression vector constructed in step (2) into BL21(DE3) competent cells to obtain a recombinant expression strain;

[0011] (4) Inducing the expression of the protein of the recombinant expression strain obtained in step (3) under certain conditions to obtain a recombinant expression tagged NPHP4 antigen polypeptide;

[0012] (5) Using the NPHP4 antigen polypeptide induced and expressed in step (4) as an antigen for rabbit immunization treatment, collecting blood to obtain a rabbit polyclonal antiserum against the NPHP4 protein, and subjecting the antiserum to affinity purification to obtain a rabbit polyclonal antibody against the NPHP4 protein.

[0013] Further, the immunization method is as follows: Mix the NPHP4 antigen polypeptide with an equal volume of complete Freund's adjuvant to obtain a first mixture and inject it into an immunized rabbit; after 28 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a second mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a third mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fourth mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fifth mixture and inject it into the immunized rabbit; the injection method is multi-point injection on the back; the immunization dose is: the first main injection is 400 μg antigen / experimental rabbit, and the second to fifth injections are all 125 μg antigen / experimental rabbit.

[0014] The present invention also provides an application of the rabbit polyclonal antibody against the above-mentioned Chlamydomonas reinhardtii NPHP4 protein in detecting the Chlamydomonas reinhardtii NPHP4 protein.

[0015] Further, the Chlamydomonas reinhardtii NPHP4 protein is detected by an immunofluorescence IF method or an immunoblotting WB method.

[0016] Further, the steps for detecting the Chlamydomonas reinhardtii NPHP4 protein by the immunofluorescence IF method are as follows:

[0017] After fixing Chlamydomonas reinhardtii cells by methanol penetration method, wash them 3 times with PBS buffer for 5 min each time. Block them at room temperature with a blocking solution for 1 h, incubate them overnight at 4°C with the purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:500; wash them 3 times with PBS buffer containing 0.5% Tween-20 for 5 min each time; incubate them with a fluorescence-labeled secondary antibody corresponding to the wavelength and species at 37°C for 1 h, then wash them 3 times with PBS buffer containing 0.5% Tween-20 for 5 min each time, then wash them once with PBS buffer without Tween-20, and finally wash them once with ddH2O. After mounting the slides, observe the localization of the NPHP4 protein in the ciliary transition zone under a fluorescence microscope, take pictures, draw graphs and analyze.

[0018] Further, the steps for detecting the Chlamydomonas reinhardtii NPHP4 protein by the immunoblotting WB method are as follows:

[0019] The Chlamydomonas cell samples frozen at -80°C were repeatedly pipetted with cell lysis buffer, and SDS buffer was added and mixed well, followed by incubation in a metal bath at 95°C for 10 min. Subsequently, SDS-PAGE electrophoresis was performed, with the stacking gel electrophoresis time being 20 min and the voltage being 80 V; the separating gel electrophoresis time was 80 min and the voltage was 120 V. Transfer membrane was carried out under the conditions of 80 min and 100 V. The PVDF membrane was blocked at room temperature for 1 h and then incubated overnight at 4°C with the purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:5000. The primary antibody was recovered, and the membrane was washed 3 times with 1×TBST, 10 min each time. The corresponding secondary antibody was incubated at room temperature for 1 h, the secondary antibody was recovered, and the membrane was washed 3 times with 1×TBST, and then developed to perform qualitative and quantitative analysis of the NPHP4 protein.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention uses the Chlamydomonas reinhardtii NPHP4 expression vector to immunize New Zealand white rabbits to prepare polyclonal antibodies. The prepared polyclonal antibodies can specifically recognize the Chlamydomonas reinhardtii NPHP4 protein, and are characterized by high titer and good specificity. In the application of the biological research field, qualitative and quantitative analysis of the NPHP4 protein in Chlamydomonas reinhardtii cells can be carried out by immunoblotting WB method, and the NPHP4 protein can also be labeled by immunofluorescence IF method to explore the localization and interaction relationship of each protein in the transition zone, thereby laying an application foundation for the in-depth exploration of ciliary phenotypic changes, ciliary gated transport, and ciliary disease pathological mechanisms using Chlamydomonas reinhardtii as a model organism. Brief Description of the Drawings

