Zearalenone hydrolase with improved trypsin resistance
A technology of zearalenone and trypsin, applied in hydrolytic enzymes, microbial-based methods, animal feed, etc.
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Embodiment 1
[0025] Example 1: Synthesis of Zearalenone Hydrolase Gene
[0026] The present invention adopts the gene of wild-type zearalenone hydrolase derived from Clonostachys rosea (GenBank registration number is KR363960.1), which is synthesized by Shanghai Jierui Gene Company (other commercial companies with full gene synthesis can also complete it).
Embodiment 2
[0027] Embodiment 2: the connection of zearalenone hydrolase gene (ZHD101) and cloning vector Taox+PgHT+BBPB
[0028] 1. The pGH plasmid containing the ZHD101 target gene synthesized by the whole gene and the cloning vector Taox+PgHT+BBPB were respectively digested with restriction endonucleases EcoRI and SpeI / XbaI at 37°C for 30min, and the digestion conditions were as follows:
[0029]
[0030] 2. The digested products were subjected to 1% agarose gel electrophoresis and the two target fragments were respectively recovered, and T 4 DNA ligase connection, the connection system is as follows:
[0031] ZHD101 digestion product 7.0 μL Cloning Vector Restriction Products 1.0 μL T 4 DNA ligase
[0032] Use DNA ligase to ligate at 16°C for 16 hours, transform the ligation product into DH5α competent cells and amplify it, then use a plasmid extraction kit to extract the plasmid, digest it with EcoRI and PstI, and run electrophoresis results showing two b...
Embodiment 3
[0033] Embodiment 3: gene fragment Paox+Pgap+SS1 is connected with cloning vector M+Taox+PgHT+PB
[0034] 1. The gene fragment Paox-+Pgap+SS1 was transferred from the cloning vector Paox+SS1+PB preserved by the inventor, and obtained by double digestion with EcoRI and SpeI endonucleases, purification and recovery;
[0035] 2. The cloning vector M+Taox+PgHT+PB is obtained from Example 2, and the connection method between the gene fragment Paox+Pgap+SS1 and the cloning vector M+Taox+PgHT+PB is the same as in Example 2.
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