Application of paralichthys olivaceus complement regulation protein PoCFH-CCP2

Abstract

The invention relates to the technical field of molecular biology, in particular to application of paralichthys olivaceus complement regulation protein PoCFH-CCP2. The invention relates to an application of paralichthys olivaceus complement regulation protein PoCFH-CCP2 in preparation of immune drugs. The paralichthys olivaceus complement regulation protein PoCFH-CCP2 adopted by the invention comes from a paralichthys olivaceus complement factor H, can be combined with edwardsiella tarda, can occupy a key binding site on the edwardsiella tarda, effectively inhibits recruitment of CFH and CFI, and can be used as an immune drug for preventing and treating Edwardsiella disease of fish.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to an application of a complement regulatory protein PoCFH-CCP2 of flounder. Background Art

[0002] The complement system plays a crucial role in immune defense and maintaining immune homeostasis. The complement system has three activation pathways: the classical pathway, the lectin pathway, and the alternative pathway. The membrane attack complex produced by complement activation has the ability to kill pathogens. To maintain precise regulation of the complement system, complement contains a comprehensive regulatory mechanism to prevent autologous tissue damage caused by excessive activation. Factor H (CFH), a soluble complement regulatory molecule, participates in the factor I (CFI)-mediated cleavage of C3b within the regulatory system, ultimately inactivating the C3 convertase and preventing abnormal complement expansion. Edwardsiella tarda (E. piscicidal) can recruit CFH from fish to its surface, which in turn recruits the hydrolytically active CFI. CFI degrades C3b bound to the bacterial surface, allowing it to evade complement toxicity in fish serum and spread systemically within the fish. Currently, research on the application of complement regulatory proteins focuses primarily on the serine protease domain of complement factor I (CFI), such as PoCFI-Tryp. Prior art (CN201911158044) shows that PoCFI-Tryp can inhibit the growth of various bacteria (such as Edwardsiella tarda, Vibrio, etc.) through its enzymatic activity, but its mechanism of action depends on Ca 2 +, and only indirectly inhibits bacteria by delaying bacterial growth, failing to directly block the pathogen's immune escape pathway. Furthermore, the broad spectrum of PoCFI-Tryp may result in less pronounced effects against specific pathogens (such as Edwardsiella tarda), and there is a lack of direct evidence of its ability to affect host survival or tissue protection.

[0003] In summary, existing research on the fish complement system faces significant limitations: First, the regulatory mechanisms of the complement system vary significantly across species, and a comprehensive map of the fish complement system has yet to be established. Second, the tissue distribution characteristics of complement effector molecules lack systematic research. Third, complement activity is significantly influenced by external factors, and its actual biological effects are highly variable. Due to these factors, the mechanisms of action and the breadth of complement systems in individual fish cannot be determined based on known reports. Therefore, further research is needed to address the gaps in the direct antibacterial application of complement regulatory proteins. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of flounder complement regulatory protein PoCFH-CCP2.

[0005] The technical solution adopted to achieve the purpose of the present invention is:

[0006] An application of the complement regulatory protein PoCFH-CCP2 of flounder, and an application of the complement regulatory protein PoCFH-CCP2 of flounder in the preparation of immune drugs.

[0007] The amino acid sequence of the flounder complement regulatory protein PoCFH-CCP2 is SEQ ID No. 1.

[0008] The primer pair for amplifying and obtaining the amino acid sequence of the complement regulatory protein PoCFH-CCP2 of flounder is:

[0009] The sequence of F1 is: 5′-GATATCATGGAACTGATAACATGTAAACTGAG-3′;

[0010] The sequence of R1 is: 5'-GATATCCCTGCACATGTACTGGACTTGT-3'.

[0011] The invention relates to an application of the flounder complement regulatory protein PoCFH-CCP2 in the preparation of immune drugs for preventing and treating Edwardsiella tarda.

[0012] The application of the flounder complement regulatory protein PoCFH-CCP2 in preventing and treating Edwardsiella tarda can increase the survival rate of the subjects to more than 60%.

[0013] An immune drug for preventing and treating Edwardsiella tarda, comprising the complement regulatory protein PoCFH-CCP2 of flounder.