[0022] Figure 1 Schematic diagram of nucleotides and amino acids of the NPHP4 polypeptide fragment selected after antigen epitope analysis;

[0023] Figure 2 SDS-PAGE detection chart of NPHP4 antigen protein expression;

[0024] Figure 3 ELISA titer detection data chart of NPHP4 antiserum and affinity purified antibody;

[0025] Figure 4 Immunoblotting detection chart of NPHP4 protein by immunoblotting WB;

[0026] Figure 5 Immunofluorescence IF detection chart of specific localization fluorescence of NPHP4 in the ciliary transition zone of Chlamydomonas reinhardtii (WT: wild type 21gr; nphp4: NPHP4 gene mutation cell line). Detailed Embodiments

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. In the following methods, unless otherwise specified, they are conventional methods.

[0028] In the following examples, the immunized rabbit breed used was 2-3-month-old New Zealand white rabbits, and laboratory SPF-grade experimental animals were used for antibody preparation to prepare immune sera (S0128-1 and S0128-2) with high titer and strong specificity.

[0029] The primary antibodies used in the following examples were S0128-1 and S0128-2 antibodies against the NPHP4 protein, and the secondary antibodies were commercial Goat-anti-rabbit HRP antibody (for WB detection) and Goat-anti-rabbit (Alexa Fluor 488) antibody (for IF detection).

[0030] In the following examples, the wild-type 21gr (WT) and nphp4 mutants were sourced from laboratory-preserved algal strains and could also be obtained from the Chlamydomonas reinhardtii Stock Center in the United States.

[0031] A method for preparing a rabbit polyclonal antibody against the NPHP4 protein of Chlamydomonas reinhardtii, comprising the following steps:

[0032] (1) Obtain the amino acid sequence suitable as an antigen fragment in the NPHP4 protein by bioinformatics analysis. Generally, the region containing the domain helps to increase the success of heterologous protein expression, and the secondary structure of the domain is complex, which helps to increase the specificity of the generated antibody;

[0033] (2) Select the cDNA fragment of NPHP4 located at 859-1058aa as the immunizing antigen polypeptide, and perform whole gene synthesis according to the codon preference of Escherichia coli; see Figure 1 , the nucleotide and amino acid sequences of the corresponding fragment, and construct the NPHP4 prokaryotic expression vector using the prokaryotic cell recombinant expression vector pSUMO.

[0034] (3) Transform the constructed plasmid into BL21(DE3) competent cells, inoculate the kanamycin-resistant LB plate medium, and grow overnight; select 6 monoclonal colonies from the transformation plate and inoculate them into 3 mL of resistant liquid medium respectively; culture at 37 °C and 220 rpm until OD 600 = 0.5-0.6, add 0.5 mM IPTG and induce expression at 20 °C for 3.5 hours; centrifuge to collect the bacteria, ultrasonically disrupt them, and detect the expression by SDS-PAGE.

[0035] (4) Analysis of the small-scale expression results: The protein was expressed in both the supernatant and the inclusion body, and soluble expression and purification could be continued.

[0036] (5) Select the strain with good small-scale expression for large-scale expression; inoculate 60 μL of the strain into 200 mL of resistant medium, and culture overnight at 37 °C with 220 rpm; the next day, add fresh resistant medium to 800 mL, and culture for 1 - 2 h until OD 600 = 0.5 - 0.6; add 200 μL of 1 M IPTG (at 28 or 37 °C) to induce expression for 3.5 h; centrifuge to collect the bacteria at 4 °C (4000 rpm, 15 min), discard the supernatant, add 30 mL of PBST to suspend the bacteria, add PMSF with a final concentration of 1 mM, and ultrasonically disrupt at 200 W for 6 min under ice bath conditions; incubate on a shaker at 4 °C for 1 h; centrifuge at 4 °C and 8000 rpm for 15 min, take the supernatant, add 400 μL of nickel column and bind overnight at 4 °C; collect the nickel column (2000 rpm, 5 min), wash the agarose beads with 1 mL of 20 mM imidazole buffer three times to wash away the miscellaneous proteins; add 300 μL of 300 mM imidazole eluent, allow the eluent to fully bind to the agarose beads for 1 h, and centrifuge to collect the supernatant; add 300 μL of eluent to the agarose beads again, elute for 1 h, and centrifuge to collect the supernatant. Combine the two eluents into one tube; dialyze and change the solution with PBS buffer; identify the molecular weight, purity, and concentration of the protein by SDS-PAGE.

