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Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof

A technology of outer membrane protein gene and Aeromonas hydrophila, applied in application, genetic engineering, bacterial antigen components, etc., can solve problems such as cell membrane instability

Inactive Publication Date: 2015-10-07
郑宗林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial variants lacking ompA and cell wall lipoproteins are spherical in shape and have unstable cell membranes

Method used

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  • Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof
  • Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof
  • Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] A prokaryotic expression protein of the Aeromonas hydrophila outer membrane protein gene, the amino acid sequence of the prokaryotic expression protein of the Aeromonas hydrophila outer membrane protein gene is SEQ ID NO.1, and the Aeromonas hydrophila The nucleotide sequence of the outer membrane protein gene is SEQ ID NO.2. The prokaryotic expression protein of the Aeromonas hydrophila outer membrane protein gene can be used as an antigen in the preparation of a vaccine preparation for preventing Aeromonas hydrophila. The vaccine preparation for preventing Aeromonas hydrophila also includes Freund's incomplete adjuvant. The volume ratio of the Freund's incomplete adjuvant to the prokaryotic expression protein of the Aeromonas hydrophila outer membrane protein gene is 1:1.

[0052] A method for preparing the prokaryotic expression protein of the outer membrane protein gene of Aeromonas hydrophila, comprising the following steps:

[0053] Step 1: Genomic DNA extractio...

Embodiment 2

[0057] Amplification, T clone and identification of embodiment 2 Aeromonas hydrophila ompA gene

[0058] Using the genomic DNA of Aeromonas hydrophila PDK06 as a template, according to the designed PCR primers, such as figure 1 As shown, a target fragment located in the interval of 1000bp-1100bp was amplified by PCR. After the PCR product was recovered and purified by gel, it was ligated with the pMD19-T vector to obtain the recombinant plasmid pMD19-T-ompA. The results of sequencing the recombinant plasmid pMD19-T-ompA showed that the ompA gene of the Aeromonas hydrophila PDK06 strain was composed of 1035 The complete open reading frame (ORF) composed of bases encodes a protein with a length of 275 amino acids, and the recombinant plasmid is confirmed to be correct by enzyme digestion and PCR.

Embodiment 3

[0059] The PCR amplification of embodiment 3 Aeromonas hydrophila ompA gene, T clone and the construction of expression vector

[0060] After being recovered by gel, it was cloned into pMD19-T vector, and then identified by single and double enzyme digestion and sequencing. The results of single and double enzyme digestion identification are as follows: figure 2 Shown: M1 is the DNA marker DL2000, 1 is the PCR amplification product of the pompA gene, 2 is the product of double digestion of pMD19-T-pompA by BamH Ⅰ and Hind Ⅲ, 3 is the product of single digestion of pMD19-T-pompA by BamH Ⅰ , M2 is a DNA marker 10000; the linearized recombinant vector is about 3600bp after single enzyme digestion, and two bands appear after double enzyme digestion, about 1000bp and 2500bp respectively, and the similarity between the sequencing result and the reference sequence for primer design is 100%. Such as image 3 Shown: M1, DNA Marker (DL2,000); 1, PCR amplification product; 2, pET-32a(...

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Abstract

The invention belongs to the biological technical field, and particularly relates to aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and an application thereof. An outer membrane protein gene segment is cloned into a prokaryotic expression vector pET-30a by the protein, a recombinant expression vector is obtained, the recombinant expression vector is converted into escherichia coli to obtain recombinant escherichia coli, the recombinant escherichia coli is inoculated into an Amp resistant LB panel to be subjected to cultivating until OD 600 reaches 0.6, IPTG is added, the final concentration reaches 1.0 mmol / L, inducible expression is carried out, and after purification is conducted, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is obtained. The aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is obtained successfully, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein can serve as vaccine which has the protecting effect on fishes, the immune effects on channel catfish by the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein pompA under the different FIA effects are compared and researched, and the basis is provided for the further research whether ompA can serve as a candidate ingredient of genetic engineering subunit vaccine or not.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a prokaryotic expression protein of the outer membrane protein gene of Aeromonas hydrophila and an application thereof. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila) widely exists in fresh water, sewage and soil in nature, and infects humans through polluted water and related products. The clinical symptoms are gastroenteritis, skin and soft tissue infection, and it is also one of the more serious pathogenic bacteria in freshwater aquaculture. Can cause hemorrhagic septicemia, hydrops, ulcers, asymptomatic septicemia and exophthalmos in fish. There are many serotypes of this bacterium, which vary with different regions and hosts, which limits the scope of use of inactivated vaccines. The key to solving this problem is to find out the common protective antigens of Aeromonas hydrophila, so as to prepare vaccines that have protective eff...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/70C12N15/31A61K39/02A61P31/04
CPCA61K39/02C07K14/195
Inventor 郑宗林黄朝芳王广军
Owner 郑宗林
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