Application of edwardsiella tarda pilin FimA

A technology for retarding Edwards and fimbriae proteins, applied in the field of molecular biology, can solve problems such as reduced ability, and achieve the effect of protecting infection and improving immune protection effect.

Inactive Publication Date: 2013-11-27
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that Edwardsiella tarda FimA has the ability to agglutinate red blood cells, and mutations in the fimA gene lead to a reduction in the ability of Edwardsiella tarda to infect cells and tissues

Method used

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  • Application of edwardsiella tarda pilin FimA
  • Application of edwardsiella tarda pilin FimA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Construction of pilin FimA expression plasmid pEtFimA:

[0018] The sequence of the pili protein FimA of the present invention has been published (GenBank accession No. YP_003295086.1).

[0019] Another pilus protein FimA can be obtained as follows: using Edwardsiella tarda TX1 as a template, PCR amplification is performed with primers F1 and R1. The PCR conditions are: 94°C for 60s to pre-denature template DNA, then 94°C for 40s, 55°C for 60s, 72°C for 60s, after 5 cycles, change to 94°C for 40s, 65°C for 60s, 72°C for 60s, after 30 cycles and then repeat at 72°C. ℃ extension reaction 7-10min. PCR products were purified with corresponding kits from Tiangen. The vector pET259 (see Zheng W, Hu Y, Zhang M, Sun L. 2010. Analysis of the expression and antioxidant property of a peroxiredoxin 6 from Scophthalmus maximus. Fish Shellfish Immunol. 29, 305–311 for the construction process of pET259.

[0020] ) was digested with SwaI to recover a 5.4kb fragment, and ligated it ...

Embodiment 2

[0023] Induced Expression and Purification of Recombinant Pili Protein FimA

[0024] The plasmid pEtFimA of the above-mentioned Example 1 was transformed into Escherichia coli BL21(DE3) (purchased from "Tiangen Biochemical Technology Co., Ltd.", Beijing) by conventional methods, and cultured in LB solid medium containing kanamycin (30ug / ml) After culturing for 18-24 hours, pick a transformant and name it BL21 / pEtFimA. Cultivate BL21 / pEtFimA overnight in LB liquid medium containing kanamycin (30ug / ml); take 1ml of overnight culture solution, and then add 100ml fresh LB liquid containing kanamycin (30ug / ml) culture medium at 37°C with shaking at 200rpm until OD 600 If the concentration is 0.6, add isopropyl-β-D-thiogalactopyranoside with a final concentration of 1mM, continue shaking culture at 18°C ​​at 160rpm for 12h, then centrifuge at 5000g, 4°C for 10min, collect the bacterial liquid, add 5ml For the lysate, shake slowly on a shaker at room temperature for 1-2 hours until...

Embodiment 3

[0026] Application of pilus protein FimA as a vaccine

[0027] Step 1) Preparation of adjuvant and vaccine mixture.

[0028] Adjuvant preparation: 5% (mass ratio) NaOH and 5% (mass ratio) Al 2 (SO 4 ) 3 Mix at a volume ratio of 2:5 and centrifuge at 10,000g for 5 minutes after mixing. The pellet was suspended in PBS to 0.2 mg / ml.

[0029] Vaccine mixture preparation: Dilute the pilus protein FimA purified in the above-mentioned Example 2 to 100ug / ml in PBS; mix the diluted vaccine protein with an equal volume of adjuvant to obtain the FimA vaccine mixture.

[0030] Preparation of control solution: mix equal volumes of PBS and adjuvant.

[0031] The composition of PBS is calculated 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.

[0032] Step 2) Immunization application of the vaccine. 70 turbots (each weighing about 12.6g) were randomly divided into 2 groups, 35 in each group. These 2 groups are name...

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Abstract

The invention relates to the field of molecular biology, and particularly relates to an application of edwardsiella tarda pilin FimA. The edwardsiella tarda pilin FimA is used as a subunit vaccine protein. Meanwhile, the edwardsiella tarda pilin FimA can be used as a vaccine adjuvant to be applied to a vaccine. When being used as the subunit vaccine, the pilin can be used for effectively protecting fish from being infected with edwardsiella tarda, when being used as the adjuvant, the pilin can be used for remarkably improving the immune protection effect of the subunit vaccine.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of the Edwardsiella tarda pilus protein FimA. Background technique [0002] Edwardsiella tarda pilus protein FimA is the main component of pilus, and its molecular weight is 18.4KDa. Because it can enhance the adsorption capacity of bacteria to the host during bacterial infection, pili play an important role in the pathogenic process of bacteria. Studies have found that Edwardsiella tarda FimA has the ability to agglutinate red blood cells, and mutations in the fimA gene lead to a reduction in the ability of Edwardsiella tarda to infect cells and tissues. Therefore, pilus protein FimA is a virulence factor, which is expected to be applied to the prevention and control of bacterial diseases. Contents of the invention [0003] The purpose of the present invention is to provide an immune application of Edwardsiella tarda pilus protein FimA. [0004] To achieve t...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61K39/39A61P31/04
Inventor 孙黎王冲胡永华孙铂光
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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