Zearalenone hydrolase with improved pepsin resistance
A technology for zearalenone, pepsin, applied in the directions of hydrolases, microorganism-based methods, biochemical equipment and methods, etc.
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Embodiment 1
[0029] Example 1: Synthesis of Zearalenone Hydrolase Gene
[0030] The present invention adopts the gene of wild-type zearalenone hydrolase 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
[0031] Example 2: Connection of Zearalenone Hydrolase Gene (ZHD101) to Cloning Vector Taox+PgHT+BBPB
[0032] 1. The pGH plasmid containing the ZHD101 target gene and the cloning vector Taox+PgHT+BBPB (constructed by the inventor's laboratory) were digested with restriction endonucleases EcoRI and SpeI / XbaI at 37°C for 30min respectively, The digestion conditions are as follows in Table 1:
[0033] Table 1:
[0034]
[0035] 2. After the digestion product was electrophoresed on a 1% agarose gel, the two target fragments were recovered respectively, and T 4 DNA ligase ligation, the ligation system is as shown in Table 2:
[0036] Table 2:
[0037] ZHD101 digestion product 7.0μL Cloning vector digestion product 1.0μL T 4 DNA ligase
1.0μL T 4 DNA ligase buffer
1.0μL ddH 2 O
1.0μL total capacity 10.0μL
[0038] Use DNA ligase at 16°C for 16 hours. The ligation product was transformed into DH5α competent cells...
Embodiment 3
[0040] Example 3: Gene fragment Paox+Pgap+SS1 is connected to cloning vector M+Taox+PgHT+PB
[0041] 1. The gene fragment Paox-+Pgap+SS1 was transferred from the cloning vector Paox+SS1+PB preserved by the inventor's research institution (Institute of Microbiology, Jinan University), and was digested with EcoRI and SpeI endonuclease and purified and recovered get. Inserting the gene fragment Paox+Pgap+SS1 into the cloning vector M+Taox+PgHT+PB can finally realize the extracellular expression of ZHD101 protein, which is beneficial to experiments such as later purification of the protein.
[0042] 2. The cloning vector M+Taox+PgHT+PB was obtained in Example 2, and the connection method of the gene fragment Paox+Pgap+SS1 and the cloning vector M+Taox+PgHT+PB was the same as that in Example 2.
[0043] After the above two steps, the gene fragment Paox+Pgap+SS1 was successfully inserted into the cloning vector M+Taox+PgHT+PB, and the expression cassette was constructed successfull...
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