Pichia pastoris engineering bacteria constitutively expressing porcine pepsinogen a and its application
A technology of constitutive expression and Pichia pastoris, applied in the field of genetic engineering, can solve problems such as safety and environmental problems, toxicity, and volatile methanol, and achieve good commercial application prospects
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Embodiment 1
[0046] Construction and Identification of Embodiment 1 Recombinant Bacteria
[0047] Based on yeast preferred codons, the porcine pepsinogen A gene (NCBI, NM_213873) was optimized to obtain the polynucleotide sequence SEQ ID NO.:1. The polynucleotide sequence shown by SEQ ID NO.:1 was artificially synthesized by Sangon Bioengineering Co., Ltd. According to the instructions provided by Invitrogen, the nucleotide sequence was cloned between the EcoR I and Not I restriction sites on the expression vector pGAPZαA (Invitrogen), to obtain the recombinant plasmid pGAPZαA-PGA (see pGAPZαA-PGA for the plasmid map). figure 1 ). Using EcoR I and Not I enzymes to carry out electrophoresis on the 1wt% agar gel containing EB after the recombinant plasmid was digested, the result showed that the size of the inserted gene sequence was correct ( figure 2 ).
[0048] Then, the recombinant vector pGAPZαA-PGA was introduced into Pichia pastoris GS115 strain (Invitrogen Company) by electropora...
Embodiment 2
[0052] Embodiment 2 uses the Pichia pastoris engineered bacterium of embodiment 1 to produce porcine pepsinogen A
[0053] The Pichia pastoris GS115-pGAPZαA-PGA constructed in Example 1 was used to ferment and produce porcine pepsinogen A, and its enzyme activity was determined.
[0054] Streak the Pichia engineered bacteria of the present invention on a YPD plate, culture it statically at 30°C for 48 hours, pick a single clone and inoculate it into a test tube containing 5mL of YPD liquid medium, and cultivate it at 30°C and 220rpm for 6 hours, Then they were all transferred to 50mL YPD liquid medium, and cultured continuously for 24h and 48h at 30°C and 220rpm. The fermentation broths of 24h and 48h were taken respectively and centrifuged at 8000rpm for 5min, and the supernatant (in which extracellular PGA was included) was taken for SDS-PAGE analysis. GenScript Biotechnology Co., Ltd. SDS-PAGE precast gel electrophoresis was selected, and the specific operation method was ...
Embodiment 3
[0066] Embodiment 3: the activation of the porcine pepsinogen A produced in embodiment 2
[0067] The enzyme activity of pepsinogen A produced in Example 2 was detected under different pH conditions (see Table 1), and the measurement results are shown in Table 2 below. Except using the following buffer to dilute the fermentation supernatant (50 times) and replace the 0.05mol / L lactic acid buffer solution to prepare 1% casein solution, the same method as the enzyme activity assay method in Example 2 was carried out. Different pH conditions were achieved by using different ratios of 0.2M disodium hydrogen phosphate-0.1M citric acid buffer solution, and the specific ratios are shown in Table 1.
[0068] Table 1 Different pH (20mL buffer system)
[0069] pH 0.2M Disodium Hydrogen Phosphate (mL) 0.1M citrate buffer (mL) pH3.0 4.11 15.89 pH3.4 5.70 14.30 pH4.0 7.71 12.29 pH4.4 8.82 11.18 pH5.0 10.30 9.70 pH5.4 11.15 8.85
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