[0014] The flounder complement regulatory protein PoCFH-CCP2 is amplified by PCR using flounder cDNA as a template and primers. The PCR product is connected to an expression vector to obtain a recombinant plasmid, which is transformed into a competent microorganism and induced and purified to obtain a recombinant protein, namely the flounder complement regulatory protein PoCFH-CCP2.

[0015] Further,

[0016] Step 1, construction of the expression vector pEtPoCFH-CCP2: using flounder cDNA as a template, PCR amplification was performed using primers, the PCR product was purified, and then ligated with the plasmid T-Simple. The ligated product was transformed into Escherichia coli, cultured on LB medium containing kanamycin for 8-12 hours, and the transformants were screened to extract the plasmid to obtain a recombinant plasmid; the recombinant plasmid was digested with EcoRV, the target fragment was recovered, ligated to pET259, and transformed into Escherichia coli. The culture was carried out on LB medium containing kanamycin for 18-24 hours, and the transformants were screened to extract the plasmid, which is the expression vector pEtPoCFH-CCP2;

[0017] Step 2: The plasmid pEtPoCFH-CCP2 of step 1 above was transformed into Escherichia coli BL21 (DE3), cultured in LB medium containing kanamycin, and the transformants were screened, namely BL21 / pEtPoCFH-CCP2, which was obtained after culture.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The Japanese flounder complement regulatory protein PoCFH-CCP2 employed in the present invention can bind to Edwardsiella tarda and, by preventing the recruitment of CFH and CFI to the bacterial surface, cause the outflow of bacterial solubles in the serum, significantly reducing the survival rate of Edwardsiella tarda in the serum. Its bactericidal action differs from that of other complement factors in that it occupies the CFH binding site of Edwardsiella tarda in fish, preventing the pathogen from escaping the complement's killing ability, thereby achieving a bactericidal effect. The Japanese flounder complement regulatory protein PoCFH-CCP2 of the present invention does not require the auxiliary action of other proteins in the serum and has an independent bactericidal effect, with a more direct and efficient action, simpler application conditions, and a wider range of applicability.

[0020] 2. The Japanese flounder complement regulatory protein PoCFH-CCP2 of the present invention specifically targets Edwardsiella tarda, has a significant immune effect, and can be used as an immune drug to protect Japanese flounder tissues and fish from infection with Edwardsiella tarda. Its preparation process is simple and poses no safety risks to the environment or fish. The obtained protein has application potential in antibacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The ability of the flounder complement regulatory protein PoCFH-CCP2 of the present invention to bind to Edwardsiella tarda, wherein, Figure 1 A is a graph showing the binding ability of the complement regulatory protein PoCFH-CCP2 of flounder to Edwardsiella detected by ELISA, B is a graph showing the binding ability of the complement regulatory protein PoCFH-CCP2 of flounder to Edwardsiella detected by Western blot, and C is a graph showing the binding ability of the complement regulatory protein PoCFH-CCP2 of flounder to Edwardsiella detected by immunofluorescence.

[0022] Figure 2 This is an analysis of the effect of the Japanese flounder complement regulatory protein PoCFH-CCP2 on complement activation, wherein: Figure 2A is a diagram showing the effect of the complement regulatory protein PoCFH-CCP2 of Japanese flounder on the bactericidal ability of Japanese flounder serum. B is a diagram showing the recruitment of CFH and CFI on the bacterial surface after serum-killed Japanese flounder complement regulatory protein PoCFH-CCP2 pretreatment by Western blot. C is a diagram showing the destruction of bacterial cell membrane and the outflow of internalized substances after serum-killed Japanese flounder complement regulatory protein PoCFH-CCP2 pretreatment observed by transmission electron microscopy.

[0023] Figure 3 The present invention shows the effects of the complement regulatory protein PoCFH-CCP2 of Japanese flounder on fish infected with Edwardsiella. A is a diagram showing the protection of Japanese flounder tissues by the complement regulatory protein PoCFH-CCP2 detected by tissue invasion method, B is a diagram showing the clinical manifestations of Japanese flounder treated with the complement regulatory protein PoCFH-CCP2 observed by characterization observation method, C is a diagram showing the protection of Japanese flounder tissues by the complement regulatory protein PoCFH-CCP2 observed by pathological section, and D is a diagram showing the lethality analysis of the protection of Japanese flounder by the complement regulatory protein PoCFH-CCP2.

