Novel dosage form
A pharmaceutical dosage form, tablet technology, applied in the field of new dosage forms, can solve the problems of high item cost, increased chance of food effect, high variability among patients, etc.
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example 1
[0486] Example 1 - Construction of the sfaA deletion mutant of Streptomyces sp. A92-308110 (DSM9954)
[0487] 1.1 Construction of sfaA deletion construct
[0488] The ~7 kb EcoRV-StuI fragment of cosmid TL3006 (SEQ ID NO. 3) including sfaA (nucleotide position 14396-21362, NCBI sequence accession number FJ809786) was excised by digestion with EcoRV and StuI, and the resulting The fragment was ligated directly into pKC1139 which had been previously digested with EcoRV and treated with shrimp alkaline phosphatase (Roche). This plasmid was designated pSGK268.
[0489] The in-frame deletion of the sfaA gene contained in this clone was performed using the Red / ET Recombination Kit provided by Gene Bridges (Cat. No. K006).
[0490] (SEQ ID NO.1)SfaA17161f5'-
[0491] CGCTCTGTGGCGCCTGGTTTCCAAGCGGCTCGCGGACCGGCACCGGCACATGCATAATTAACCCTCACTAAAGGGCG-3’
[0492] (SEQ ID NO.2) SfaA17825r5'-
[0493] TGGATGTATCGTCGCAGGACGCCCAGAATTCACCTGCGACGTCCTCCAGATGCATTAATACGACTCACTATAGGGCTC-3’
[04...
proportion 1
[0499]Mix the prepared E.coli ET12567pUZ8002pSGK271 and Biot-4370 in a ratio of 1:1 (250 μL of each strain) and 1:3 (100 μL of E.coli) and immediately spread it on the R6 plate and transfer to a 37°C incubator . After approximately 2 hours of incubation, the plates were overlaid with 2 ml sterile water containing nalidixic acid, resulting in a final in-plate concentration of 25 μg / L. Plates were returned to the 37°C incubator overnight before overlaying with 2ml of sterile water containing apramycin, resulting in a final in-plate concentration of 20-25 μg / L. The post-conjugant (Ex-conjugant) clones that appeared after ~4-7 days were patched into ISP4 medium containing apramycin (25 μg / L) and nalidixic acid (25 μg / L), and incubated at 37° C. incubate. Once proper hyphal growth was observed, the strains were re-patched to ISP4 medium containing apramycin (25 μg / L) at 37°C and allowed to sporulate. Strains were then subcultured three times by patching onto ISP4 (without antibi...
example 2
[0505] Example 2 - Additional methods for constructing sfaA deletion constructs
[0506] Other methods can be used to generate sfaA deletion mutants. Examples include sfaA insertional inactivation mutants (eg Example 12 from WO2010 / 034243). This strain was generated as described in WO2010 / 034243 and was given the name BIOT-4452.
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