Purified anthelmintic compositions and related methods
A composition and compound technology, which can be used in drug combinations, anti-infective drugs, pharmaceutical formulations, etc., can solve the problems of difficult application of preparations to humans and animals, difficult to handle spores, and low efficiency.
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Embodiment 1
[0142] Example 1: Generation of IBaCC
[0143] To produce Cry5B in sporulation-deficient cells, Bacillus thuringiensis (Bt) strain 4D8 lacked any crystal protein expression (Identification of Bacillus thuringiensis subsp. kurstaki strain HD1-Like bacteria from environmental and human samples after aerial spraying of Victoria, British Columbia, Canada , with Foray 48B. Appl EnvironMicrobiol. 2001 Mar;67(3):1035-43), and in which the major spoOA regulator of sporulation was deleted by homologous recombination (spoOA-). This composition is also known as Cry5B-BaCC (Bacillus with Cytosolic Crystal).
[0144] To inactivate Cry5B-BaCC, the transformed Bacillus thuringiensis 4D8 strain was cultured in three-fold concentrated Luria supplemented with 10 μg / mL erythromycin and 200 μg / mL kanamycin in a 2 L baffled flask. - Aerobic proliferation in Bertani Broth (LB) at 30° C. with shaking in a volume of 200 mL for 48 hours. The transformed Bacillus thuringiensis cells were centrifuge...
Embodiment 2
[0145] Example 2: Purification of Cry5B from IBaCC using homogenate
[0146] Image 6 A flow diagram of the process used to purify Cry5B crystal protein from IBaCC is shown. To purify a large amount of Cry5B protein from IBaCC, a 10 L fermentation harvest was performed ( Image 6 , step 610). Ten liters of BaCC were grown to 250 to 350 OD / ml. The BaCC is then concentrated to 0.5L to 1L using centrifugation (8000 xg, 30 minutes) and then inactivated by adding 1 ml / L carvacrol and stirring at room temperature for 15 minutes (step 612) to generate IBaCC. Other methods of concentrating the BaCC prior to the inactivation step can be used, such as ultrafiltration or diafiltration.
[0147] The IBaCC was homogenized 5 to 7 times at 15,000 psi (step 614), and the resulting lysate was centrifuged to concentrate the bacterial lysate (step 616). Other methods of concentrating IBaCC can be used, such as ultrafiltration or diafiltration. The resulting pellet is resuspended in 0.5 L...
Embodiment 3
[0150] Example 3: Purification of Crv5B from autolyzed IBaCC
[0151] Some strains of BaCC (eg strain 4D8) autolyse at the end of their growth cycle. This autolysis can eliminate or reduce the need for a homogenization step after inactivation with the antimicrobial compound. Therefore, referring to Image 6 The purification flow chart of the above, if the autolyzed strain of bacteria is used, the homogenization step 614 can be eliminated or the number of times can be reduced. In some such embodiments, a 10 L fermentation harvest of autolyzed BaCC ( Image 6 , step 610). Ten liters of BaCC can be grown to 250 to 350 OD / ml. The BaCC can then be concentrated to 0.5 L to 1 L using centrifugation and then inactivated by adding 1 ml / L carvacrol and stirring at room temperature for 15 minutes (step 612 ) to generate IBaCC. Since IBaCC is autolyzed before and / or after inactivation, no homogenization is required. Other methods of concentrating the BaCC prior to the inactivation...
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