[0037] (6) Analysis of the large-scale expression results: The results are as Figure 2 shown. The target protein is expressed in the soluble supernatant, with a total amount of 3 mg and an expected molecular weight of 42 kDa, and can be used for immunization.

[0038] The immunization method is as follows: Mix the above NPHP4 antigen polypeptide with an equal volume of complete Freund's adjuvant to obtain the first mixture and inject it into the immunized rabbits (S0128 - 1 and S0128 - 2); after 28 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain the second mixture and inject it into the immunized rabbits (S0128 - 1 and S0128 - 2); after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain the third mixture and inject it into the immunized rabbits (S0128 - 1 and S0128 - 2); after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain the fourth mixture and inject it into the immunized rabbits (S0128 - 1 and S0128 - 2); after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain the fifth mixture and inject it into the immunized rabbits (S0128 - 1 and S0128 - 2); the injection method is multiple-point injection on the back; the immunization dose is: the first main injection is 400 μg of antigen per experimental rabbit, and the subsequent booster injection is 125 μg of antigen per experimental rabbit. Blood is collected after 7 days. If the blood collection is normal, collect the antiserum.

[0039] The antiserum was processed as follows: 1 mg of purified protein was covalently linked to a bromo-hydrogen-activated Sepharose 4B column by a conventional method to prepare an affinity purification column; 10 mL of antiserum was incubated with the affinity purification column overnight; pre-washed with pH 5.0 HCl to remove heterologous antibodies; eluted with 0.15 M glycine buffer at pH 2.5 and quickly neutralized with 10×PBS buffer to prepare an affinity-purified antibody; dialyzed and exchanged with PBS buffer; the concentration of the purified antibody was detected by the Bradford method; the titers of the antiserum and the affinity-purified antibody were detected by indirect ELISA; the ELISA titer detection results of the affinity-purified antibody were as follows: as Figure 3 shown. Figure 3 Among them, the dilution ratios of the antiserum to the purified antibody were 1:20K, 1:40K, 1:80K, 1:160K, 1:320K, 1:640K, and 1:1280K. The experimental results showed that both purified antibodies S0128-1 and S0128-2 could perform immunoaflinity reactions with the designed antigen to obtain concentrated polyclonal antibodies: the concentrations of the two polyclonal antibodies were 2.00 mg / mL. They had specificity and a relatively high titer, and thus the purified antibodies could be used for subsequent experiments.

[0040] Affinity purification result: The yield of the affinity-purified antibody was normal.

[0041] RabbitNo. Antiserum titer Titer of affinity-purified antibody Volume of affinity-purified antibody Concentration of affinity-purified antibody S0128-1 1280K 1280K 5mL 2.00mg / mL S0128-2 640K 1280K 5mL 2.00mg / mL

[0042] Using the polyclonal antibodies S0128-1 and S0128-2 as described above, the NPHP4 protein can be qualitatively and quantitatively analyzed by immunoblotting (WB). The steps are as follows:

[0043] a) Collect wild-type 21gr (WT) and nphp4 mutants in good condition and control their concentrations at 1×10 7 cells / mL; aliquot 1 mL per tube, centrifuge at 7000 rpm for 1 min, discard the supernatant, and freeze the cell pellet in an -80°C refrigerator for more than 24 h;

[0044] b) After the cell lysis buffer and 2×SDS Buffer thaw on ice, take out the samples and add 50 μL of cell lysis buffer to the samples on ice and pipette repeatedly to disperse the cell pellet, then add 50 μL of 2×SDS, pipette and mix well, and place in a 95°C metal bath. Take out after 10 min;

