Rabbit polyclonal antibody of chlamydomonas reinhardtii NPHP4 protein as well as preparation method and application of rabbit polyclonal antibody

Abstract

The invention discloses a rabbit polyclonal antibody of chlamydomonas reinhardtii NPHP4 protein as well as a preparation method and application of the rabbit polyclonal antibody. The amino acid sequence of the NPHP4 protein is shown as SEQ ID NO. 1. The preparation method comprises the following steps: analyzing and designing a chlamydomonas reinhardtii NPHP4 protein antigen epitope, and selecting 859-1058aa amino acid fragments to construct a prokaryotic cell recombinant expression vector; transferring the recombinant expression vector into a prokaryotic expression cell to obtain a recombinant expression strain, and inducing antigen protein expression; carrying out rabbit immunization by taking the purified antigen protein as an immunogen to obtain NPHP4 protein rabbit polyclonal antiserum, and further carrying out purification treatment on the NPHP4 protein rabbit polyclonal antiserum; the purified polyclonal antibody can be used for labeling verification of a cilia transition region of Chlamydomonas reinhardtii by an IF method, and the result proves that the polyclonal antibody can be used for specific fluorescence labeling of the cilia transition region; and the chlamydomonas reinhardtii NPHP4 protein can also be qualitatively and quantitatively analyzed by an immunoblotting WB method.

Description

Technical Field

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

[0002] Chlamydomonas reinhardtii is the most commonly used model organism in laboratory research. This single-cell eukaryotic green algae is called "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 diverse functions in cell biology. The complex ultrastructure of cilia has evolved in conjunction with their multiple roles, including motility and sensing. Despite the diverse functions, there is a universally conserved structural core, consisting of the microtubule-based axoneme with nine-axis symmetry. The architecture and symmetry of the axoneme are determined by the mother centriole or basal body from which it emanates. After the basal body is properly anchored to the plasma membrane, the developing cilium must establish a selective barrier to sequester molecules that are essential for its construction, as it lacks an autonomous translational machinery. This region, which connects the basal body and the nascent cilium, is known as 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 critical ciliary zone. Notably, the importance of the TZ in scientific research has been highlighted by the increasing number of cilium-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. It 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 ciliary and cytoplasmic compartments. The various phenotypic consequences of NPHP4 mutations in humans and other organisms are likely due to protein mislocalization caused by defects in the transition zone. In order to solve the scientific problem of studying the important function of NPHP4 in cilia, it is particularly important to seek an antibody that can specifically recognize the NPHP4 protein in Chlamydomonas reinhardtii and its preparation method. Summary of the invention

[0005] The invention provides a rabbit polyclonal antibody of Chlamydomonas reinhardtii NPHP4 protein and a preparation method and application thereof. The polyclonal antibody can specifically identify the Chlamydomonas reinhardtii NPHP4 protein and can be used to study the positioning marker of the ciliary transition zone. In the application in the field of biological research, the NPHP4 protein in the Chlamydomonas reinhardtii cell can be qualitatively and quantitatively analyzed by immunoblotting WB method, and the NPHP4 protein can be labeled by immunofluorescence IF method to explore the positioning and mutual relationship of various proteins in the transition zone, thereby laying an application foundation for the study of the ciliary phenotype change of Chlamydomonas reinhardtii as a model organism and the ciliary gating transport and other related regulatory mechanisms.

[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 shown in the sequence table SEQ ID NO.1.

[0007] The present invention also provides a method for preparing a rabbit polyclonal antibody against the Chlamydomonas reinhardtii NPHP4 protein, comprising the following steps:

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

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

[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 the recombinant expression tag NPHP4 antigen polypeptide;

[0012] (5) using the NPHP4 antigen polypeptide induced to be expressed in step (4) as an antigen to perform rabbit immunization, obtaining rabbit polyclonal antiserum against NPHP4 protein by blood collection, affinity purifying the antiserum, and obtaining rabbit polyclonal antibody against NPHP4 protein.

[0013] Further, the immunization treatment method is: mixing the NPHP4 antigen polypeptide with an equal volume of complete Freund's adjuvant to obtain a first mixed solution and injecting it into the immunized rabbit; 28 days later, mixing the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a second mixed solution and injecting it into the immunized rabbit; 7 days later, mixing the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a third mixed solution and injecting it into the immunized rabbit; 7 days later, mixing the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fourth mixed solution and injecting it into the immunized rabbit; 7 days later, mixing the NPHP4 antigen polypeptide with an equal volume of incomplete Freund's adjuvant to obtain a fifth mixed solution and injecting it into the immunized rabbit; the injection method is multi-point injection on the back; the immunization amount 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 Chlamydomonas reinhardtii NPHP4 protein in detecting the Chlamydomonas reinhardtii NPHP4 protein.

