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Pseudomonas plecoglossicida HtpG antigen and preparation method of polyclonal antibody of Pseudomonas plecoglossicida HtpG antigen

A Pseudomonas mutans and polyclonal antibody technology, applied in the biological field, to achieve the effect of strong immunogenicity of antibodies and simple methods

Pending Publication Date: 2021-05-25
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies are essential scientific reagents in the process of molecular signal transduction research, and there are no commercially available Pseudomonas mutans HtpG antigens and antibodies on the market

Method used

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  • Pseudomonas plecoglossicida HtpG antigen and preparation method of polyclonal antibody of Pseudomonas plecoglossicida HtpG antigen
  • Pseudomonas plecoglossicida HtpG antigen and preparation method of polyclonal antibody of Pseudomonas plecoglossicida HtpG antigen
  • Pseudomonas plecoglossicida HtpG antigen and preparation method of polyclonal antibody of Pseudomonas plecoglossicida HtpG antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A method for preparing Pseudomonas mutans HtpG antigen and its polyclonal antibody in this example, wherein the specific implementation steps of constructing the recombinant plasmid are as follows: according to the codon preference of Escherichia coli, artificially synthesize the HtpG gene and insert it into pET-32 In the expression vector, insert gene nucleic acid sequence such as figure 1 Shown in SEQ ID NO: 1; the pET-32 vector is ampicillin-resistant and contains Trx tags and 6xHis tags. The Trx thioredoxin tag is beneficial to enhance the soluble expression of the protein, the molecular weight is 20kDa, and it is located at the N-terminal of the target gene in the pET-32 vector. Transform the constructed recombinant plasmid into Escherichia coli DH5α, use an LB plate containing 100 μg / ml ampicillin, incubate at 37°C for 12 hours, pick a single colony and shake it backward, extract the plasmid and sequence it, and the sequenced one is the desired recombinant plasmid...

Embodiment 2

[0030] The preparation method of a kind of Pseudomonas mutans HtpG antigen and its polyclonal antibody of the present example, wherein the specific implementation steps of small sample expression are as follows: transform the recombinant plasmid constructed as described in Example 1 into BL21 DE3 competent cells, inoculate resistant LB plate medium, grow for 24 hours; select 6 single clones on the transformed plate, inoculate 3ml of resistant liquid medium respectively; culture at 37°C, 220RPM until the OD600nm value is between 0.5-0.6, add 0.5mM IPTG at 20°C to induce expression 3.5 hours; the cells were collected by centrifugation, sonicated, and the expression was detected by SDS-PAGE. The detection results showed that the protein was expressed in both the supernatant and the inclusion body, and the soluble expression and purification could be continued.

Embodiment 3

[0032]A method for preparing Pseudomonas mutans HtpG antigen and its polyclonal antibody of this example, wherein the specific implementation steps of large-scale expression are as follows: select the well-expressed bacterial strain of the small sample described in Example 2 and inoculate 60 μl to 200ml of resistance medium , 37°C, 220RPM culture for 24 hours; add fresh resistance medium to 800ml, culture for 1-2h until the OD600nm value is between 0.5-0.6; add 200μl of 1M IPTG (28°C or 37°C) to induce expression for 3.5h; Collect the cells by centrifugation at 4°C (66 rpm × 15min), discard the supernatant, add 30ml PBST to suspend the cells, add a final concentration of 1mM PMSF, and ultrasonically break at 200W for 6min in an ice bath; incubate at 4°C for 1h; 133 Centrifuge at high speed for 15 min at rpm, take the supernatant, add 400 μl nickel column to combine at 4°C for 24 hours; collect nickel column (33 rpm × 5 min), wash beads with 20 mM imidazole washing solution, and...

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Abstract

The invention discloses a Pseudomonas plecoglossicida H tpG antigen and a preparation method of a polyclonal antibody of the Pseudomonas plecoglossicida H tpG antigen. The method includes: the following steps: firstly, artificially synthesizing an H tpG gene according to codon preference of escherichia coli, inserting the H tpG gene into a pET-32 expression vector, and selecting positive clone for sequencing verification; then, transforming the constructed plasmid into BL21DE3 competent cells, performing culturing, inducing, splitting and centrifuging to obtain protein, and detecting the expression condition of the protein by SDS-PAGE; further, selecting a strain with good sample expression for inoculation, culture, induction, splitting decomposition and purification, obtaining protein, and identifying the molecular weight, purity and concentration of the protein through SDS-PAGE; furthermore, taking the obtained H tpG protein as an antigen for animal immunization, collecting and purifying antiserum, and obtaining the H tpG antibody. The HtpG antigen of pseudomonas proteinans is obtained, and the corresponding HtpG polyclonal antibody is prepared. The titer of the antiserum and the affinity purification antibody is detected by an indirect ELISA method, and the result shows that the yield of the affinity purification antibody is normal. The method is simple, and the prepared antibody is high in immunogenicity.

Description

technical field [0001] The invention relates to an antibody in the field of biotechnology and a preparation method thereof, in particular to a preparation method of a Pseudomonas mutans HtpG antigen and a polyclonal antibody thereof. Background technique [0002] Pseudomonas mutans is a common opportunistic pathogen in aquaculture. Studies have shown that many disease outbreaks in aquaculture are caused by Pseudomonas mutans. Pseudomonas mutans can proliferate in aquaculture water and spread disease through water diffusion. The research group Hu Jiao et al. isolated Pseudomonas mutans NZBD9 from diseased large yellow croakers cultured in artificial cages. There were no special changes on the body surface of the sick large yellow croaker, but obvious white spots were found in the internal organs. This visceral white spot disease has the characteristics of long incubation period, high mortality, and strong temperature dependence. The pathogenic temperature is about 18°C. It...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/31C07K16/12C07K16/06
CPCC12N15/70C07K14/21C07K16/1214C07K16/06C07K16/065C12N2800/22C07K2319/21C07K2319/35
Inventor 章幼玉黄力行王佳佳何荣超鄢庆枇
Owner XIAMEN UNIV
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