Opsonization of bacteroides by the alternative complement pathway reconstructed from isolated plasma proteins.
Opsonization of bacteroides by the alternative complement pathway reconstructed from isolated plasma proteins.
复制标题
由分离的血浆蛋白重建的替代补体途径对拟杆菌的调理作用。
DOI：
10.1084/jem.165.3.777
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Bjornson,HS
中科院分区：
文献类型：
--
作者：
Bjornson,AB;Magnafichi,PI;Schreiber,RD;Bjornson,HS
Materials and MethodsBacterial Strains and Growth Conditions. B. fragilis 1365 and B. thetaiotaomicron 1343 were clinical isolates used in our previous studies (10). Stock cultures were maintained in thioglycolate medium (Difco Laboratories, Detroit, MI) in frozen form at-70 C. Cultures were thawed and inoculated into broth consisting of equal parts of trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD) and brain-heart infusion broth (Difco Laboratories) supplemented with 0.05% thioglycolate (Difco Laboratories). For preparation of radiolabeled bacteria, broth was supplemented with 10'UCi/ml ofL-[sH] amino acid mixture (New England Nuclear, Boston, MA). Cultures were incubated for 20 h at 37 C in an anaerobic glove box (Coy Manufacturing Products, Ann Arbor, MI), which was monitored to contain 85% nitrogen, 10% hydrogen, 5% carbon dioxide, and not more than 20 parts per million of oxygen. Bacteria were washed and resuspended in HBSS (MA Bioproducts, Walkersville, MD) containing 0.1% gelatin (Difco Laboratories)(HBG)'; this solution was boiled for 10 min before use to remove oxygen. Isolation of Complement Proteins and IgM. C3 was prepared from human plasma by the method of Tack et al.(12). Trace contaminants were removed by chromatography on anti-IgG Sepharose 4B, anti-IgA Sepharose 4B, and anti-C5 Sepharose 4B equilibrated with isotonic veronal-buffered saline (VBS), pH 7.4. Antisera to IgG (7-chain-specific), IgA (-y-chain-specific), and C5 were obtained from Behring Diagnostics, LaJolla, CA, and IgG fractions of these antisera were prepared by ammonium sulfate precipitation (13). The IgG fractions were coupled to Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) by cyanogen bromide activation (14).Factor B was prepared by a minor modification of the method of Gotze and Muller-Eberhard (15). Human serum was precipitated with 40% ammonium sulfate (wt/vol) at 4 C, and the precipitate was discarded. The ammonium sulfate concentration in the supernatant was raised to 68%. The resulting precipitate was solubilized and then chromatographed according to the original procedure, except DEAF Sephacel (Pharmacia Fine Chemicals) was substituted for DEAE-cellulose. As a final step in the purification procedure, active fractions from the DEAE Sephacel column were chromatographed on anti-IgG Sepharose 4B.