[0015] Furthermore, the Chlamydomonas reinhardtii NPHP4 protein was detected by immunofluorescence IF method or immunoblotting WB method.

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

[0017] After the Chlamydomonas reinhardtii cells were fixed by methanol permeation method, they were washed 3 times with PBS buffer for 5 minutes each time. They were blocked with blocking solution at room temperature for 1 hour, and incubated with purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:500 at 4°C overnight; washed 3 times with PBS buffer containing 0.5% Tween-20 for 5 minutes each time; incubated with fluorescent labeled secondary antibodies of corresponding wavelength and species at 37°C for 1 hour, and then washed 3 times with PBS buffer containing 0.5% Tween-20 for 5 minutes each time, and then washed once with PBS buffer without Tween-20, and finally washed once with ddH2O. After sealing, the localization of NPHP4 protein in the ciliary transition zone was observed under a fluorescence microscope, and photographed, mapped and analyzed.

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

[0019] The Chlamydomonas cell samples frozen at -80°C were repeatedly blown with cell lysis buffer, and SDS buffer was added and mixed, and then placed in a 95°C metal bath for 10 minutes; then SDS-PAGE electrophoresis was performed, wherein the concentrated gel electrophoresis time was 20 minutes and the voltage was 80V; the separation gel electrophoresis time was 80 minutes and the voltage was 120V; the membrane was transferred, the conditions were 80 minutes and 100V; the PVDF membrane was blocked at room temperature for 1 hour, and then incubated with purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:5000 at 4°C overnight; the primary antibody was recovered, and the membrane was washed 3 times with 1×TBST, each time for 10 minutes; the corresponding secondary antibody was used for incubation at room temperature for 1 hour, the secondary antibody was recovered, and the membrane was washed 3 times with 1×TBST, and then developed, and the NPHP4 protein was qualitatively and quantitatively analyzed.

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

[0021] The present invention utilizes the Chlamydomonas reinhardtii NPHP4 expression vector to immunize New Zealand white rabbits to prepare polyclonal antibodies, and the prepared polyclonal antibodies can specifically identify the Chlamydomonas reinhardtii NPHP4 protein, and are characterized by high titer and good specificity. In the application of biological research, the NPHP4 protein in the Chlamydomonas reinhardtii cell can be qualitatively and quantitatively analyzed by immunoblotting WB method, and the NPHP4 protein can be labeled by immunofluorescence IF method to explore the location and mutual relationship of each protein in the transition zone, thereby laying an application foundation for the in-depth exploration of the ciliary phenotype changes, ciliary gated transport and ciliary disease pathological mechanism of Chlamydomonas reinhardtii as a model organism. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 This is the expression diagram of NPHP4 antigen protein detected by SDS-PAGE;

[0024] Figure 3 This is the data diagram of ELISA titer test of NPHP4 antiserum and affinity purified antibody;

[0025] Figure 4 This is a Western blot detection image of NPHP4 protein by Western blot;

[0026] Figure 5 This is an immunofluorescence IF fluorescence detection image of the specific localization of NPHP4 in the ciliary transition zone of Chlamydomonas reinhardtii (WT: wild type 21gr; nphp4: NPHP4 gene mutant cell line). DETAILED DESCRIPTION

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

[0028] The immunized rabbits used in the following examples are 2-3 month old New Zealand white rabbits, and the antibodies are prepared in laboratory SPF grade experimental animals, thereby preparing immune sera (S0128-1 and S0128-2) with high titer and strong specificity.

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

[0030] The wild-type 21gr (WT) and nphp4 mutant used in the following examples are from laboratory-preserved algae species and can also be obtained from the American Chlamydomonas Germplasm Resource Center.

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

[0032] (1) Using bioinformatics analysis to obtain amino acid sequences in NPHP4 protein that are suitable as antigen fragments, generally, regions containing structural domains help increase the success of exogenous protein expression, and complex secondary structures of structural domains help increase the specificity of the generated antibodies;

[0033] (2) The cDNA fragment of NPHP4 located at 859-1058aa was selected as the immune antigen polypeptide, and the whole gene was synthesized according to the codon preference of Escherichia coli; Figure 1 , the corresponding nucleotide and amino acid sequences of the fragment, and the NPHP4 prokaryotic expression vector was constructed using the prokaryotic cell recombinant expression vector pSUMO.

