Cyprinid herpesvirus II type truncated polypeptide and application thereof in preparation of cyprinid herpesvirus II type vaccine

Abstract

The invention discloses a cyprinid herpesvirus II type truncated polypeptide and an application of the cyprinid herpesvirus II type truncated polypeptide in preparation of a cyprinid herpesvirus II type vaccine, and the truncated polypeptide CyHV-2 ORF104 is a linear polypeptide with the length of 9 amino acids and is as shown in SEQ ID NO: 1. A synthesized linear peptide fragment and keyhole limpet hemocyanin (KLH) are subjected to cross-linking and sterile treatment and then are mixed with an adjuvant in proportion to obtain the CyHV-2 ORF104 polypeptide vaccine, carassius auratus gibelio is subjected to intraperitoneal injection, it is found that the CyHV-2 ORF104 polypeptide vaccine can effectively improve the survival rate of the carassius auratus gibelio after challenge, safety tests prove that after the carassius auratus gibelio is immunized, no adverse reaction is generated, and the safety of the carassius auratus gibelio is improved. The CyHV-2 infection of the carassius auratus gibelio can be effectively prevented and treated.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular vaccinology, and specifically relates to a truncated polypeptide of carp herpesvirus type II and an application thereof in preparing a carp herpesvirus type II vaccine. Technical Background

[0002] Crucian carp are susceptible to infection by Cyprinid herpesvirusⅡ (CyHV-2) during breeding, which in turn causes crucian carp hematopoietic organ necrosis. The virus is a double-stranded DNA virus with a regular icosahedral structure, which can harm crucian carp at all stages, including broodstock, fry, and adult fish, and the mortality rate to the host can reach over 90%. In the prevention and treatment of crucian carp hematopoietic organ necrosis, traditional methods such as water quality control and enhancing fish immunity are still the main means. Vaccines, as a drug that can specifically activate the host immune system, have always been the focus and difficulty of research in the field of anti-infection.

[0003] At present, in the direction of crucian carp CyHV-2 vaccine, research has been carried out including inactivated vaccines, nucleic acid vaccines, and recombinant protein subunit vaccines, but there is no peptide vaccine targeting very specific antigenic epitopes. Peptide vaccines not only have higher sequence specificity, but also minimize the risk of allergic or autoimmune reactions and have higher safety. In addition, peptide vaccines also have the advantages of low production cost and short production cycle, which is an important direction for future vaccine research.

[0004] The present invention is based on the CyHV-2ORF104 antigen peptide sequence (NCBI Reference Sequence: YP_007003923.1) expressed by crucian carp, which is truncated to construct a CyHV-2ORF104 polypeptide vaccine that can provide a relative immune protection rate of up to 76%. It not only has a similar protective effect to other reported CyHV-2 vaccines, but also has the advantages of high safety, easy large-scale production and storage. In summary, the CyHV-2ORF104 polypeptide vaccine constructed by the present invention is suitable for promotion as a new polypeptide vaccine for crucian carp CyHV-2 because of its high protection rate, high safety, easy large-scale production and storage. Summary of the invention

[0005] The object of the present invention is to provide a truncated polypeptide of carp herpesvirus type II, wherein the polypeptide is shown as SEQ ID NO.1.

[0006] Another object of the present invention is to provide an application of a truncated polypeptide of carp herpesvirus type II in the preparation of a carp herpesvirus type II vaccine. The synthesized linear peptide segment is cross-linked with keyhole limpet hemocyanin (KLH) and aseptically treated, mixed with an adjuvant, and intraperitoneally injected into a hybrid silver crucian carp. It was found that it can effectively improve the survival rate of the hybrid silver crucian carp after CyHV-2 infection.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A truncated polypeptide of carp herpesvirus type II, wherein the polypeptide is shown as SEQ ID NO.1.

[0009] A gene encoding the above polypeptide.

[0010] An expression vector for expressing the above polypeptide.

[0011] A recombinant microorganism expressing the above polypeptide. A complex containing the above polypeptide.

[0012] The use of the above polypeptide, gene, expression vector, recombinant microorganism or complex in the preparation of drugs for treating or preventing carp herpes virus type II infection.

[0013] The application of the above polypeptide, gene, expression vector, recombinant microorganism or complex in the preparation of carp herpes virus type II vaccine.

