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Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein

A technology of Aeromonas hydrophila and outer membrane protein gene, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., and 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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  • Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein
  • Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein
  • Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein

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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 BamHI and HindIII, 3 is the product of single digestion of pMD19-T-pompA by BamHI, M2 is DNA marker 10000; the linearized recombinant vector is about 3600bp in single enzyme digestion, and two bands appear in 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(+)-pompA d...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein. The method comprises the following steps that the protein clones an outer membrane protein gene segment into a prokaryotic expression vector pET-30a to obtain a recombinant expression vector, the recombinant expression vector is converted into Escherichia coli to obtain recombinant Escherichia coli, the recombinant Escherichia coli is inoculated to an Amp-resistant LB plate to be cultured for OD600 for 0.6 h, IPTG is added to reach the final concentration being 1.0 mmol / l for inducible expression, and the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is obtained after purification is performed. According to the method, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein is successfully constructed, the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein can be used as a vaccine capable of protecting fish, the immune effects of the aeromonas hydrophila outer membrane protein gene prokaryotic expression protein pompA for channel catfish under different FIA effects are compared and researched, and a foundation is laid for further researching the pompA and researching whether the pompA can be used as the candidate component of the 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 method for preparing prokaryotic expression protein of Aeromonas hydrophila outer membrane protein gene. 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 effects on diffe...

Claims

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Application Information

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