[0024] Figure 4 The comparative examples of the present invention provide the ability of PoCFH-CCP1 and PoCFH-CCP3 to bind to Edwardsiella tarda, respectively. Figure A shows the binding ability of the complement regulatory proteins PoCFH-CCP1 and PoCFH-CCP3 of Japanese flounder to Edwardsiella by ELISA, Figure B shows the binding ability of the complement regulatory proteins PoCFH-CCP1 and PoCFH-CCP3 of Japanese flounder to Edwardsiella by Western blot, and Figure C shows the binding ability of the complement regulatory proteins PoCFH-CCP1 and PoCFH-CCP3 of Japanese flounder to Edwardsiella by immunofluorescence. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] The following examples describe that Edwardsiella tarda was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on January 9, 2008, with the deposit number CGMCC No. 2330, and was obtained as a gift from Professor Sun Li of the Institute of Oceanology, Chinese Academy of Sciences.

[0027] Example 1

[0028] Preparation of complement regulatory protein PoCFH-CCP2 from flounder

[0029] (1) Construction of the expression plasmid pEtPoCFH-CCP2 for the complement regulatory protein PoCFH-CCP2 of flounder

[0030] The flounder complement protein PoCFH-CCP2 of the present invention is obtained through a prokaryotic protein expression system, and its sequence is the amino acid sequence in SEQ ID No. 1 in the sequence table.

[0031] The sequence listing SEQ ID No. 1 is:

[0032] ELITCKLSLPPVYGTEYEPATVNVFSPGGTVRVTCGDRHWISDPLETSAVVSCKSDGQWSLRPICKEVTCSNRRHYTLRQWSVYYGEQKKFGDRVTYQCKDGYKSTGGATSAVCTRGGWKPDPLCQEITCDRHE IPNTDIVNSKYTYKYRERANYVCKNGYKGQFHLTCDRNGWSRPPQCKEITCDRHEIPNTDIVNSKYTYKYREQVNYVCKNGYKGQFHLTCDEYGWSGEAQCKESQCEHQDISDADIVSNDRESYSHNEQVQYMCR

[0033] Using flounder cDNA as a template, PCR amplification was performed with primers F1 and R1. The PCR product was purified and ligated with plasmid T-Simple. After transformation into Escherichia coli, the cells were cultured on LB medium containing kanamycin for 8-12 hours, and transformants were screened to extract the plasmid to obtain a recombinant plasmid. The recombinant plasmid was digested with EcoRV, the target fragment was recovered, ligated to a pET259 vector, and transformed into Escherichia coli. The cells were cultured on LB medium containing kanamycin for 18-24 hours, and transformants were screened to extract the plasmid, which is the expression vector pEtPoCFH-CCP2.

[0034] The LB composition is calculated by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride, and 97.5% distilled water.

[0035] The primers F1 and R1 are respectively:

[0036] F1, 5'-GATATCATGGAACTGATAACATGTAAACTGAG-3';

[0037] R1, 5'-GATATCCCTGCACATGTACTGGACTTGT-3'.

[0038] (2) Construction and expression of recombinant Escherichia coli; Induced expression and purification of the complement regulatory protein PoCFH-CCP2 from flounder

[0039] The plasmid pEtPoCFH-CCP2 was transformed into Escherichia coli BL21 (DE3) (purchased from Quanshijin Biotechnology Co., Ltd., Beijing) and cultured on LB solid medium containing kanamycin (50 μg / ml) for 18-24 hours. Transformants were picked and named BL21 / pEtPoCFH-CCP2. BL21 / pEtPoCFH-CCP2 was cultured overnight in LB liquid medium containing kanamycin (50 μg / ml); 1 ml of the overnight culture was added to 200 ml of fresh LB liquid medium containing kanamycin (50 μg / ml) and cultured at 37°C with shaking at 150 rpm until the OD 600 The suspension was centrifuged at 10,000 g for 10 minutes at 4°C, and 5 ml of lysis buffer was added. The suspension was shaken slowly on a shaker at room temperature for 1-2 hours until the suspension became clear. The suspension was centrifuged at 10,000 g for 30 minutes at 4°C, and the supernatant was recovered. The protein in the supernatant was purified using a Ni-NTA affinity chromatography column.

