Antrodia camphorata immunomodulatory protein ACA1 and a coding gene thereof and application thereof
An immunomodulatory activity, protein technology, applied in application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0052] This embodiment provides an optimized artificially synthesized ACA1 gene, the specific sequence of which is shown in sequence 1 in the sequence listing, and the protein sequence corresponding to the gene is shown in sequence 2 in the sequence listing. The sequence before optimization in this example is based on the DNA sequence provided by the NCBI database, which is the natural DNA for the synthesis of ACA1, and the optimized DNA was optimized and synthesized according to the expression characteristics of Escherichia coli. Compared with the natural polynucleotide of ACA1 (GenBank: AY569691.1), the optimized DNA sequence has little obvious homology.
[0053] The genes before and after optimization were connected to the E. coli expression vector pET28 to obtain recombinant vectors. The above recombinant vectors verified by sequencing were transformed into competent cells of E. coli expression strains by heat shock, and the corresponding resistant LB plates were coated. 37...
Embodiment 2
[0056] This embodiment provides a method for preparing protein, which specifically includes the following steps:
[0057] S1: Optimizing the gene, constructing the prokaryotic expression vector and transforming: artificially synthesize the optimized mature ACA1 gene, and connect it to the expression vector pET28 to obtain the recombinant expression vector pET28 / ACA1. The vector construction is as follows: figure 1 shown. The main vector construction steps are as follows:
[0058] (1) Digest the recombinant vector with BamH I and Hind III to obtain the target fragment. The reaction system is as follows (all endonucleases and buffers used are purchased from Dalian TAKARA Company):
[0059] Vector ACA1 / pUC 15 μL
[0060] 10×H buffer 5 μL
[0061] BamH I 5U
[0062] Hind III 5U
[0063] Sterile water to 50μL
[0064] (2) Digest pET28 with BamH I and Hind III to obtain vector fragments. The reaction system is as follows (endonucleases and buffers used are purchased from Dalia...
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