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

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Problems solved by technology

In the research process of protein biological function and related molecular regulation mechanism, the u

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

Experimental program
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Example Embodiment

[0025]Example 1

[0026]A method for preparing a modified fake skeleton transcription factor Lexa polyclonal antibody in this example, wherein the specific embodiment of the recombinant plasmid is constructed is as follows: Deformation pseudocensis Lexa gene sequence obtained from the previous genome sequence, manually synthesized Lexa gene And insert it into a PET-32 expression vector, inserted into a gene nucleic acid sequence such asfigure 1 SEQ ID NO: 1 showed; PET-32 vector is ampic resistance, containing TRX tags and 6xHIS tags. The TRX sulfur oxygen label facilitates reinforcing protein soluble expression, the molecular weight of approximately 20 kDa, located in the N-end of the target gene in the PET-32 vector. The constructed recombinant plasmid was converted into E. coli DH5α, using LB plate containing 100 μg / ml ampicillin, 37 ° C for 12 hours, and the filamental rock, extract plasmid and sequencing, sequencing, the desired recombination Plasmid.

Example Embodiment

[0027]Example 2

[0028]A method of preparing a modified homophilic antibody in the present example, wherein the specific embodiment of the sample expression is as follows: the constructing recombinant plasmids described in Example 1 transformed BL21 DE3 sensitive state cells, inoculation resistance LB Tablet medium, growing 24 hours; 6 monoclons of transformed plates were selected, and 3 ml of resistant liquid medium was seeded; 37 ° C, 220 rpm was cultured to OD600NM0.5-0.6, and 0.5 mM IPTG was induced induced by 3.5 hours; centrifugation The bacteria, ultrasonic crushing, and SDS-PAGE detection expression. The test results show that the protein is expressed in the upper part of the liquid and the inclusion body, and soluble expression is purified.

Example Embodiment

[0029]Example 3

[0030]The method of preparing a variant homophilic antibody in the present example, wherein the specific embodiment of the specific expression is as follows: Selecting the small species of a small expression of a small expression of a small expression in which 60 μL to 200 ml of resistant medium, 37 ° C, cultured for 24 hours; fresh resistance medium to 800 ml, incubation for 1-2 hours, to OD600NM0.5-0.6; 200 μL of 1M IPTG (28 ° C or 37 ° C) induced expression of 3.5 hours; 4 ° C centrifugation The bacteria (66 rpm), precipitate and discarded the upper cleaner, add 30 mlpbst suspension, add 1 mm PMSF, and the ultrasonic wave 200W under ice bath is 6 min; shake the bed at 4 ° C for 1 hour; 133 rpm The second high speed is centrifuged for 15 minutes, take the upper part to the 400 μl of nickel column 4 ° C for 24 hours; collect the nickel column (33 rpm), 20 mm imidazole wash solution to wash the beads, wash it off the miscellaneous protein (1 ml × 3 times) Add 300 μL o...

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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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