[0040] The lysis buffer contained 10 mM NaH2PO4, 10 mM Tris, and 8 M urea at a final concentration of pH 8.0.

[0041] Example 2

[0042] (1) The ability of the complement regulatory protein PoCFH-CCP2 of Japanese flounder to bind to Edwardsiella tarda was detected by ELISA. The detection steps were as follows: Edwardsiella tarda was inoculated into 5 ml of liquid LB medium and cultured at 28°C until OD 600 About 0.8. Dilute to 10 with coating solution 9 CFU / ml was used as a bacterial diluent, and the complement regulatory protein PoCFH-CCP2 purified in Example 1 was gradiently diluted in PBS to 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, and 80 μg / ml, which were the dilutions of PoCFH-CCP2.

[0043] The above bacterial dilutions were mixed with different concentrations of flounder complement regulatory protein PoCFH-CCP2 dilutions or PBS (control group) and incubated for 2 hours. 5% skim milk powder, mouse anti-His antibody and goat anti-mouse IgG antibody were added in sequence, and TMB was used for color development. The absorbance was measured at 450 nm.

[0044] The results showed that the complement regulatory protein PoCFH-CCP2 of flounder has the ability to bind Edwardsiella tarda ( Figure 1(A) The coating solution composition is: 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6.

[0045] Depend on Figure 1 As shown in Figure A, the complement regulatory protein PoCFH-CCP2 of flounder can bind to Edwardsiella tarda, and the binding ability increases with the increase of protein concentration.

[0046] (2) Western Blot was used to detect the ability of the complement regulatory protein PoCFH-CCP2 of Japanese flounder to bind to Edwardsiella tarda. The detection steps were as follows: Edwardsiella tarda was inoculated into 5 ml of liquid LB medium and cultured in the conventional way until OD 600 The concentration of Edwardsiella tarda was about 0.8, and Edwardsiella tarda was diluted to 10 10 CFU / ml, the complement regulatory protein PoCFH-CCP2 purified in Example 1 was gradiently diluted in PBS to 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, and 160 μg / ml, which was the dilution solution of rPoCFH-CCP2.

[0047] After incubating the above bacterial solution with protein diluent, the solution was lysed with lysis buffer, and the lysate was subjected to SDS-PAGE and membrane transfer. Figure 1 As shown in B, the results showed that the complement regulatory protein PoCFH-CCP2 of flounder can bind to Edwardsiella tarda, and the binding ability increases with the increase of protein concentration.

[0048] (3) Immunofluorescence assay was used to detect the ability of the complement regulatory protein PoCFH-CCP2 of flounder to bind to Edwardsiella tarda. The assay steps were as follows: Edwardsiella tarda was inoculated into 5 ml of liquid LB medium and cultured in a conventional manner until OD 600 The concentration of Edwardsiella tarda was about 0.8, and Edwardsiella tarda was diluted to 10 10 CFU / ml is the bacterial dilution solution. The protein purified in Example 1 was gradiently diluted to 80 μg / ml in PBS to obtain the PoCFH-CCP2 dilution solution.

[0049] The above protein dilution and bacterial dilution were added dropwise to the slide for incubation, and mouse anti-his antibody and FITC-labeled goat anti-mouse IgG were added in sequence, stained with DAPI, and observed under a confocal microscope. Figure 1 As shown in middle C, the complement regulatory protein PoCFH-CCP2 of flounder can bind to Edwardsiella tarda.

[0050] Depend on Figure 1As shown in Figures A, B, and C, the complement regulatory protein PoCFH-CCP2 of flounder can bind to Edwardsiella tarda, and the binding ability increases with the increase of protein concentration.

