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Application of bacterial filamentous hemagglutinin Fha recombinant protein

A technology of hemagglutinin and recombinant protein, which is applied in the application field of bacterial filamentous hemagglutinin Fha recombinant protein, and can solve the problems of no effective immune prevention and control methods

Active Publication Date: 2016-11-23
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, there is no effective immune control method against this bacteria

Method used

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  • Application of bacterial filamentous hemagglutinin Fha recombinant protein
  • Application of bacterial filamentous hemagglutinin Fha recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Preparation of Fha Recombinant Protein rFha

[0018] Step 1) Construction of the expression vector pEtFha of rFha:

[0019] The sequence of Pseudomonas fluorescens Fha has been reported (GenBank accession number. WP_014719704.1). The sequence contains a haemagglutination activity domain consisting of amino acids 1-684.

[0020] The construction of pEtFha is as follows: Pseudomonas fluorescens TSS1 is used as a template, and the hemagglutination activity domain of Fha is amplified by primers F1 / R1. The PCR conditions were: 94°C for 60s to pre-denature template DNA, then 94°C for 40s, 62°C for 60s, 72°C for 60s, 30 cycles. PCR products were purified with corresponding kits from Tiangen. The expression vector pET259 (see HuYH, Zheng WW, Sun L. Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus) for the construction process of pET259. Fish Shellfish Immunol 2010; 28:678-86) with restriction enzyme SwaI After digestion, connect...

Embodiment 2

[0028] Immunization application of rFha as a vaccine

[0029] Step 1) Preparation of adjuvant and vaccine mixture.

[0030] Preparation of adjuvant control solution: mix 5% (mass ratio) NaOH and 5% (mass ratio) Al 2 (SO 4 ) 3 Mix in a 2:5 volume ratio, and centrifuge the mixture at 10,000g for 5 minutes. Suspend the precipitate in PBS to 0.2mg / ml, which is the adjuvant. Mix equal volumes of PBS and adjuvant, which is the adjuvant control solution.

[0031] Vaccine mixture preparation: the rFha purified in the above-mentioned Example 1 was diluted to 200ug / ml in PBS, and the diluted protein was mixed with the adjuvant control solution in equal volumes to obtain the rFha vaccine mixture.

[0032] The composition of the 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.

[0033] Step 2) Immunization application of the vaccine. 60 turbots (each weighing about 14 g) were randomly divided into 2 gr...

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Abstract

The invention relates to the field of molecular vaccinology, in particular to application of bacterial filamentous hemagglutinin Fha recombinant protein. The bacterial filamentous hemagglutinin Fha recombinant protein is used for preparing immune vaccines of pseudomonas fluorescens. An Fha recombinant protein preparation method includes: adopting pseudomonas fluorescens TSS1 as a template and adopting F1 / R1 as a primer to carry out PCR (polymerase chain reaction) amplification, and connecting PCR products with a carrier pET259 to obtain plasmid pEtFha, and transforming escherichia coli BL21(DE3) to express the Fha recombinant protein. The Fha recombinant protein serving as a vaccine is effective in protection of scophthalmus maximus from infection of pseudomonas fluorescens.

Description

technical field [0001] The invention relates to the field of molecular vaccinology, in particular to the application of a bacterial filamentous hemagglutinin Fha recombinant protein. Background technique [0002] Filamentous hemagglutinin (Fha) is an adhesin produced by bacteria and plays an important role in the process of bacterial adhesion to the surface of host cells. Fha can either be expressed on the bacterial outer membrane or secreted into the environment around the bacteria. Functionally, Fha can mediate bacterial adhesion to host cells, promote bacterial biofilm formation and autoagglutination. In addition, Fha also has immunomodulatory effects. Fha is also an important immunogen, which has a good immune protective effect against Bordetella bronchiseptica in mouse experimental models. [0003] Pseudomonas fluorescens is a Gram-negative bacterium that occurs widely in nature. Pseudomonas fluorescens is an important pathogenic bacteria, which can not only infect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/104A61P31/04C12N15/31C12N15/70
CPCA61K39/104C07K14/21C12N15/70C12N2800/101
Inventor 孙黎孙园园
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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