3 alpha hydroxysteroid dehydrogenase mutant and application thereof in total bile acid detection
A steroid dehydrogenase and mutant technology, which is applied in the field of human serum total bile acid detection kits, can solve the problems of low enzyme protein yield, low enzyme stability, complex extraction process, etc.
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Embodiment 1
[0028] Example 1. Construction of a Pichia pastoris library expressing 3α-HSD mutants using the error-prone PCR method
[0029] Nucleotide mutations were introduced in vitro into the 3α-HSD gene of Comamonas testosterone using error-prone PCR. The reaction conditions for error-prone PCR are as follows:
[0030] Wherein, the sequences of the upstream primer F and the downstream primer R are:
[0031] F: 5'-TCAAGATC CCTAGG ATGACTGGTCCCCTGATCTAT-3';
[0032] R: 5'-AATTCCAGT GCGGCCGC TCAGTTCAGTGGCACGGCTTC-3'.
[0033] PCR amplification conditions: 94°C for 3 min; 94°C for 1 min, 59°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min.
[0034] After the error-prone PCR amplification product is purified by the DNA purification kit, the restriction endonucleases AvrⅡ and NotI digest the error-prone PCR amplification product and the plasmid pPIC9K, respectively, and connect to obtain the expression plasmid containing the mutant gene, which is transformed into E .coli JM109...
Embodiment 2
[0036] Example 2. Construction of a Pichia pastoris library expressing 3α-HSD mutants using the DNA Shuffling method
[0037]The mutation sites in the 3α-HSD mutant constructed by error-prone PCR were randomly combined by DNA Shuffling method. The conditions for DNA shuffling are as follows: extract the genome of the Pichia pastoris library expressing Comamonas testosteronei 3α-HSD mutant constructed by the error-prone PCR method, digest it with DNase I for 30 min, extract the digested product with phenol-chloroform to remove the protein, and dissolve it in 30 μL sterile water. Using the genome as a template, proceed as follows:
[0038] Step 1: PCR reaction system:
[0039] Taq (2.5U) 0.5 μL
[0040] 5×buffer (Mg 2+ plus) 10μL
[0041] Genome 0.5 μL
[0042] dNTP (25mmol / L) 4 μL
[0043] dd H2O 34.5 μL
[0044] PCR amplification conditions: 94°C for 3 min; 10 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min; 72°C for 10 min.
[0045] Step 2: Add 1 μL eac...
Embodiment 3
[0047] Example 3, Screening of mutants capable of stably expressing 3α-HSD activity
[0048] Copy the Pichia library stored on the MD plate to the MM plate, culture at 30°C for 2 days, and use the Pichia expressing the parent Comamonas testosteronei 3α-HSD as the control bacteria.
[0049] Plate initial screening: Add 200 μL of methanol to the cover of the MM plate every 12 hours to induce the expression of recombinant 3α-HSD. After 2-3 days of induction, heat the plate at 65°C for 60 minutes, cool to room temperature, and pour 18 mL evenly onto the plate. 0.2M Tris-Cl at pH 8.0; 0.25% NTB (nitrotetrazolium blue); 10mmol NAD 2ml; (100μ / ml); diaphorase (diaphorase); 2% Triton X-100 ; 20mmol androsterone 1ml; the balance is water, and the colonies whose red color is darker than that of the control within 2 minutes are the strains of primary screening purpose.
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