Clam dopa decarboxylase gene and its encoded protein and application
The technology of dopa decarboxylase and barium decarboxylase of clams is applied in the fields of molecular biology and bioengineering, which can solve the problems of obtaining dopa decarboxylase gene and the like, and achieve the effect of making up for the low amount of natural protein
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0017] The clam dopa decarboxylase gene has the sequence shown in SEQ ID NO.1 in the sequence table.
[0018] SEQ ID NO.1 (The part in bold italics is the coding frame sequence, which can encode protein)
[0019]
[0020]
[0021] The cDNA sequence cloning of clam dopa decarboxylase among the present invention comprises the following steps:
[0022] 1) extraction of clam total RNA and purification of mRNA;
[0023] 2) Clam cDNA library construction;
[0024] 3) Large-scale determination of EST sequences of clam cDNA library;
[0025] 4) Homology analysis of clam EST sequences and screening of dopa decarboxylase gene fragments;
[0026] 5) The full sequence of clam dopa decarboxylase obtained by RACE amplification and the verification of the full sequence.
[0027] The specific operation is as follows:
[0028] 1. Extraction of clam total RNA and purification of mRNA: Total RNA was extracted from clam larvae using Trizol reagent from Invitrogen Company, and mRNA was ...
Embodiment 2
[0054] Embodiment 2. Obtaining of clam dopa decarboxylase recombinant protein
[0055] According to the cDNA sequence of the coding frame corresponding to SEQ ID NO.1, design specific primers BamH I-F and Sal I-R containing restriction endonuclease BamH I and SalI restriction sites, and amplify the gene encoding dopa decarboxylase by PCR The gene fragment was then cloned into the pGEX-4T-1 expression vector by enzyme digestion, transformed into Escherichia coli BL21, and after sequencing confirmed that the expression frame was correct, the positive clone was inoculated into the LB medium containing ampicillin (100mg / ml), Incubate at 37°C with shaking until O.D. 600 = 0.4-0.6, add IPTG to a final concentration of 1 mM and induce for 4 hours to collect the cells by centrifugation. Bacteria were treated with ultrasonic wave 200W for 30-60 minutes under the condition of ice bath (1 second each time, 1 second interval). Centrifuge to remove the supernatant, wash the precipitate (...
Embodiment 3
[0067] Example 3. ELISA detection of clam dopa decarboxylase recombinant protein
[0068] The protein concentration determination kit 2-D Quant Kit (GE Healthcare, UK) was used to measure the concentration of the recombinant dopa decarboxylase-GST fusion protein, and then the dopa decarboxylase ELISA detection kit (Qiyi Biotechnology (Shanghai) Co., Ltd. Co., Ltd.) detected clam dopa decarboxylase recombinant protein, and detected the content of dopa decarboxylase contained in it. The results showed that every mg of recombinant dopa decarboxylase-GST fusion protein contained about 594pg of dopa decarboxylase.
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More