[0051] Example 3

[0052] The test steps for the effect of complement regulatory protein PoCFH-CCP2 on complement activation in flounder were as follows: Edwardsiella tarda was inoculated into 5 ml of liquid LB medium and cultured in the conventional manner until OD 600 About 0.8, diluted to 10 with HBSS 7 CFU / ml is the bacterial dilution solution. The protein purified in Example 1 was gradiently diluted to 80 μg / ml in HBSS to obtain the PoCFH-CCP2 dilution solution.

[0053] (1) Effect of the complement regulatory protein PoCFH-CCP2 of Japanese flounder on the bactericidal ability of serum. Detection steps: The above bacterial dilution was incubated with an equal volume of protein dilution or label protein rTrx (control group) or PBS at 22°C for 1 hour. After incubation, the above mixture was incubated with an equal volume of heat-treated or untreated Japanese flounder serum at 22°C for 2 hours, and the dilution plate was counted ( Figure 2 The survival rate was calculated as follows: (number of cells treated with serum / number of cells treated with heat-treated serum) × 100%. Figure 2 As shown in middle A, treatment with the complement regulatory protein PoCFH-CCP2 of flounder significantly reduced the survival rate of Edwardsiella tarda in serum.

[0054] (2) Western blot was used to detect the recruitment of CFH and CFI on the bacterial surface after pretreatment with serum-killed complement regulatory protein PoCFH-CCP2. The detection steps were as follows: the above bacterial dilution was incubated with an equal volume of protein dilution or label protein rTrx (control group) or PBS at 22°C for 1 hour. After incubation, the above mixture was incubated with an equal volume of flounder serum at 22°C for 2 hours, lysed with lysis buffer, and the lysate was subjected to SDS-PAGE and transferred to a membrane. A group of PoCFH-CCP2 cells that were not treated with serum and Edwardsiella tarda were set as controls. Figure 2 As shown in middle B, the results showed that the complement regulatory protein PoCFH-CCP2 of flounder inhibited the recruitment of PoCFH and PoCFI to the bacterial surface of Edwardsiella tarda.

[0055] (3) Transmission electron microscopy was used to observe the destruction of bacterial cell membranes and the outflow of endosomes after serum-killed flounder complement regulatory protein PoCFH-CCP2 pretreatment. The detection steps were as follows: the above bacterial dilution was incubated with an equal volume of protein dilution or label protein rTrx (control group) or PBS at 22°C for 1 hour. After incubation, the bacteria were observed using transmission electron microscopy, as shown in Figure 2. Figure 2 As shown in middle C, the results showed that the complement regulatory protein PoCFH-CCP2 of flounder can cause the lysis and death of Edwardsiella tarda in serum.

[0056] Example 4

[0057] Detection steps of the complement regulatory protein PoCFH-CCP2 protecting the tissues and individuals of the otter: Inoculate Edwardsiella tarda into 5 ml of liquid LB medium and culture in the conventional way until the OD 600 About 0.8, diluted to 10 with PBS 8 CFU / ml represents the bacterial dilution. The purified flounder complement regulatory protein PoCFH-CCP2 from Example 1 was serially diluted in PBS to 80 μg / ml, creating the PoCFH-CCP2 dilution. This bacterial dilution was incubated with an equal volume of protein dilution, rTrx (control), or PBS at 22°C for 1 hour. Flounder (11 ± 0.7 g) were randomly divided into three groups (N = 90) and injected intramuscularly with 100 μl of the above mixture.