[0045] c) Place the prepared gel in the electrophoresis tank, pour in the electrophoresis buffer, remove the comb, and sequentially add 1.5 μL of protein Marker and 10 μL of the sample to the sample wells, then cover the electrophoresis tank lid, turn on the power supply, and perform vertical electrophoresis. The electrophoresis time for the stacking gel is 20 min and the voltage is 80 V; the electrophoresis time for the separating gel is 80 min and the voltage is 120 V;

[0046] d) Cut a suitable PVDF membrane according to the target band size, make marks, and activate it with methanol for 30 s for standby; cut the gel at a suitable position according to the protein Marker, place the gel on filter paper, cover the gel with the activated membrane, and use a roller to drive away air bubbles during the process to make the membrane and the gel fit tightly. Place the transfer device in the electrophoresis tank, connect the power supply, and perform membrane transfer on ice. The general membrane transfer conditions are 80 min, 100 V, and 350 mA;

[0047] e) After the membrane transfer is completed, transfer the membrane to the blocking solution and block it at room temperature for 1 h;

[0048] f) After blocking, put the prepared primary antibodies (S0128-1 and S0128-2 antibodies), 1:5000, in the antibody incubation box, place the corresponding membrane in the corresponding primary antibody, and incubate overnight at 4 °C;

[0049] g) Recover the primary antibody, wash the membrane 3 times with 1×TBST, 10 min each time. During the membrane washing process, pay attention not to dirty the membrane;

[0050] h) Incubate the secondary antibody: Add the corresponding secondary antibody (Goat-anti-rabbit HRP antibody), 1:5000, to the antibody incubation box. After incubating at room temperature for 1 - 2 h, recover the secondary antibody, and also wash the membrane three times with 1×TBST, 10 min each time, for the development reaction;

[0051] i) Add solution A and solution B to the developing box in equal proportions and mix well. Place the membrane to be developed in the developing box, repeatedly rinse the membrane with the mixed developing solution, and after rinsing evenly, place it on the imaging instrument for color development. After completion, save the color development results for analysis ( Figure 4 ). Figure 4 In the WB detection of wild-type (WT) and nphp4 in cell samples, the arrow indicates the position of the target NPHP4 protein, with a molecular weight of approximately 210 kDa; α-tubulin is used as a loading control. The results show that there is a band in the wild-type cell protein, indicating that the NPHP4 antibody can specifically recognize the endogenously expressed NPHP protein in cells, while in the mutant cell line nphp4, there is no corresponding band due to the absence of the NPHP4 protein, demonstrating the specificity of the antibody in detecting intracellular NPHP4 protein.

[0052] Using the polyclonal antibodies S0128-1 and S0128-2 as described above, the ciliary transition zone of Chlamydomonas reinhardtii can be labeled by immunofluorescence (IF). The steps are as follows:

[0053] A) Collect wild-type 21gr (WT) and NPHP4 gene mutant cell lines in good condition, and control their concentrations at 5×10 6cells / mL; Take 1 mL of each and transfer it to an EP tube. Centrifuge at 6000 rpm for 1 min at room temperature, and discard the supernatant.

[0054] B) Add approximately 20 - 50 μL of MT buffer to resuspend the cells to an appropriate concentration for loading.

[0055] C) Pipette approximately 20 μL of the above cell suspension onto an adhesion slide with pre-drawn wells, and let it stand for 20 min to allow the slide to adsorb the cells.

[0056] D) Take out methanol that has been pre-cooled to -20 °C from the refrigerator. After washing off the excess cell suspension on the slide with a vacuum pump, place the slide in methanol and keep it at -20 °C for 20 min.

[0057] E) Take out the slide. After the residual methanol has dried, add an appropriate amount of PBS Buffer to each well and wash 3 times, 5 min each time. During this process, the number of cells can be observed under a microscope. If there are a lot of cells, the unattached cells can be washed away by repeatedly pipetting. Note that each subsequent step should not dry the wells.

[0058] F) After washing 3 times, add an appropriate amount of immunohistochemical goat serum blocking solution to each well and block for 1 h.

[0059] G) Aspirate the blocking solution, and add 20 μL of diluted S0128 - 1 or S0128 - 2 antibody (1:500, mixed with ac - tubulin 1:200) to each well, and incubate overnight at 4 °C.