[0014] The complex described above is preferably a complex in which a truncated polypeptide of carp herpesvirus type II is cross-linked with keyhole limpet hemocyanin (KLH).

[0015] Compared with existing vaccines, the present invention has the following advantages:

[0016] Traditional inactivated vaccines, nucleic acid vaccines and recombinant protein subunit vaccines have certain protective effects, but they all have problems such as unknown specific antigen epitope information and unclear immune action mechanisms. At the same time, the vaccine components are relatively complex and there are potential safety risks. The polypeptide vaccine provided by the present invention not only has high specificity and high protective effects, but also has simple vaccine components, is safe and reliable, and is easy to mass produce and store. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the survival rate of the immunized crucian carp after being challenged with poison in Example 2.

[0018] Figure 2 It is the antibody titer of the immunized crucian carp serum in Example 3.

[0019] Figure 3 It is the survival rate of the immunized crucian carp after being challenged with poison in Example 3.

[0020] Figure 4 This is the viral load of the head kidney tissue of the immunized crucian carp after infection in Example 3. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the reagents described in the present invention are purchased from commercial channels or are conventionally prepared reagents in the art. The technical solutions described are conventional solutions in the art unless otherwise specified.

[0022] Embodiment 1:

[0023] Obtaining the polypeptide vaccine of carp herpesvirus type II (the present invention or polypeptide vaccine):

[0024] The applicant truncates the CyHV-2ORF104 antigen peptide sequence (NCBI Reference Sequence: YP_007003923.1) expressed by crucian carp and obtains the polypeptide sequence shown in SEQ ID NO.1, i.e., MMSEIILGV, i.e., the truncated polypeptide of carp herpesvirus type II, which is also referred to as CyHV-2ORF104 polypeptide in the present invention.

[0025] Embodiment 2:

[0026] Application of truncated polypeptides of carp herpesvirus type Ⅱ in the preparation of carp herpesvirus type Ⅱ vaccine:

[0027] (1) Experimental plan

[0028] Experimental animals: Crucian carp (15±2g) were purchased from the Jingzhou Yaowan Base of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0029] Challenge strain: Cyprinid herpesvirus type 2 strain isolated from diseased crucian carp (Chen Xu, Yiqun Li, Zidong Xiao, Jie Yang, Mingyang Xue, Nan Jiang, Yan Meng, Wenzhi Liu, Yuding Fan*, and Yong Zhou*. Proteomic and phosphoproteomic analyses reveal gibel car presponses to cyprinid herpesvirus 2 infection. Journal of Proteome Research. 2022, 21(8): 1961-1973.)

[0030] Experimental groups: Crucian carp were divided into 4 groups: blank group, negative control group (Poly(I:C)), low-dose immunization group and high-dose immunization group, with 50 fish in each group.

[0031] Experimental conditions: water temperature was 25±1℃, aerated tap water was used for breeding, and compound feed was fed twice a day during the experiment.

[0032] Vaccine preparation: Chemically synthesize the truncated polypeptide of carp herpesvirus type II (sequence as shown in SEQ ID NO.1), desalt and remove endotoxin, and freeze-dry for use. Use PBS solution to prepare a poly(I:C) solution with a concentration of 0.1 mg / mL, take 10 mL of the prepared poly(I:C) solution to dissolve 2 mg and 0.6 mg of the synthetic CyHV-2ORF104 polypeptide, respectively, and the final concentrations of the polypeptide are 0.2 mg / mL and 0.06 mg / mL, respectively. That is, it corresponds to high and low doses of CyHV-2ORF104 polypeptide vaccines.

[0033] Immunization method: intraperitoneal injection, the immunization dose is 150μL / tail, that is, the total amount of polypeptide in the low-dose immunization group is 9μg / tail, and the total amount of polypeptide in the high-dose immunization group is 30μg / tail. Two weeks after the first immunization, a second booster immunization is performed, and the immunization dose is the same as the first one.

[0034] Virus attack method: 14 days after the second immunization, use a concentration of 10 6 The high- and low-dose immunization groups and the negative control group were subjected to intraperitoneal injection of CyHV-2 strains of 150 μL / tail. The fish were continuously observed for 14 days after the infection, and the survival of the fish in each group was counted.

[0035] (2) Biosafety evaluation of peptide vaccines on crucian carp

[0036] After the first immunization and within 14 consecutive days after the second immunization, the immunized crucian carp had no obvious stress symptoms and no mortality occurred. At the same time, the vitality and food intake of the crucian carp did not decline or decrease.

