Sun Y, Clinkenbeard K D
Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.
Am J Vet Res. 1998 Jul;59(7):851-5.
To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification.
One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf.
Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated.
Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1 M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1% glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium.
Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT.
筛选在4种无血清培养基中生长的溶血巴斯德菌培养物的上清液，以获得最大量的白细胞毒素（LKT）产生和最低的蛋白质浓度，作为纯化LKT的最佳来源。
一株最初从小牛肺炎肺中分离出的A型1血清型溶血巴斯德菌。
溶血巴斯德菌在脑心浸液（BHI）肉汤、酵母胰蛋白胨肉汤、RPMI-1640培养基和麦科伊改良5A培养基中培养。测量培养物生物量和蛋白质浓度、LKT活性以及培养上清液中的LKT浓度。评估培养基pH值以及添加金属阳离子和葡萄糖对溶血巴斯德菌生长速率和培养上清液参数的影响。
在含有0.1 M磷酸盐（pH 6.8）的BHI肉汤或RPMI-1640培养基中培养的溶血巴斯德菌产生的LKT浓度最高。向RPMI-1640培养基中添加0.36 mM FeCl3或1.0 mM MgSO4可进一步提高培养上清液中LKT的比活性，但添加1%葡萄糖并不能增强LKT的产生。在添加MgSO4的RPMI-1640培养基中产生的白细胞毒素与在添加血清蛋白的培养基中相当。
尽管BHI肉汤在溶血巴斯德菌生长和LKT产生方面优于RPMI-1640培养基，但与RPMI-1640培养基相比，其较高的蛋白质浓度和较低的LKT比活性使其成为不太理想的培养基。在pH 6.8的磷酸盐缓冲且添加MgSO4的RPMI-1640培养基中，生长速率和LKT产生且蛋白质含量最低时最为理想。该培养基可作为纯化LKT的培养上清液来源。
