Preparation method of pseudomonas protegens transcription factor LexA polyclonal antibody

A Pseudomonas mutans and polyclonal antibody technology, applied in the biological field, achieves the effect of simple method and strong immunogenicity

Pending Publication Date: 2021-04-30
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research process of protein biological function and related molecular regulation mechanism, the use of antibodies is inevitable, but there is no commercial Pseudomonas mutans LexA antibody on the market

Method used

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  • Preparation method of pseudomonas protegens transcription factor LexA polyclonal antibody
  • Preparation method of pseudomonas protegens transcription factor LexA polyclonal antibody
  • Preparation method of pseudomonas protegens transcription factor LexA polyclonal antibody

Examples

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Embodiment 1

[0026] A method for preparing a polyclonal antibody against Pseudomonas mutans transcription factor LexA in this example, wherein the specific implementation steps for constructing a recombinant plasmid are as follows: artificially synthesize the LexA gene according to the sequence of the Pseudomonas mutans LexA gene obtained by previous genome sequencing and insert it into the pET-32 expression vector, wherein the gene nucleic acid sequence is inserted 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 about 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 back, extract the plasmid an...

Embodiment 2

[0028] A method for preparing a polyclonal antibody against Pseudomonas mutans transcription factor LexA in this example, wherein the small sample expression specific implementation steps are as follows: transform the recombinant plasmid constructed as described in Example 1 into BL21 DE3 competent cells, and inoculate resistant LB Plate medium, grow for 24 hours; select 6 single clones from the transformed plate, inoculate 3ml of resistant liquid medium respectively; culture at 37°C, 220RPM to OD600nm0.5-0.6, add 0.5mM IPTG at 20°C to induce expression for 3.5 hours; centrifuge The cells were collected, 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

[0030]A method for preparing a polyclonal antibody against Pseudomonas mutans transcription factor LexA in this example, wherein the specific implementation steps for large-scale expression are as follows: select a small-scale well-expressed strain as described in Example 2 and inoculate 60 μl to 200 ml of resistant medium, Cultivate at 37°C for 24 hours; add fresh resistant medium to 800ml, incubate for 1-2 hours, to OD600nm0.5-0.6; add 200μl of 1M IPTG (28°C or 37°C) to induce expression for 3.5 hours; collect by centrifugation at 4°C Bacteria (66 rpm × 15min), precipitate and discard the supernatant, add 30ml PBST to suspend the bacteria, add a final concentration of 1mM PMSF, and ultrasonically break at 200W for 6min in an ice bath; incubate at 4°C for 1 hour on a shaker; 133rpm Centrifuge at high speed for 15 minutes, take the supernatant, add 400μl nickel column to combine at 4°C for 24 hours; collect the nickel column (33 rpm x 5min), wash the beads with 20mM imidazole w...

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Abstract

The invention discloses a preparation method of a pseudomonas protegens transcription factor LexA polyclonal antibody. The method comprises the following steps: artificially synthesizing an LexA gene according to the LexA gene sequence of Pseudomonas mutans obtained by early-stage genome sequencing, inserting the LexA gene into a pET-32 expression vector, and selecting positive clone for sequencing verification; transforming the constructed plasmid into a BL21DE3 competent cell, culturing, inducing, splitting and centrifuging to obtain protein, and detecting the expression condition of the protein by SDS-PAGE; 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 LexA protein as an antigen for animal immunization, collecting and purifying antiserum, and obtaining the LexA antibody. 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 has strong 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 polyclonal antibody against Pseudomonas mutans transcription factor LexA. Background technique [0002] Large yellow croaker (Pseudosciaena crocea) is the main economic fish unique to China's coastal waters and the marine fish with the largest single species breeding volume at present. It is also one of China's eight advantageous export aquatic products. At present, the national annual breeding volume of large yellow croaker exceeds 3 billion, the aquaculture output is nearly 200,000 tons, and the direct output value exceeds 10 billion yuan. It has become one of the important pillar industries of my country's mariculture industry. However, with the rapid development of large yellow croaker aquaculture in recent years, various diseases of large yellow croaker frequently broke out. Among them, "visceral white sp...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/06C07K1/22C07K14/21C12N15/31C12N15/70
CPCC07K16/1214C07K16/065C07K14/21C12N15/70
Inventor 黄力行章幼玉王佳佳何荣超鄢庆枇
Owner JIMEI UNIV
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