Immunoadjuvant of oral vaccine for tilapia and use thereof

An oral vaccine and immune adjuvant technology, applied in the application field of tilapia recombinant secreted heat shock protein 70 protein, can solve problems such as weak specificity and pertinence

Inactive Publication Date: 2012-10-10
GUANGXI INST OF FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (3) Animal and plant sources: including propolis, glycyrrhizin, concanavalin A, etc. The use of these immune adjuvants or immune enhancers can enhance the non-specific or specific immunity of fish to varying degrees, but specific and less targeted
[0011] Although heat shock protein 70 has been widely studied in huma...

Method used

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  • Immunoadjuvant of oral vaccine for tilapia and use thereof
  • Immunoadjuvant of oral vaccine for tilapia and use thereof
  • Immunoadjuvant of oral vaccine for tilapia and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Expression and purification of tilapia heat shock protein 70

[0123] (1) Cloning of tilapia heat shock protein 70 gene

[0124] ①Heat stress treatment:

[0125] After the test fish were temporarily raised in a 500L large water tank for 1 week (the water temperature was about 25°C, and the air pump was inflated), they were placed in a water temperature of 39°C for 10 minutes for heat stress, and then placed at room temperature for 6 hours.

[0126] ②Total RNA extraction:

[0127] The surface of the test fish was scrubbed and disinfected with 75% alcohol cotton ball, placed on a sterile tray, and blood was collected from the tail vein with a 5ml syringe rinsed with heparin sodium solution. Mix the blood of 3 fish of the same strain into 1 part. Take 0.2ml of mixed blood (about 2.0×10 7 blood cells), add 1ml Trizol reagent, shake vigorously until the liquid is completely transparent, and act at room temperature for 5 minutes; add 0.2ml chloroform and shake ...

Embodiment 2

[0168] Example 2: Activity identification of recombinant secreted and expressed tilapia heat shock protein 70 protein

[0169] (1) Tilapia heat shock protein 70 protein: obtained according to the expression and purification steps in Example 1.

[0170] (2) Antigen preparation: use Streptococcus tilapia, the main disease pathogen of tilapia culture, strain number CMS005, inoculate on a rabbit blood plate, and incubate at 28°C for 24 hours. Pick a single colony and inoculate it in a 250mL Erlenmeyer flask containing 50mL of TSB medium, shake at 150r / min, and culture at 28°C for 24h. Add formaldehyde to make the final concentration 0.25%, 150r / min, 28°C static inactivation for 24h. The 2kD ultrafiltration was concentrated to 1 / 10 of the original volume, and the ultrafiltration product was centrifuged at 10,000 g for 10 minutes to obtain the thallus and extracellular product (ECP) antigens with a molecular weight greater than 2kD. Resuspend the thallus with PBS and adjust the co...

Embodiment 3

[0172] Example 3: Tilapia heat shock protein 70 improves the immune function of peritoneal macrophages

[0173] (1) Tilapia heat shock protein 70 protein was obtained according to the steps of expression and purification in Example 1.

[0174] (2) Preparation of tHsp70-antigen complex According to the method in Example 2 for the combination of recombinant secreted and expressed fish heat shock protein 70 and antigen in vitro. A total of 12 test groups were set up in the experiment: tHsp70-thallus complex group (thallus amount was 1.0×10 6 cell), tHsp70 was set at 3 concentrations of 100 μg / mL, 10 μg / mL and 1 μg / mL; in the tHsp70-ECP complex group (the amount of ECP was 1 μg), tHsp70 was set at 3 concentrations of 100 μg / mL, 10 μg / mL and 1 μg / mL ; tHsp70 stimulation group set 100 μg / mL, 10 μg / mL, 1 μg / mL 3 concentrations; another thallus (1.0 × 10 6 cell), ECP (1 μg) and no stimulant as 3 control groups, and 3 experimental parallel wells were set up for each group.

[0175] ...

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Abstract

The invention discloses an immunoadjuvant of an oral vaccine for tilapia and a use thereof. The immunoadjuvant of an oral vaccine for tilapia is a recombinant tilapia heat shock protein 70 (tHSP70). A tHSP70 gene is cloned by specific upstream and downstream primers of HSP70; a tHSP70 yeast expression system is constructed; and a tHSP70 protein is expressed and purified. Through combined utilization of the tHSP70 protein and an oral tilapia streptococcus vaccine, NO release and phagocytosis activity of macrophages of the tilapia enterocoelia are improved obviously; proliferation activity of peripheral blood lymphocytes is improved; the expression of immune-related genes is adjusted; and an immune protection rate of an oral tilapia streptococcus vaccine is improved. Through the use of the immunoadjuvant of an oral vaccine for tilapia, the defect of poor immune effects of the existing oral vaccine for tilapia is overcome and application of a vaccine in an aquaculture industry is promoted.

Description

technical field [0001] The invention relates to an immune adjuvant for an oral vaccine for tilapia, in particular to the application of a tilapia recombinant secreted heat shock protein 70 protein. Background technique [0002] With the rapid development of the aquaculture industry, in the past 20 years, the epidemic diseases of aquaculture species in my country have occurred frequently, and the economic losses have been serious. According to incomplete statistics, the annual incidence of diseases in aquaculture in the country is more than 50%, and the loss rate About 20%, the annual economic loss amounts to tens of billions of yuan, and there is an upward trend, and the disease has become one of the major restrictive factors for the development of aquaculture. In 1942, Duff succeeded in oral immunization of steelhead trout with inactivated Aeromonas salmonicida (A.salmonicida) for the first time, creating a history of vaccine application in fish. After that, vibrio disease ...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K38/17A61P37/04A61P31/00A61P31/04
Inventor 陈明王瑞梁万文王秋华李超黄婷雷爱莹李莉萍甘西陈晓汉
Owner GUANGXI INST OF FISHERIES
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