[0034] (3) The constructed plasmid was transformed into BL21(DE3) competent cells, inoculated with kanamycin-resistant LB plate medium, and grown overnight; six single clones from the transformation plate were selected and inoculated with 3 mL of resistant liquid medium respectively; cultured at 37°C and 220 rpm until OD 600 =0.5-0.6, add 0.5mM IPTG at 20℃ to induce expression for 3.5 hours; collect the bacteria by centrifugation, disrupt by ultrasonication, and detect the expression by SDS-PAGE.

[0035] (4) Analysis of sample expression results: The protein is expressed in both the supernatant and inclusion bodies, and soluble expression purification can be continued.

[0036] (5) Select the strain with good expression in the small sample for large sample expression; inoculate 60 μL of the strain into 200 mL of resistance culture medium and culture overnight at 37°C and 220 rpm; the next day, add fresh resistance culture medium to 800 mL and culture for 1-2 h until OD 600 =0.5-0.6; add 200 μL of 1M IPTG (28 or 37°C) to induce expression for 3.5h; collect the bacteria by centrifugation at 4°C (4000rpm, 15min), discard the supernatant, add 30mL PBST to suspend the bacteria, add PMSF with a final concentration of 1mM, and break it with ultrasound at 200W for 6min under ice bath conditions; incubate on a shaker at 4°C for 1h; centrifuge at 4°C and 8000rpm for 15min, take the supernatant, add 400 μL nickel column and bind overnight at 4°C; collect the nickel column (2000rpm, 5min), wash the agarose ball with 1mL of 20mM imidazole buffer, wash three times to wash away foreign proteins; add 300 μL 300mM imidazole eluent was used to allow the eluent to fully combine with the agarose beads for 1 hour, and the supernatant was collected by centrifugation; 300μL of eluent was added to the agarose beads again, and the eluent was eluted for 1 hour, and the supernatant was collected by centrifugation. The two eluents were combined into one tube; the PBS buffer was dialyzed for replacement; and the protein molecular weight, purity and concentration were identified by SDS-PAGE.

[0037] (6) Analysis of expression results of large samples: Figure 2 As shown, the target protein is expressed in soluble supernatant, with a total amount of 3 mg and an estimated molecular weight of 42 kDa, which can be used for immunological treatment.

[0038] The immunization treatment method is as follows: the NPHP4 antigen polypeptide is mixed with an equal volume of complete Freund's adjuvant to obtain a first mixed solution and injected into the immunized rabbits (S0128-1 and S0128-2); 28 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a second mixed solution and injected into the immunized rabbits (S0128-1 and S0128-2); 7 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a third mixed solution and injected into the immunized rabbits (S0128-1 and S0128-2). 28-2); 7 days later, the NPHP4 antigen polypeptide was mixed with an equal volume of incomplete Freund's adjuvant to obtain a fourth mixed solution and injected into the immune rabbits (S0128-1 and S0128-2); 7 days later, the NPHP4 antigen polypeptide was mixed with an equal volume of incomplete Freund's adjuvant to obtain a fifth mixed solution and injected into the immune rabbits (S0128-1 and S0128-2); the injection method was multi-point injection on the back; the immunization dose was: 400 μg antigen / rabbit for the first main injection, and 125 μg antigen / rabbit for subsequent booster injection. Blood was collected 7 days later, and the blood collection was normal, and antiserum was collected.

[0039] The antiserum was treated as follows: 1 mg of purified protein was covalently linked to a Sepharose 4B column activated with hydrogen bromide by conventional methods to prepare an affinity purification column; 10 mL of antiserum was incubated with the affinity purification column overnight; pH 5.0 HCl was pre-washed to remove impurities; pH 2.5, 0.15 M glycine buffer was eluted, and 10× PBS buffer was quickly neutralized to prepare affinity purified antibodies; PBS buffer was dialyzed for replacement; the concentration of purified antibodies was detected by Bradford method; the titer of antiserum and affinity purified antibodies was detected by indirect ELISA; the titer of affinity purified antibodies ELISA test results were as follows: Figure 3 shown. Figure 3 The dilution ratio of antiserum to purified antibody was 1:20K, 1:40K, 1:80K, 1:160K, 1:320K, 1:640K, 1:1280K. The experimental results showed that the purified antibodies S0128-1 and S0128-2 could react with the designed antigens to obtain concentrated polyclonal antibodies: the concentration of the two polyclonal antibodies was 2.00 mg / mL. They have specificity and high titer, and the purified antibodies can be used for subsequent experiments.

[0040] Affinity purification results: The yield of affinity purified antibodies was normal.

