Gene for efficiently expressing hyaluronic acid hydrolase and expression method thereof
A high-efficiency expression technology of hyaluronic acid, which is applied in the field of genetic engineering, can solve the problems of reports and no recombinant expression of hyaluronan hydrolase, and achieve the effect of reducing costs
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Embodiment 1
[0071] Example 1 contains codon-optimized hyaluronan hydrolase gene (HYAL_OM opt ) construction of expression system
[0072] Based on the full-length gene sequence (Genbank: FX985505.1) of the hyaluronan hydrolase in the NCBI database, the codon optimization was carried out according to the codon preference of Pichia pastoris. Such as figure 1 As shown, the optimized gene sequence is shown in SEQ ID NO.1, which has a homology of 73.2% with the wild-type gene sequence. The amino acid sequence encoded by the optimized gene is shown in SEQ ID NO.2, which is consistent with the amino acid sequence encoded by the wild-type gene. The codon-optimized hyaluronan hydrolase sequence was commissioned to Nanjing GenScript Biotechnology Co., Ltd. to synthesize the whole gene, and cloned into the Pichia pastoris expression vector pPIC9K between the EcoRI and NotI restriction sites for recombinant expression Vector pPIC9K-HYAL_OM opt . After DNA sequencing comparison, the recombinant s...
Embodiment 2
[0073] Example 2 contains highly expressed hyaluronan hydrolase gene (ΔN24HYAL_OM opt ) construction of expression system
[0074] With the above pPIC9K-HYAL_OM opt The recombinant expression vector is used as a template, primers are designed, and a signal peptide sequence at the N-terminal of the full-length hyaluronic acid hydrolase gene is cut off to obtain a genetically engineered hyaluronan hydrolase gene fragment.
[0075] Wherein the primer sequence is as follows:
[0076] Upstream primer F: 5'-CCGGAATTCATGAAGACACTACGCGGCTC-3' (SEQ ID NO.6);
[0077] Downstream primer R: 5'-ATTTGCGGCCGCTCAATGATGATGATGGTGGTGATGAA GGGTGAACTTCTT-3' (SEQ ID NO.7);
[0078] It should be noted that the GAATTC sequence in the upstream primer is the introduced EcoRI restriction enzyme site, the GCGGCCGC sequence in the downstream primer is the introduced NotI restriction enzyme site, and the reverse complementary sequence of the ATGATGATGATGGTGGTG sequence in the downstream primer encodes 6...
Embodiment 3
[0080] Example 3 Heterologous expression of hyaluronan hydrolase by recombinant Pichia pastoris
[0081] For the obtained recombinant engineering bacteria P.pastoris GS115 / pPIC9K-HYAL_OM opt and P. pastorisGS115 / pPIC9K-ΔN24HYAL_OM opt Shake flask fermentation was carried out separately. The fermentation steps are as follows: pick a single clone and inoculate it in 40 mL of YPD medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L), and culture at 30 and 200 rpm for 24 hours. Transfer to 40mL initial expression medium BMGY (yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 -4 g / L, glycerol 10mL / L), 30,200rpm for 24h. Collect the thalli by centrifugation, wash the thalli with physiological saline, and replace it with 40 mL induction expression medium BMMY (yeast extract 10 g / L, peptone 20 g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 -4 g / L, methano...
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