[0058] After 6, 12 and 24 hours, kidneys, spleens and blood were collected from the flounder and counted by dilution plate counting ( Figure 3 In order to observe the clinical manifestations of flounder, the skin pathological symptoms of flounder were photographed every day on days 1, 2, 3, and 5 after infection ( Figure 3 Middle B). For histopathological analysis, kidneys, spleens, and intestines of flounder were collected 5 days after infection, fixed with 4% paraformaldehyde, and sliced ​​and stained with HE. The slices were processed and observed ( Figure 3 To determine the mortality of fish, flounder were infected as described above and the mortality of fish was recorded every day ( Figure 3 The results showed that in the presence of the complement regulatory protein PoCFH-CCP2, the bacterial loads in the blood, kidneys, and spleen of flounder infected with Edwardsiella tarda were significantly lower than those in fish infected with Edwardsiella tarda in the presence of rTrx or PBS ( Figure 3 Clinical observations showed that the control and rTrx groups of flounder showed significant white ulcers and skin ulcers on the surface of the fish, accompanied by congestion and bleeding. In contrast, the surface condition of the flounder in the rPoCFH-CCP2 group was almost unchanged compared to the uninfected group ( Figure 3 Middle B); Histopathological analysis showed that the complement regulatory protein PoCFH-CCP2 of flounder reduced the damage of Edwardsiella tarda to the intestine, spleen and kidney tissues ( Figure 3 Middle C); Mortality analysis showed that treatment with the complement regulatory protein PoCFH-CCP2 significantly increased the survival rate of flounder ( Figure 3 Middle D).

[0059] Depend on Figure 3 It can be seen that rPoCFH-CCP2 can be used as a drug to protect the tissues and body of flounder.

[0060] Comparative Example 1

[0061] (1) Constructing and obtaining the expression plasmids pEtPoCFH-CCP1 and pEtPoCFH-CCP3 of complement regulatory proteins PoCFH-CCP1 and PoCFH-CCP3 of flounder according to the description of Example 1;

[0062] PoCFH-CCP1 and PoCFH-CCP3 were obtained through a prokaryotic protein expression system, and their sequences were the amino acid sequences shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing, respectively.

[0063] PoCFH-CCP1 amino acid sequence SEQ ID No. 2 is:

[0064] KNCTLEQFLNGDLFDSNFDTTGLEDNYPSGKQIRVSCSIGYSGFFKLHCDGGVWKSKSTKCQPRSCGHPGDAPFADFHLEIGEDFVFGSQVKFTCHKGYQMVSRSNRRRCLAEGWDGVVPVC EAQQCRALPVNDNVQVIGDPEEANFGNVVRFRCKSSSHMLFGSQEVYCDENGEWSGGAPKCDEIKCLVTEIENGNVLGDTQEYKEILHFECEPGFKPTEARSPVCTKLGMRADWSPTPAC

[0065] PoCFH-CCP3 amino acid sequence table SEQ ID No.3 is:

[0066] KNNRRFTIVCENGVWTGIENCSGCPNAEVSHGFYVGPYNDTLYYTCDEGYKLVTDGWWAQAECHDGVWSGLDLCIANNRCGKLPVIPNGGVKYPGSNYEEGQRVTITCNKGYRVQVKQLTCHNGEWKSHESSPICAPLANPCSPPPKVKDAVVENLYQREFLSGSEVTYQCRHNHTTEADTTIRCNDGNWETHNIVCAPVPEK

[0067] Using flounder cDNA as a template, PCR amplification was performed with primers F2 and R2 and F3 and R3, respectively. The construction of expression vectors pEtPoCFH-CCP1 and pEtPoCFH-CCP3 was carried out as described in Example 1.

[0068] The primers F2 and R2 are respectively:

[0069] F2, 5'-GATATCATGAAAAATTGTACACTTGAACAGT-3';

[0070] R2, 5'-GATATCACATGCAGGAGTGGGGCTCC-3'.

[0071] The primers F3 and R3 are respectively:

[0072] F3, 5'-GATATCATGAAAAATAACAGACGTTTCACTAT-3';

[0073] R3, 5'-GATATCCTTTTCTGGAACAGGTGCAC-3'.

[0074] (2) Construction and Expression of Recombinant Escherichia coli The induced expression and purification of the complement regulatory proteins PoCFH-CCP1 and PoCFH-CCP3 of flounder were carried out as described in Example 1.

[0075] Detection of the corresponding proteins obtained above:

[0076] (1) The ability of PoCFH-CCP1 and PoCFH-CCP3 to bind to Edwardsiella tarda was tested by ELISA. The detection steps were carried out in the same manner as described in Example 2. The results showed that PoCFH-CCP1 and PoCFH-CCP3 did not have the ability to bind to Edwardsiella tarda. Figure 4 Middle A).