[0041] Rabbit No. Antiserum titer Affinity Purified Antibody Titer Affinity Purified Antibody Volume Affinity Purification Antibody Concentration 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 described above, the NPHP4 protein can be qualitatively and quantitatively analyzed by immunoblotting (WB), as follows:

[0043] a) Collect wild-type 21gr (WT) and nphp4 mutants in good condition and control their concentrations at 1×10 7 / mL; 1mL was dispensed into each tube, centrifuged at 7000rpm for 1min, the supernatant was removed, and the cell pellet was frozen in a -80℃ refrigerator for more than 24h;

[0044] b) After the cell lysis buffer and 2×SDS Buffer are thawed on ice, take out the sample and add 50 μL of cell lysis buffer on ice, pipette repeatedly to disperse the cell precipitate, then add 50 μL of 2×SDS, pipette and mix well, place in a 95°C metal bath, and take out after 10 minutes;

[0045] c) Place the prepared gel in the electrophoresis tank, pour in the electrophoresis solution, remove the comb, add 1.5 μL protein marker and 10 μL sample to the spotting wells in sequence, cover the electrophoresis tank, turn on the power supply, and perform vertical electrophoresis. The electrophoresis time for the concentrated gel is 20 minutes, the voltage is 80V; the electrophoresis time for the separation gel is 80 minutes, the voltage is 120V;

[0046] d) Cut the appropriate PVDF membrane according to the size of the target band, mark it, and activate it with methanol for 30 seconds for later use; select the appropriate position according to the protein marker to cut the gel, put the gel on the filter paper, and cover the gel with the activated membrane. During the process, use a roller to drive away the bubbles so that the membrane and gel fit tightly. Put the transfer device into the electrophoresis tank, turn on the power, and transfer the membrane on ice. The transfer conditions are generally 80 minutes, 100V, and 350mA;

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

[0048] f) After blocking, the prepared primary antibodies (S0128-1 and S0128-2 antibodies) were placed in the antibody incubation box at 1:5000, and the corresponding membranes were placed in the corresponding primary antibodies and incubated overnight at 4°C;

[0049] g) Recover the primary antibody and wash the membrane three times with 1×TBST, 10 min each time. Be careful not to dirty the membrane during the washing process;

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

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

[0052] Using the polyclonal antibodies S0128-1 and S0128-2 described above, immunofluorescence (IF) can be used to label the ciliary transition zone of Chlamydomonas reinhardtii 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 6 / mL; take 1mL of each into EP tube, centrifuge at 6000rpm for 1min at room temperature, and discard the supernatant;

[0054] B) Add about 20-50 μL MTbuffer to resuspend the cells to an appropriate cell concentration for loading;

[0055] C) Take about 20 μL of the above cell solution and drop it onto the adhesive slide with holes drawn on it, and let it stand for 20 minutes to allow the slide to absorb the cells;

[0056] D) Take out the methanol that has been pre-cooled to -20℃ from the refrigerator, use a vacuum pump to wash away the excess cell fluid on the slide, then put it into methanol, and then keep it at -20℃ for 20 minutes;

[0057] E) Take out the slide, wait for the residual methanol to dry, add appropriate amount of PBS buffer to each well, wash 3 times, 5 minutes each time; during this process, the number of cells can be observed under a microscope. If there are many cells, the unabsorbed cells can be washed away by repeated blowing with a pipette. Note that each subsequent step must not dry out the wells;

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

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

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

[0061] I) aspirate the secondary antibody (Goat-anti-rabbit (Alexa Fluor 488) antibody) in the well, wash three times with PBS buffer containing 0.5% Tween-20, each washing time is 5 minutes, then wash once with PBS buffer without Tween-20, and finally wash once with ddH2O;

[0062] J) Aspirate the ddH2O in the wells and add 10 μL Fluoromount-G (containing DAPI) to each well to prevent fluorescence quenching; finally, attach a cover slip to seal the slide and fix the cover slip with nail polish. After drying in the dark for 2 hours, fluorescence photography can be performed ( Figure 5 ). Figure 5 In the figure, IF detection of cell samples wild-type 21gr (WT) and nphp4, the arrow is the subcellular localization of the target NPHP4 protein, located at the base of the cilia, and the acetylated microtubule ac-tubulin is used as a reference for the cilia. The results show that the NPHP4 antibody can specifically recognize the NPHP4 protein in the cell cilia (located at the base of each cilia, the point at the arrow), while in the mutant cell line nphp4, due to the absence of NPHP4 protein, there is no corresponding green dot signal at the base of the cilia, indicating that when using this antibody for IF, the detection of intracellular NPHP4 protein is highly specific and can be used to mark the transition zone at the base of the cilia.

