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Alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase antibody preparation and detection method

A technology of methylglutaryl coenzyme and reductase, applied in the field of molecular biology

Inactive Publication Date: 2015-10-28
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the key enzymes of the biosynthesis of triterpenoids in Alisma alisma

Method used

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  • Alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase antibody preparation and detection method
  • Alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase antibody preparation and detection method
  • Alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase antibody preparation and detection method

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Experimental program
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Embodiment 1

[0065] Using homologous cloning method and RACE technology to clone the full-length cDNA of Alisma 3-hydroxy-3-methylglutaryl-CoA reductase gene, polyclonal antibody preparation method, prokaryotic expression and purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene Diacyl-CoA reductase protein was used to prepare polyclonal antibody in immunized rabbits.

[0066] The full-length cDNA of Alisma 3-hydroxy-3-methylglutaryl-CoA reductase gene was cloned by using the homologous cloning method and RACE technology above, and the full-length AoHMGR was cloned on the basis of the conserved region fragment of the AoHMGR gene obtained earlier. Alisma RNA was extracted by the kit method, cDNA was obtained by reverse transcription with Oligo dT18 primers, the fragment cloning of the conserved region of the AoHMGR gene was carried out, and RACE and full-length cDNA amplification were performed.

[0067] The methods for cloning the conserved region of the above-mentioned AoHM...

Embodiment 2

[0074]Purification is to prepare an antigen affinity purification column by coupling the protein with agarose medium, mix the obtained antiserum with PBS in equal amounts, and slowly load the sample, and elute with glycine elution buffer after the antigen and antibody are combined to obtain The desired purified antibody was immediately dialyzed in PBS at 4°C overnight, and the purity, concentration and titer were determined the next day.

[0075] The method for determining the concentration of the purified antibody comprises the following steps:

[0076] (1) Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution according to 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and mix well.

[0077] (2) Take an appropriate amount of BSA protein standard as needed, add deionized water to dilute to 1 mg / mL (stock solution 5 mg / mL), and mix well.

[0078] Note: In principle, whatever solution t...

Embodiment 3

[0090] Take the purified 3-hydroxy-3-methylglutaryl-CoA reductase protein, and immunize 2-2.5Kg New Zealand white rabbits by subcutaneous injection, 400μg / time, once every 2-3 weeks, and immunize 4 times in total ;Blood testing, determine the titer of the antiserum against the protein by indirect ELISA method, wait for the titer to be greater than 1:50,000 for final blood collection to prepare antiserum, and purify polyclonal antibodies.

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PUM

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Abstract

The invention relates to alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase coded by genes of an amino acid sequence in a sequence table SEQ ID No.2 or a base sequence in a sequence table SEQ ID No.1. The invention further relates to a prokaryotic expression of the alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase, a method for obtaining purified albumen and a preparation method of a polyclonal antibody. The alisma 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase is obtained, the polyclonal antibody of the albumen is prepared, an ELISA detection method and a Western blot detection method of the albumen are obtained, a basis is laid for further AoHMGR functional analysis development, and an important basis is provided for original terpane type triterpenoid bioengineering application.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to the preparation and detection method of the key enzyme gene 3-hydroxy-3-methylglutaryl-CoA reductase antibody in the biosynthetic pathway of Alisma proterane-type triterpenoids. Background technique [0002] Alismatis Rhizoma (Alismatis Rhizoma) is the dried tuber of Alisma orientalis, a plant of the family Alismaceae. It has the functions of diuresis, dampness, and heat release. The original production area is Fujian. Its main medicinal ingredients are Protostane-type tetracyclic triterpenoids, which have obvious anti-hyperlipidemia, blood pressure-lowering, anti-HIV1 and other effects, especially significant anti-cancer activity, which indicates its wide application and development However, its resource distribution is narrow and its content is low. It only exists in a few plant groups such as Alisma, which cannot meet the needs of utilization. Bioengineering is one of the effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C07K16/40C07K16/06
CPCC12N9/0006C07K16/06C07K16/40C12Y101/01088
Inventor 谷巍吴启南巢建国徐飞李思蒙田方耿超
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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