[0060] H) Aspirate the primary antibody (S0128 - 1 and S0128 - 2 antibodies) in the wells, and wash 3 times with PBS buffer containing 0.5% Tween - 20, 5 min each time. After aspirating the washing solution, add the corresponding diluted secondary antibody (Goat - anti - rabbit (AlexaFluor 488) antibody), 1:500, to each well and let it stand at 37 °C for 1 h.

[0061] I) Aspirate the secondary antibody (Goat - anti - rabbit (Alexa Fluor 488) antibody) in the wells, and wash 3 times with PBS buffer containing 0.5% Tween - 20, 5 min each time, then wash once with PBS buffer without Tween - 20, and finally wash once with ddH2O.

[0062] J) Aspirate ddH2O in the suction holes and add 10 μL of Fluoromount-G (containing DAPI) to each hole to prevent fluorescence quenching; finally, cover the slide and seal it, and fix the cover slip with nail polish. After drying in the dark for 2 h, fluorescence photography can be performed. Figure 5 ) Figure 5 In the IF detection of cell sample wild type 21gr (WT) and nphp4, the arrow indicates the subcellular localization of the target NPHP4 protein, which is located at the base of the cilium. Acetylated microtubule ac-tubulin is used as a cilium reference. The results show that the NPHP4 antibody can specifically recognize the NPHP4 protein in the cilia of cells (located at the base of each cilium, the dot at the arrow). However, in the mutant cell line nphp4, since there is no NPHP4 protein, there is no corresponding green dot signal at the base of the cilium, indicating that when using this antibody for IF, the detection of intracellular NPHP4 protein has high specificity and can be used to label the transition zone at the base of the cilium.

[0063] Through the above steps, the results show that the NPHP4 rabbit polyclonal antibody prepared by the present invention has the characteristics of high titer and good specificity. It can not only qualitatively and quantitatively analyze the NPHP4 protein in Chlamydomonas reinhardtii cells by immunoblotting WB method, but also label the NPHP4 protein by immunofluorescence IF method to explore the localization and interaction of each protein in the transition zone.

Claims

1. A rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein, characterized in that, The polyclonal antibody can specifically recognize the NPHP4 protein in the ciliary transition zone of Chlamydomonas reinhardtii; the amino acid sequence of the NPHP4 protein is shown in SEQ ID NO.1 in the sequence listing; the method for preparing the rabbit polyclonal antibody against the Chlamydomonas reinhardtii NPHP4 protein comprises the following steps: (1) Obtaining the amino acid sequence suitable as an antigen fragment in the NPHP4 protein by bioinformatics analysis, selecting the cDNA fragment of NPHP4 located at 859-1058 aa as the immunogenic antigen polypeptide, and performing total gene synthesis according to the codon preference of Escherichia coli; the cDNA fragment of NPHP4 located at 859-1058 aa is shown in SEQ ID NO.2 in the sequence listing, and the corresponding amino acid sequence is shown in SEQ ID NO.3 in the sequence listing; (2) Using the NPHP4 antigen cDNA fragment synthesized in step (1) and the prokaryotic cell recombinant expression vector pSUMO to construct the NPHP4 prokaryotic expression vector; (3) Transforming the NPHP4 prokaryotic expression vector constructed in step (2) into BL21(DE3) competent cells to obtain a recombinant expression strain; (4) Inducing the expression of the protein of the recombinant expression strain obtained in step (3) under certain conditions to obtain the recombinant expression tagged NPHP4 antigen polypeptide; (5) Using the NPHP4 antigen polypeptide induced and expressed in step (4) as an antigen for rabbit immunization treatment, obtaining the rabbit polyclonal antiserum against the NPHP4 protein by blood collection, and affinity purifying the antiserum to obtain the rabbit polyclonal antibody against the NPHP4 protein.

