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Preparing and detecting method for bacterium phosphopantetheinyl transferase antibody

A bacterial phosphopantetheinyl, phosphopantetheinyl technology, applied in the field of molecular biology

Inactive Publication Date: 2016-04-20
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports related to the preparation and detection methods of bacterial phosphopantetheinyl transferase antibodies

Method used

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  • Preparing and detecting method for bacterium phosphopantetheinyl transferase antibody
  • Preparing and detecting method for bacterium phosphopantetheinyl transferase antibody
  • Preparing and detecting method for bacterium phosphopantetheinyl transferase antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The expression and purification of bacterial phosphopantetheinyl transferase protein is carried out according to the following steps:

[0028] (1) Cloning enzyme protein gene sequence

[0029] Primers F1 and R1 were designed by comparing the homologous gene sequences of phosphopantetheinyl transferases of strains such as Shewanellasp.SC2A and Shewanellasp.Ac10 provided by GnenBank (see Table 1). The amplification uses the genome of Shewanellasp.Ac10 as a template, and the amplification reaction system is as follows: 5 μl of 10×PCR reaction buffer, 25mmol / LMgSO 4 2μl, 5μl of 10mmoldNTP, 1μl of each 10nmol / L primer, 80ng of genomic DNA template, 1μl of KoD-Plus DNA polymerase, and distilled deionized water to 50μl. The reaction conditions were: pre-denaturation at 95°C for 5 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, and 68°C for 1.30 min; extension at 68°C for 10 min; 15°C for 10 min. The PCR amplified products were subjected to 1% agarose gel electrophoresis, rec...

Embodiment 2

[0039] The method for preparing an antibody by bacterial phosphopantetheinyl transferase comprises the following steps:

[0040] (1) preparing higher-purity phosphopantetheinyl transferase;

[0041] (2) Using higher-purity phosphopantetheinyl transferase as antigenic protein, adopting conventional polyclonal antibody preparation method to prepare antiserum;

[0042] (3) The antiserum is subjected to antibody purification treatment.

[0043] In step (1), the preparation method of higher-purity phosphopantetheinyl transferase is to take the purified phosphopantetheinyl transferase for polyacrylamide gel electrophoresis, and then perform gel electrophoresis. Staining, after staining, cut out the phosphopantetheinyl transferase protein in polyacrylamide gel, place the cut gel piece in a dialysis bag, and conduct further electrophoresis in an electrophoresis tank to separate out the protein completely. And remove the gel block, concentrate to obtain higher purity antigenic protei...

Embodiment 3

[0047] The detection methods of polyclonal antibody derived from rabbits for phosphopantetheinyl transferase include determination of purified antibody concentration, ELISA titer determination, and Western Blotting antibody specificity determination.

[0048] The method for determining the concentration of the purified antibody comprises the following steps:

[0049] (1) Preparation of CBB staining solution: According to the number of samples, dilute 5×CBB staining solution and mix well.

[0050] (2) Take an appropriate amount of BSA standard protein as needed, prepare standard samples with final concentrations of 1mg / ml, 0.75mg / ml, 0.5mg / ml, 0.25mg / ml and 0.125mg / ml, and mix well. The standard sample uses PBS as solvent.

[0051] (3) Take a microtiter plate and add reagents according to Table 2.

[0052] Table 2 Reagents for ELISA plate

[0053] Hole number

A

B

C

D

E

F

Standard concentration (mg / ml)

0

0.125

0.25

0.5 ...

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Abstract

The invention relates to a phosphopantetheinyl transferase. The phosphopantetheinyl transferase has a base sequence of a sequence table SEQ ID No.1, or has an amino acid sequence of a sequence table SEQ ID No.2. The invention further relates to a prokaryotic expression method for the phosphopantetheinyl transferase, a method for obtaining purified protein and a preparing method for a polyclonal antibody. According to the phosphopantetheinyl transferase antibody, an external-source phosphopantetheinyl transferase is obtained, and the polyclonal antibody of the protein and a Western blotting detecting method of the protein are obtained, the foundation is laid for further developing functional analysis of phosphoric-acid pantetheine in a bacterium, and the important basis is provided for a polyketide synthase system in the bacterium body in bioengineering.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to the preparation and detection method of an enzyme protein phosphopantetheinyl transferase antibody necessary in the biosynthetic pathway of eicosapentaenoic acid in bacteria. Background technique [0002] Bacteria Shewanellasp.Ac10 is a low-temperature bacterium isolated from seawater, which can grow well at low temperature and produce eicosapentaenoic acid. Eicosapentaenoic acid has the functions of anti-oxidation, anti-aging, and prevention of cardiovascular and cerebrovascular diseases. It can also greatly reduce the risk of cancer. These benefits to humans have caused scientists to pay great attention to eicosapentaenoic acid, indicating that it has broad application value. It only exists in plants and marine organisms and is necessary for human life, but humans and animals cannot synthesize it. Therefore, the use of biosynthesis is an alternative route to produce eicosapenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07K16/40G01N33/573
CPCC07K16/40C12N9/12G01N33/573
Inventor 宫春杰张华山
Owner HUBEI UNIV OF TECH
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