[0077] (2) Western Blot was used to detect the ability of the complement regulatory protein PoCFH-CCP2 of Japanese flounder to bind to Edwardsiella tarda. The detection steps were carried out as described in Example 2. The results showed that PoCFH-CCP1 and PoCFH-CCP3 could not bind to Edwardsiella tarda ( Figure 4 Middle B).

[0078] (3) Immunofluorescence assay was used to detect the ability of PoCFH-CCP1 and PoCFH-CCP3 to bind to Edwardsiella tarda. The assay was performed as described in Example 2. The results showed that PoCFH-CCP1 and PoCFH-CCP3 could not bind to Edwardsiella tarda. Figure 4 Middle C).

[0079] Depend on Figure 4 As can be seen in Figures A, B, and C, PoCFH-CCP1 and PoCFH-CCP3 cannot bind to Edwardsiella tarda.

[0080] In summary, it can be seen that only a specific fragment PoCFH-CCP2 in the complement regulatory protein of flounder derived from the complement factor H of flounder has an immune effect specifically against Edwardsiella tarda. It can occupy the key binding sites on Edwardsiella tarda, effectively inhibit the recruitment of CFH and CFI, and can be used as an immune drug to prevent and treat Edwardsiella tarda disease in fish; furthermore, it can be used as an immune drug to protect flounder tissues and fish bodies from infection with Edwardsiella tarda. Its preparation process is simple and poses no safety risks to the environment or fish bodies. The obtained protein has application potential in anti-bacterial infection.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of the complement regulatory protein PoCFH-CCP2 of flounder, characterized in that: Application of flounder complement regulatory protein PoCFH-CCP2 in the preparation of immune drugs.

2. The use of the flounder complement regulatory protein PoCFH-CCP2 according to claim 1, characterized in that: The amino acid sequence of the flounder complement regulatory protein PoCFH-CCP2 is SEQ ID No.

1.

3. The use of the flounder complement regulatory protein PoCFH-CCP2 according to claim 2, characterized in that: The primer pair for amplifying and obtaining the amino acid sequence of the complement regulatory protein PoCFH-CCP2 of flounder is: The sequence of F1 is: 5′-GATATCATGGAACTGATAACATGTAAACTGAG-3′; The sequence of R1 is: 5'-GATATCCCTGCACATGTACTGGACTTGT-3'.

4. The use of the flounder complement regulatory protein PoCFH-CCP2 according to any one of claims 1 to 3, characterized in that: The invention relates to an application of the flounder complement regulatory protein PoCFH-CCP2 in the preparation of immune drugs for preventing and treating Edwardsiella tarda.

5. An immune drug for preventing and treating Edwardsiella tarda, characterized in that: Contains the complement regulatory protein PoCFH-CCP2 of flounder.

6. The immune drug for preventing and treating Edwardsiella tarda according to claim 5, characterized in that: The flounder complement regulatory protein PoCFH-CCP2 is amplified by PCR using flounder cDNA as a template and primers. The PCR product is connected to an expression vector to obtain a recombinant plasmid, which is transformed into a competent microorganism and induced and purified to obtain a recombinant protein, namely the flounder complement regulatory protein PoCFH-CCP2.

7. The immune drug for preventing and treating Edwardsiella tarda according to claim 6, characterized in that: Step 1, construction of the expression vector pEtPoCFH-CCP2: using flounder cDNA as a template, PCR amplification was performed using primers, the PCR product was purified, and then ligated with the plasmid T-Simple. The ligated product was transformed into Escherichia coli, cultured on LB medium containing kanamycin for 8-12 hours, and the transformants were screened to extract the plasmid to obtain a recombinant plasmid; the recombinant plasmid was digested with EcoRV, the target fragment was recovered, ligated to pET259, and transformed into Escherichia coli. The culture was carried out on LB medium containing kanamycin for 18-24 hours, and the transformants were screened to extract the plasmid, which is the expression vector pEtPoCFH-CCP2; Step 2: The plasmid pEtPoCFH-CCP2 of step 1 above was transformed into Escherichia coli BL21 (DE3), cultured in LB medium containing kanamycin, and the transformants were screened, namely BL21 / pEtPoCFH-CCP2, which was obtained after culture.

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