[0037] (3) Determination of protection ability of immune crucian carp after challenge

[0038] The high-dose and low-dose immunization groups, negative control group, and blank group were all raised under the same conditions, and the survival and death of crucian carp within 14 days after infection were counted, and the relative immune protection rate was calculated. Figure 1 As shown in the figure, no death occurred in the blank group; fish in the negative control group began to die on the 4th day after the infection, and all died on the 9th day. However, the survival rates of crucian carp in the high- and low-dose immunization groups increased to varying degrees within 14 days of the infection, with overall survival rates of 30% and 13%, respectively ( Figure 1 ). The results showed that under the condition of poly(I:C) as adjuvant, the simple CyHV-2ORF104 polypeptide vaccine could better protect crucian carp.

[0039] Embodiment 3:

[0040] Application of truncated peptide of carp herpesvirus type Ⅱ coupled with KLH in the preparation of carp herpesvirus type Ⅱ vaccine:

[0041] 1) Conjugation of truncated polypeptide of carp herpesvirus type II with keyhole limpet hemocyanin (KLH):

[0042] The polypeptide shown in SEQ ID NO. 1 was artificially synthesized and an additional cysteine ​​was added to its carboxyl terminus. Weigh KLH (5 mg) and dissolve it in PBS to a concentration of 10 mg / mL; weigh 2.5 mg of the cross-linking agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) and completely dissolve it in ultrapure water to a concentration of 5 mg / mL; after KLH and SMCC are completely dissolved, mix them, seal them with a sealing film, and react on a rotating shaker at room temperature for 1 hour; place the mixed reaction KLH-SMCC in a dialysis bag, dialyze in a PBS solution for half an hour to remove reaction byproducts and excess SMCC molecules; dissolve 5 mg of the synthetic polypeptide in 1 mL of PBS, add it to the dialyzed KLH-SMCC solution, and react on a rotating shaker at room temperature for 4 hours; transfer the coupled polypeptide KLH solution into a dialysis bag, dialyze in PBS to remove reaction byproducts, and dialyze four times, each time for half an hour; finally, transfer the dialyzed solution into a new centrifuge tube, and use PBS to adjust the volume of KLH+ polypeptide to 1 mg / mL for the following examples.

[0043] 2) Application of the truncated polypeptide of carp herpesvirus type II coupled with keyhole limpet hemocyanin (KLH) in the preparation of carp herpesvirus type II polypeptide vaccine:

[0044] (1) Experimental plan

[0045] Experimental animals: Crucian carp (15±2g) were purchased from the Jingzhou Yaowan Base of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0046] Challenge strain: Cyprinid herpesvirus type 2 strain isolated from diseased crucian carp (Chen Xu, Yiqun Li, Zidong Xiao, Jie Yang, Mingyang Xue, Nan Jiang, Yan Meng, Wenzhi Liu, Yuding Fan*, and Yong Zhou*. Proteomic and phosphoproteomic analyses reveal gibel car presponses to cyprinid herpesvirus 2 infection. Journal of Proteome Research. 2022, 21(8): 1961-1973.)

[0047] Experimental groups: Crucian carp were divided into 3 groups: blank group, negative control group (PBS) and immune group, with 50 fish in each group.

[0048] Experimental conditions: water temperature was 25±1℃, aerated tap water was used for breeding, and compound feed was fed twice a day during the experiment.

[0049] Vaccine preparation: The preparation method of the polypeptide vaccine for the first immunization is as follows: Step 1) The prepared coupled polypeptide is mixed with Freund's complete adjuvant in a volume ratio of 1:1, and then stirred 4 times with an ultrasonic crusher (150W, working for 10s, interval of 5s) to obtain a stable emulsion, and then repeatedly aspirated and mixed with a syringe until the emulsion drops on the water surface without spreading.

[0050] The preparation method of the polypeptide vaccine for secondary booster immunization is as follows: Step 1) The prepared coupled polypeptide is mixed with Freund's incomplete adjuvant in a volume ratio of 1:1, and then stirred 4 times with an ultrasonic crusher (150W, working for 10s, interval of 5s) to obtain a stable emulsion, and then repeatedly aspirated and mixed with a syringe until the emulsion drops on the water surface without spreading.