2. A method for preparing a rabbit polyclonal antibody against the Chlamydomonas reinhardtii NPHP4 protein as described in claim 1, characterized in that, Comprising the following steps: (1) Obtaining the amino acid sequence suitable as an antigen fragment in the NPHP4 protein by bioinformatics analysis, selecting the cDNA fragment of NPHP4 located at 859-1058 aa as the immunogenic antigen polypeptide, and performing total gene synthesis according to the codon preference of Escherichia coli; the cDNA fragment of NPHP4 located at 859-1058 aa is shown in SEQ ID NO.2 in the sequence listing, and the corresponding amino acid sequence is shown in SEQ ID NO.3 in the sequence listing; (2) Using the NPHP4 antigen cDNA fragment synthesized in step (1) and the prokaryotic cell recombinant expression vector pSUMO to construct the NPHP4 prokaryotic expression vector; (3) Transforming the NPHP4 prokaryotic expression vector constructed in step (2) into BL21(DE3) competent cells to obtain a recombinant expression strain; (4) Inducing the expression of the protein of the recombinant expression strain obtained in step (3) under certain conditions to obtain the recombinant expression tagged NPHP4 antigen polypeptide; (5) Using the NPHP4 antigen polypeptide induced and expressed in step (4) as an antigen for rabbit immunization treatment, obtaining the rabbit polyclonal antiserum against the NPHP4 protein by blood collection, and affinity purifying the antiserum to obtain the rabbit polyclonal antibody against the NPHP4 protein.

3. The preparation method of a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein according to claim 2, characterized in that, The immunization method is as follows: Mix the NPHP4 antigen polypeptide with an equal volume of complete Freund's adjuvant to obtain a first mixture and inject it into an immunized rabbit; after 28 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a second mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a third mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fourth mixture and inject it into the immunized rabbit; after 7 days, mix the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fifth mixture and inject it into the immunized rabbit; the injection method is multi-point injection on the back; the immunization dose is: the first main injection is 400 μg antigen per experimental rabbit, and the second to fifth injections are all 125 μg antigen per experimental rabbit.

4. Application of the rabbit polyclonal antibody against the Chlamydomonas reinhardtii NPHP4 protein as described in claim 1 in detecting the Chlamydomonas reinhardtii NPHP4 protein.

5. The application according to claim 4, characterized in that, Detect the Chlamydomonas reinhardtii NPHP4 protein by immunofluorescence IF method or immunoblotting WB method.

6. The application according to claim 5, wherein The steps for detecting the Chlamydomonas reinhardtii NPHP4 protein by immunofluorescence IF method are as follows: After fixing the Chlamydomonas reinhardtii cells by methanol penetration method, wash them 3 times with PBS buffer for 5 minutes each time; block them at room temperature with a blocking solution for 1 h, incubate them overnight at 4 °C with the purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:500; wash them 3 times with PBS buffer containing 0.5% Tween-20 for 5 minutes each time; incubate them with a fluorescence-labeled secondary antibody corresponding to the wavelength and species at 37 °C for 1 h, then wash them 3 times with PBS buffer containing 0.5% Tween-20 for 5 minutes each time, then wash them once with PBS buffer without Tween-20, and finally wash them once with ddH2O. After mounting the slides, observe the localization of the NPHP4 protein in the ciliary transition zone under a fluorescence microscope, take pictures, draw graphs and analyze.

7. The application according to claim 5, wherein The steps for detecting the NPHP4 protein of Chlamydomonas reinhardtii by Western Blot (WB) are as follows: The Chlamydomonas cell samples frozen at -80 °C are repeatedly pipetted with cell lysate, and SDS buffer is added and mixed well, followed by incubation in a metal bath at 95 °C for 10 min; Subsequently, SDS-PAGE electrophoresis is carried out, with the stacking gel electrophoresis time being 20 min and the voltage being 80 V; The separating gel electrophoresis time is 80 min and the voltage is 120 V; Transfer membrane is carried out under the conditions of 80 min and 100 V; The PVDF membrane is blocked at room temperature for 1 h and then incubated overnight at 4 °C with the purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:5000; The primary antibody is recovered, and the membrane is washed 3 times with 1×TBST, 10 min each time; The corresponding secondary antibody is incubated at room temperature for 1 h, the secondary antibody is recovered, and the membrane is washed 3 times with 1×TBST, and then developed to qualitatively and quantitatively analyze the NPHP4 protein.

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