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Streptococcus iniae trivalent DNA vaccine and construction method thereof

A DNA vaccine, Streptococcus iniae technology, applied in the field of vaccinology, can solve problems such as lack of vaccines

Inactive Publication Date: 2012-10-10
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, due to the lack of effective vaccines, the prevention and treatment of Streptococcus dolphin disease in my country mainly relies on the use of antibiotics and other drugs

Method used

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  • Streptococcus iniae trivalent DNA vaccine and construction method thereof
  • Streptococcus iniae trivalent DNA vaccine and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of Trivalent DNA Vaccine S3D

[0031] 1) Construction of plasmid pCNF: Streptococcus iniae G26 was used as a template and F1 / R1 was used as primers for PCR amplification. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 94°C for 40s, 50°C for 60s, and 72°C for 60s , after 5 cycles, change to 94°C for 40s, 62°C for 60s, 72°C for 60s, and after 25 cycles, extend the reaction at 72°C for 10min. The PCR product was purified with Tiangen DNA Product Purification Kit. Plasmid pCN3 (see Jiao XD, Zhang M, Hu YH, Sun L. Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens. Vaccine 2009; 27:5195 202. for the construction process of pCN3) was digested with SmaI, and the 5.4kb fragment was recovered. Ligate it with the above-mentioned purified PCR product with T4 DNA ligase at room temperature for 2-4 hours, transform Escherichia coli DH5α with the ligation solution and culture it on LB solid medium containing am...

Embodiment 2

[0039] Vaccine application

[0040] Step 1) Preparation of vaccine preparation solution. Dilute the above DNA vaccine S3D in PBS to a final concentration of 150 μg / ml, which is the vaccine preparation solution.

[0041] The composition of PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

[0042] Step 2) Vaccination. 100 flounder (each weighing about 11.4g) were randomly divided into 4 groups, 25 in each group. These 4 groups are named A, B, C and D respectively. Each fish in groups A and C was injected intraperitoneally with 100ul of the vaccine preparation solution in step 1) above, and each fish in groups B and D (control group) was injected intraperitoneally with 100ul PBS.

[0043] Step 3) Preparation of Streptococcus iniae suspension. Cultivate Streptococcus iniae G26 in LB medium to an OD600 of 0.9, then centrifuge (5000g, 4°C, 10min), collect the bacteria, and suspend it in PBS to a fi...

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Abstract

The invention relates to the field of vaccinology, in particular to a Streptococcus iniae trivalent DNA vaccine and to a construction method thereof. The method comprises the steps of using Streptococcus iniae G26 as a template, allowing the primers of F1 / R1, F2 / R2 and F3 / R3 to carry out PCR amplification respectively, allowing the product and a vector pCN3 plasmid to be subjected to connection and transformation, and allowing the plasmid to be subjected to equivalent volume mixing to obtain the trivalent DNA vaccine. The efficiency of the protection of the vaccine obtained by the invention against Streptococcus iniae infection is more than 90% in one month or two months after immunization.

Description

technical field [0001] The invention relates to the field of vaccinology, in particular to a Streptococcus iniae trivalent DNA vaccine and a construction method thereof. Background technique [0002] Streptococcus iniae is a Gram-positive bacterium that can infect a variety of cultured seawater and freshwater fish, including flounder, tilapia, grouper, and American redfish. considered to be a major fish pathogen. In addition, Streptococcus iniae is a zoonotic pathogen that can infect humans through sick animals, so the research on its immune control is of great significance. At present, due to the lack of effective vaccines, the prevention and treatment of streptococcal dolphin disease in my country mainly relies on the use of drugs such as antibiotics. In this case, the development of an effective vaccine against this bacteria has become an urgent need for the aquaculture industry. Contents of the invention [0003] The purpose of the present invention is to provide a ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/116A61K39/09A61P31/04C12N15/70C12N1/21C12R1/19C12R1/46
Inventor 孙黎孙云胡永华
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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