[0051] Immunization method: intraperitoneal injection, the immunization dose is 100μL / tail, that is, the immunization dose is 50μg / tail. Two weeks after the first immunization, a second booster immunization is performed, and the immunization dose is the same as the first time.

[0052] Virus attack method: 14 days after the second immunization, use a concentration of 10 6 The immune group and the negative control group were challenged with the CyHV-2 strain of cfu / tail by intraperitoneal injection, 150 μL / tail. The fish were continuously observed for 14 days after the challenge, and the survival of the fish in each group was counted.

[0053] (2) Biosafety evaluation of peptide vaccines on crucian carp

[0054] After the first immunization and within 14 consecutive days after the second immunization, the immunized crucian carp had no obvious stress symptoms and no mortality occurred. At the same time, the vitality and food intake of the crucian carp did not decline or decrease.

[0055] (3) Antibody level detection after immunization of crucian carp

[0056] 14 days after the first immunization and 14 days after the second immunization, 3 fish were randomly selected from the immunization group and the negative control group, respectively, and whole blood was collected from the tail vein. The blood was kept at 4°C overnight and centrifuged at 4000r / min for 10min to prepare sample serum of the immunization group and the negative control group 14 days after the first and second immunizations.

[0057] The serum prepared above was used for ELISA serum antibody titer detection, using KLH-coupled CyHV-2ORF104 polypeptide as antigen, the polypeptide was diluted with antigen coating solution to a final content of 500 ng / 100 μL (well), and then coated in a 96-well plate dedicated to ELISA; 5% (w / v) BSA was used. Incubate at 37℃ for 2h for blocking; after washing, add the test serum diluted with PBS at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 100μL per well, incubate at 37℃ for 3h; after washing, add mouse anti-carp IgM antibody at a ratio of 1:1000 to each well, incubate at 37℃ for 2h; after washing, add secondary antibody goat anti-mouse IgG, incubate at 37℃ for 2h; after washing again, add 100μL of color development solution to each well, react for 30min, and then add 50μL of stop solution (10% concentrated H2SO4) to each well to stop the reaction; finally, use an enzyme reader to read the OD value at a wavelength of 450nm. Results Figure 2 The results showed that after immunization with polypeptide vaccine, crucian carp produced high levels of serum antibodies, and the antibody titer increased significantly after the second immunization.

[0058] (4) Determination of protection ability of immune crucian carp after challenge

[0059] The immune group, negative control group and blank group were all raised under the same conditions, and the survival and death of crucian carp within 14 days after infection were counted, and the relative immune protection rate was calculated. Figure 3 As shown, no death occurred in the blank group; fish in the negative control group began to die on the fourth day after the virus attack, and all died on the eighth day, while the crucian carp in the polypeptide immunization group had a higher survival rate within 14 days of the virus attack, and the overall survival rate reached 76%, proving that the CyHV-2ORF104 polypeptide vaccine can better protect crucian carp.

[0060] (5) Determination of viral load in tissues of immune crucian carp after challenge

[0061] Three days after the virus attack, the head and kidney tissues of crucian carp in the immune group, negative control group and blank control group were collected, and the viral load in each group of samples was determined by digital PCR. The sequences of digital PCR primers and probes are as follows:

[0062] Primer and probe sequences:

[0063]

[0064] The results are as follows Figure 4 As shown, no virus content was detected in the blank group. Compared with the negative control group, the viral load in the head and kidney tissues of the immunized crucian carp was significantly reduced, indicating that the vaccine better protected the crucian carp in the immunized group.

Claims

1. A truncated polypeptide of carp herpesvirus type II, the polypeptide being shown in SEQ ID NO.

1.

2. A gene encoding the polypeptide according to claim 1.

3. An expression vector for expressing the polypeptide according to claim 1.

4. A recombinant microorganism expressing the polypeptide of claim 1.

5. A complex comprising the polypeptide according to claim 1.

6. Use of the polypeptide according to claim 1, the gene according to claim 2, the expression vector according to claim 3, the recombinant microorganism according to claim 4 or the complex according to claim 4 in the preparation of a drug for treating or preventing carp herpesvirus type II infection.

7. Use of the polypeptide, gene, expression vector, recombinant microorganism or complex according to claim 1 in the preparation of carp herpesvirus type II vaccine.

8. The composite according to claim 5, characterized in that The polypeptide according to claim 1 is cross-linked with keyhole limpet hemocyanin to obtain the polypeptide.

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