A kind of glycosaminoglycan lyase and its coding gene and application
A technology of glycosaminoglycans and coding genes, which is applied in the field of genetic engineering and achieves the effect of broad application prospects
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Embodiment 1
[0037] Example 1. Obtaining glycosaminoglycan lyase
[0038] Pick the Vibriosp. FC509 strain and inoculate it into 100mL liquid culture medium, culture it on a shaker for 12 hours at a temperature of 28℃ and a rotation speed of 200rpm, add chondroitin sulfate to make the mass concentration reach 0.01%, continue Cultivate for 48h; centrifuge the culture solution to collect the supernatant medium, precipitate the supernatant with ammonium sulfate with a final concentration of 80%, and collect the precipitate. The precipitate is dialyzed with PBS buffer to remove the ammonium sulfate to prepare glycosaminoglycan lyase.
[0039] The above-mentioned Vibriosp. FC509 was deposited at the General Microbiology Center of the China Microbial Culture Collection and Management Committee on March 12, 2014, with the deposit number CGMCCNO.8913, address: No. 3, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology.
[0040] The components per li...
Embodiment 2
[0043] Example 2. Extraction of genomic DNA of Vibriosp. FC509 strain
[0044] The Vibriosp. FC509 strain was inoculated into the liquid medium (same as Example 1), and cultured with shaking at 30°C and 200 rpm to OD 600 =0.8; Take 40mL of the culture broth, centrifuge for 25min at 12,000rpm, collect the bacterial pellet, wash with 20mL of lysozyme buffer (10mM Tris-HClpH8.0), centrifuge at 12,000rpm for 25min, collect the bacterial pellet ;
[0045] Add 12.0 mL of lysozyme buffer to each tube to obtain about 14.0 mL of bacterial solution. Add 560 μL of lysozyme with a concentration of 20 mg / mL to the final concentration of about 800 μg / mL; after ice bath for 1.0 h Incubate at 37°C for 2h until the solution is viscous; add 0.82mL of 10wt% SDS, 60μL of 100mg / mL proteinase K solution, water bath at 52°C for 1.0h; add Tris-equilibrated phenol / chloroform / isoamyl alcohol (volume ratio 25 : 24:1) 15mL, gently invert and mix until fully emulsified; centrifuge at 10,000g, 4℃ for 10min, tr...
Embodiment 3
[0046] Example 3. Genome scanning and sequence analysis of Vibriosp. FC509 strain.
[0047] The large-molecular-weight genomic DNA prepared in Example 2 was sequenced (Mega Biotech). The sequencing results were analyzed with the software on NCBI (National Center for Biotechnology Information, http: / / www.ncb1.nlm.nih.gov / ). The NCBI analysis software used is OpenReadingFrameFinder (ORFFinder, http: / / www.ncb1.nlm.nih.gov / gorf / gorf.html) and BasicLocalAlignmentSearchTool (BLAST, http: / / blast.ncb1.nlm.nih.gov / Blast.cgi).
[0048] The results of NCBI analysis showed that the genome of the Vibriosp.FC509 strain carries a glycosaminoglycan lyase gene hcdase, the coding region of the hcdase gene is 2436 bp long, and its nucleotide sequence is shown in SEQ ID NO.1. It has 49% homology with the 384 amino acids of hyaluronatelyase gene in the whole genome sequence of Vibriofischeri ES114 (NCBI registration number: YP_206952.1).
[0049] The glycosaminoglycan lyase HCDase encoded by the hcda...
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