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A dopa decarboxylase homologue with strong catalytic activity and its preparation and application

A technology of dopa decarboxylase and homologues, applied in the field of molecular biology, can solve the problems of developmental delay, cognitive impairment, inability to resist external physical and pathogenic attacks, etc.

Active Publication Date: 2020-10-20
INNER MONGOLIA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current research results show that dopa decarboxylase can participate in various biological mechanisms, and participate in the synthesis of melanin in mammals. At the same time, the lack of dopa decarboxylase can lead to developmental delay, cognitive impairment, mental disorder and other diseases; Insects participate in the process of growth and development and epidermis formation, and the lack of dopa decarboxylase will lead to soft exoskeleton and cannot resist external physical and pathogenic attacks

Method used

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  • A dopa decarboxylase homologue with strong catalytic activity and its preparation and application
  • A dopa decarboxylase homologue with strong catalytic activity and its preparation and application
  • A dopa decarboxylase homologue with strong catalytic activity and its preparation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1 Preparation of recombinant Procambarus clarkii dopa decarboxylase homologue

[0028] (1) The total RNA of the mixed tissue samples of Procambarus clarkii hepatopancreas, gills and intestines was extracted by using the animal total RNA extraction kit, and the total RNA samples of the mixed tissues of Procambarus clarkii were obtained.

[0029] (2) Using the total RNA sample of Procambarus clarkii obtained in step (1) as a template, using the specific primer 5'-CTACTGCAAAAGCGGGAG-3' as the back primer of the reverse transcription synthesis reaction, using a one-step RT-PCR amplification kit The reverse transcription synthesis of cDNA was carried out to obtain the cDNA samples of mixed tissue samples of Procambarus clarkii.

[0030] (3) Design and amplify the front and back primers Pc-ddc-i-F and Pc-ddc-i-R of the expression fragment (mature peptide) of the Pc-ddc-i coding region of the Procambarus clarkii homologue gene Pc-ddc-i:

[0031] Pc-ddc-i-F: 5′-tact...

Embodiment 2

[0040] Example 2 Experiment of the Catalytic Activity in Vitro of Recombinant Procambarus clarkii Dopa Decarboxylase Homologue rPc-DDCi

[0041] The concentration obtained in Example 1 was 200 μg·mL -1 The objective recombinant protein rPc-DDCi was used to catalyze the conversion of levodopa to dopamine in vitro. First, make a standard curve of dopamine concentration: prepare a set of dopamine standard solutions with concentrations of 1000, 333.33, 111.11, 37.04, and 12.35 pg / mL, and use a microplate reader to measure the absorbance at a wavelength of 450 nm. Afterwards, the absorbance value was used as the abscissa, and the logarithm of the concentration value was used as the ordinate to make a standard curve of absorbance versus dopamine concentration. Afterwards, the experiment of the target recombinant protein rPc-DDCi catalyzing levodopa was carried out: ① Take 0.591 g of levodopa and 0.0106 g of pyridoxal phosphate, and dissolve them in 100 mL of PBS. ②Adjust the pH to...

Embodiment 3

[0043] Example 3 The Experiment of Recombinant Procambarus clarkii Dopa Decarboxylase Homologue rPc-DDCi to Promote Hemocyte Encapsulation Activity in Vitro

[0044] The concentration obtained in Example 1 was 200 μg·mL -1 The purpose of the recombinant protein rPc-DDCi is to carry out the detection experiment on the hemocyte encapsulation activity of Procambarus clarkii in vitro. The first step is to process the agarose microspheres: draw 200 μL of agarose microspheres from the His-tag gel column into a 1.5 mL sterilized centrifuge tube, then add 1 mL of 1×PBS, and then turn it upside down or vortex Wash for 5-10 min, then centrifuge at 1500 rpm for 1 min. Repeat the above steps 4 times. The second step is to dilute the agarose microspheres: resuspend the treated agarose microspheres with 1mL 1×PBS, take a drop of the respinned microsphere liquid and place it on a glass slide, and observe the microspheres under a 40X microscope. If the concentration of microspheres is too ...

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Abstract

The invention discloses a dopamine decarboxylase homologue with strong catalytic activity as well as preparation and application thereof and belongs to the technical field of molecular biology. The dopamine decarboxylase homologue disclosed by the invention is derived from procambarus clarkii and has an amino acid sequence shown as SEQ ID NO. 1; the preparation comprises the following steps: extracting total RNA (Ribonucleic Acid) of the procambarus clarkii as a template and carrying out reverse transcription to obtain cDNA (complementary Deoxyribonucleic Acid); taking the cDNA as a template and carrying out c (Polymerase Chain Reaction) amplification to obtain a nucleotide segment containing a dopamine decarboxylase homologue encoding sequence; carrying out enzyme digestion, connection and conversion on an amplification product and an expression vector to construct escherichia coli engineering bacteria capable of expressing a recombinant homologue; and carrying out induction culture on the escherichia coli engineering bacteria to express a recombinant dopamine decarboxylase homologue. The dopamine decarboxylase homologue provided by the invention has very strong catalytic activity, has the effects of enhancing a blood cell encapsulation capability of the procambarus clarkii and the anti-oxidization capability of organisms and also has a very good bacterium-inhibition effect and a good application prospect.

Description

technical field [0001] The invention relates to a dopa decarboxylase homologue (isoform) with strong catalytic activity and its preparation and application, belonging to the technical field of molecular biology. Background technique [0002] After the long-term pressure of natural selection, the animal body has gradually formed an efficient immune system that can resist the harm of external pathogens to the body itself. The structure of this system is relatively complex, and the internal connections are also very ingenious. The process of immunity is that the animal body uses its own immune system to resist the invasion of the body by external pathogenic microorganisms, so as to effectively protect itself and avoid the occurrence of various diseases. The immune system of animals has gradually evolved two more complex anti-infection mechanisms, innate immunity and acquired immunity, in the process of constantly fighting against pathogens. The two have different functions and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P13/00A23K20/189A61K38/51A61P31/00A61P25/16A61P25/22A61P25/24
CPCA23K20/189A61K38/00A61P25/16A61P25/22A61P25/24A61P31/00C12N9/88C12N15/70C12P13/001C12Y401/01028
Inventor 杜志强沈秀丽任乾王凯
Owner INNER MONGOLIA UNIV OF SCI & TECH
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