百日咳杆菌有效免疫原的发酵培养

为扩大生产,采用５００立升发酵罐培养无细胞百日咳菌苗,发现随着培养时间的延续,细胞浓度增高,培养液的ｐＨ值上升,ＰＴ、ＦＨＡ活性、血凝效价逐渐增加、Ｏ２溶压下降、ＣＯ２溶压上升。ｐＨ值达８２时,ＰＴ活性最高为３００ＥＵ／ｍｌ,较现用扁瓶培养方法高５倍。ｐＨ值继续上升时,ＰＴ活性开始下降。ＦＨＡ活性及血凝效价具有相似的变化。通过测定培养液ｐＨ值以确定收获时间,可获得富含ＰＴ、ＦＨＡ、且活性均保持较高水平的培养液     

Physicochemical and immunobiological properties of the dialysate antigen of Bordetella pertussis

The study of the physicochemical and immunobiological properties of B. pertussis dialysate antigen indicates that the antigen has a complex composition and possesses hemagglutinating and lymphocytosis-promoting activity, which permits further studies with a view to developing diagnostic and prophylactic preparations on the basis of this antigen.     

Isolation of hemolytic cultures of Bordetella pertussis intended for the production of acellular pertussis preparations and the characterization of their biological properties

The heterogeneity of the population of B. pertussis laboratory strains with respect to the capacity of individual clones to lyse erythrocytes has been established. The complete or partial reduction of the antigens under study in nonhemolytic bacteria has been shown. The use of hemolytic cultures for the preparation of acellular pertussis vaccine makes it possible to increase the content of B. pertussis toxin in these vaccines.     

Ultrastructure of freshly isolated strains of Bordetella pertussis

The study of the electronograms of B. pertussis strains isolated in the foci of pertussis revealed the existence of the morphological variants of these cells, differing in the character of the cell wall, the state of the cytoplasm, the presence of amorphous inclusions of medium electron-optical density. The morphological variants did not correlate with the character of B. pertussis colonies isolated from blood-charcoal agar. The ultrastructure of the cells belonging to the second morphological variant was similar to that of the cells from the museum strain, altered by tetracycline treatment in the course of the experiment.     

Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.     

Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.     

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization.

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.     

Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin

Summary Large quantities of purified Bordetella pertussis toxin were required to develop an investigational toxoid vaccine. To overcome the difficulties associated with the large scale growth of Bordetella pertussis in submerged culture, we employed an aeration strategy using compressed oxygen and an antifoam agent. The microorganism grown in this way in 1001 batches for 20–24 h yielded 4–6 mg·1^-1 pertussis toxin.