[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 perform qualitative and quantitative analysis of 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 relationship 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 the sequence table SEQ ID NO.

1.

2. A method for preparing a rabbit polyclonal antibody against the Chlamydomonas reinhardtii NPHP4 protein according to claim 1, characterized in that: The following steps are involved: (1) using bioinformatics analysis to obtain an amino acid sequence in the NPHP4 protein that is suitable for being used as an antigen fragment, selecting a cDNA fragment of NPHP4 located at 859-1058aa as an immune antigen polypeptide, and performing full gene synthesis according to the codon preference of Escherichia coli; the cDNA fragment of NPHP4 located at 859-1058aa is shown in the sequence listing SEQ ID NO.2, and the corresponding amino acid sequence is shown in the sequence listing SEQ ID NO.3; (2) constructing a NPHP4 prokaryotic expression vector using the NPHP4 antigen cDNA fragment synthesized in step (1) and the prokaryotic cell recombinant expression vector pSUMO; (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 tag NPHP4 antigen polypeptide; (5) using the NPHP4 antigen polypeptide induced to be expressed in step (4) as an antigen to perform rabbit immunization, obtaining rabbit polyclonal antiserum against NPHP4 protein by blood collection, affinity purifying the antiserum, and obtaining rabbit polyclonal antibody against NPHP4 protein.

3. The method for preparing a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein according to claim 2, characterized in that: The immunization treatment method is as follows: the NPHP4 antigen polypeptide is mixed with an equal volume of complete Freund's adjuvant to obtain a first mixed solution and the mixture is injected into the immunized rabbit; 28 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a second mixed solution and the mixture is injected into the immunized rabbit; 7 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a third mixed solution and the mixture is injected into the immunized rabbit; 7 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a fourth mixed solution and the mixture is injected into the immunized rabbit; 7 days later, the NPHP4 antigen polypeptide is mixed with an equal volume of incomplete Freund's adjuvant to obtain a fifth mixed solution and the mixture is injected into the immunized rabbit; the injection method is multi-point injection at the back; the immunization amount is as follows: 400 μg antigen / experimental rabbit is injected in the first main injection, and 125 μg antigen / experimental rabbit is injected in the second to fifth injections.

4. Use of a rabbit polyclonal antibody against Chlamydomonas reinhardtii NPHP4 protein as claimed in claim 1 in detecting Chlamydomonas reinhardtii NPHP4 protein.

5. The use according to claim 4, characterized in that: The Chlamydomonas reinhardtii NPHP4 protein was detected by immunofluorescence IF or immunoblotting WB.

6. The use according to claim 5, characterized in that: The steps for detecting Chlamydomonas reinhardtii NPHP4 protein by immunofluorescence IF method are as follows: After the Chlamydomonas reinhardtii cells were fixed by methanol permeation method, they were washed 3 times with PBS buffer, each time for 5 minutes; they were blocked with blocking solution at room temperature for 1 hour, and incubated with purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:500 at 4°C overnight; they were washed 3 times with PBS buffer containing 0.5% Tween-20, each time for 5 minutes; they were incubated with fluorescent-labeled secondary antibodies of corresponding wavelengths and species at 37°C for 1 hour, and then washed 3 times with PBS buffer containing 0.5% Tween-20, each time for 5 minutes, and then washed once with PBS buffer without Tween-20, and finally washed once with ddH2O. After sealing, the localization of NPHP4 protein in the ciliary transition zone was observed under a fluorescence microscope, and photographed, mapped and analyzed.

7. The use according to claim 5, characterized in that: The steps for detecting Chlamydomonas reinhardtii NPHP4 protein by immunoblotting WB method are as follows: The Chlamydomonas cell samples frozen at -80°C were repeatedly blown with cell lysis buffer, and SDS buffer was added and mixed, and then placed in a 95°C metal bath for 10 minutes; then SDS-PAGE electrophoresis was performed, wherein the concentrated gel electrophoresis time was 20 minutes and the voltage was 80V; the separation gel electrophoresis time was 80 minutes and the voltage was 120V; the membrane was transferred, the conditions were 80 minutes and 100V; the PVDF membrane was blocked at room temperature for 1 hour, and then incubated with purified NPHP4 rabbit polyclonal antibody at a dilution ratio of 1:5000 at 4°C overnight; the primary antibody was recovered, and the membrane was washed 3 times with 1×TBST, each time for 10 minutes; the corresponding secondary antibody was used for incubation at room temperature for 1 hour, the secondary antibody was recovered, and the membrane was washed 3 times with 1×TBST, and then developed, and the NPHP4 protein was qualitatively and quantitatively analyzed.

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