PATHOGENESIS OF EXPERIMENTAL CHOLERA - PREPARATION AND ISOLATION OF CHOLERAGEN AND CHOLERAGENOID
PATHOGENESIS OF EXPERIMENTAL CHOLERA - PREPARATION AND ISOLATION OF CHOLERAGEN AND CHOLERAGENOID
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DOI：
10.1084/jem.130.1.185
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发表时间：
1969-01-01
影响因子：
15.3
通讯作者：
LOSPALLUTO, JJ
中科院分区：
文献类型：
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作者：
FINKELSTEIN, RA;LOSPALLUTO, JJ
材料和方法scholeragen在容量为12升的Virtis 40-100型发酵罐中以9或10升的syncase培养基(4)生产，其中接种了大约105个活细胞/ml的霍乱弧菌菌株569 B Inaba。8小时加入Antifoam A （Dow Coming Corp., Midland, michigan）。在30℃强通风（7.6升/雨）条件下孵育20-24小时，并用磁力搅拌器以50（0700转/分）的速度搅拌后，将温度提高到37℃，并通过添加0.05至0.1%的β -丙内酯（BPL, Betaprone, Fellows- testagar, Div. Fellows Medical Mfg）杀死大部分生物体。Co.，底特律，密歇根州)，并进一步孵育2小时。细胞在Sorvali RC2B冷冻离心机中以16000 rpm和约100 ml/min的流速连续离心除去，该离心机配备了Szent-Gyorgi和Blum系统。加入硫酸铵（70 g/100 ml）沉淀霍乱原和其他蛋白质，也能有效杀死残留的活弧菌。沉淀物在室温下放置过夜后，使用“篮式”离心机（Virtis Co., Inc., Gardiner， NY）用优质滤纸进行连续离心。第二次沉淀是在上清液中形成的，在进一步放置一天后，通过传统的纸过滤收集并添加到第一次沉淀中。经冷水和蒸馏水透析后，该制剂具有冻干性。当四种这样的制剂积累后，将粉末通过悬浮液混合在大约200毫升的0.01 st PO4缓冲液中，pH为7.5，并添加到i公斤湿重的二乙胺乙基纤维素（DEAEC， Whatman微颗粒DE 52）中，该缓冲液先前已与相同的缓冲液平衡。搅拌1小时后，通过烧结玻璃漏斗过滤除去多余的液体，用0.1 xf PO4缓冲液（pH 6.0）洗涤去除霍乱原。这个过程留下了相当数量的不相关的蛋白质和棕色色素吸收到DEAEC（然后再循环使用）。首先，用（NH4）~ SO4沉淀、透析、冻干等方法浓缩含胆素洗脱液。在后来的实验中，它通过Diaflo UM-10膜（Amicon公司，列克星敦，马萨诸塞州）进行硝化过滤。浓缩物在5 × 125 cm琼脂糖柱（rioo - gel a -5m， 200-400目，Bio-Rad Laboratories, Richmond, Calif.）上进行凝胶过滤，使用Tris-EDTA缓冲液pH为7.5(0.05~三羟甲基胺甲烷，0.001~乙二胺四乙酸添加二钠盐，0.003 st nanans, 0.2~（NaC1）作为洗脱液，流速约为60 ml/hr。含有霍乱原的部分，通过Ouchterlony琼脂凝胶双重扩散试验与特异性抗霍乱原兔血清鉴定，在280 m/~的吸光度显示的晚出现组分中，汇集，通过超滤浓缩，并通过串联的两个2.5 X 90 cm Sephadex G-75柱（Pharmacia Fine Chemicals, Piseataway， NJ）重新色谱。在后一种情况下，以0.4 st甲酸铵（含0.003 st NaNa）作为洗脱缓冲液，流速为20 mlfur。含霍乱原抗原的部分再次用乌氏沉淀法鉴定，用超滤池和浓缩。根据Andrews（13）的方法，通过测定已知分子量标记物的洗脱体积，定期校准凝胶过滤柱。
Materials and MethodsCholeragen was produced in 9 or 10 liter volumes of syncase medium (4) in a Virtis Model 40-100 fermenter of 12 liter capacity inoculated with approximately 105 viable cells/ml of Vibrio cholerae strain 569 B Inaba. Antifoam A (Dow Coming Corp., Midland, Mich.) was added at 8 hr. After incubation at 30 C for 20-24 hr with vigorous aeration (7.6 liter/rain.) and stirring by means of a magnetic agitator at 50 (0700 rpm, the temperature was raised to 37 C and the bulk of the organisms killed by the addition of 0.05 to 0.1% beta-proplolactone (BPL, Betaprone, Fellows-Testagar, Div. Fellows Medical Mfg. Co., Detroit, Mich.) and further incubation for 2 hr. The ceils were removed by continuous flow centrifugation at 16,000 rpm and a flow rate of approximately 100 ml/min, in a Sorvali RC2B refrigerated centrifuge fitted with the Szent-Gyorgi and Blum system. The choleragen and other proteins were precipitated by the addition of ammonium sulfate (70 g/100 ml), which also served effectively to kill any residual viable vibrios. The precipitate was harvested, after standing overnight at room temperature, by continuous flow centrifugation using a" basket" centrifuge (Virtis Co., Inc., Gardiner, NY) with fine grade filter paper. A second precipitate, which formed in the supernate on further standing for a day, was harvested by conventional filtration through paper and added to the first. After dialysis in the cold vs. distilled water, the preparation was lyophili~. ed. When four such preparations had been accumulated, the powders were pooled by suspension in approximately 200 ml of 0.01 st PO4 buffer, pH 7.5, and added to i kg wet weight of diethylaminoethyl cellulose (DEAEC, Whatman microgranular DE 52) which had previously been equilibrated with the same buffer. After stirring for 1 hr, the excess fluid was removed by filtration through a sintered glass funnel and choleragen was einted by washing with 0.1 xf PO4 buffer, pH 6.0. This procedure leaves a considerable amount of unrelated protein and brown pigment absorbed to the DEAEC (which was then recycled for subsequent use). At first, the choleragen-containing eluate was concentrated by (NH4)~ SO4 precipitation, dialysis, and lyophilization. In later experiments it was concentrated by nitrafiltration through Diaflo UM-10 membranes (Amicon Corp., Lexington, Mass.). The concentrate was then subjected to gel filtration on a 5 X 125 cm column of Agarose (Rio-Gel A-5m, 200-400 mesh, Bio-Rad Laboratories, Richmond, Calif.) using Tris-EDTA buffer pH 7.5 (0.05~ tris-hydroxymethylaminomethane, 0.001~ ethylenediaminetetraacetic add disodium salt, 0.003 st NaNs, 0.2~(NaC1) as eluant at a flow rate of approximately 60 ml/hr. The choleragen-containing fractions, identified by Ouchterlony agar gel double diffusion tests with specific anti-choleragen rabbit serum, in a late appearing component as indicated by its absorbance at 280 m/~, were pooled, concentrated by ultrafiltration, and rechromatographed through two 2.5 X 90 cm columns of Sephadex G-75 (Pharmacia Fine Chemicals, Piseataway, NJ) connected in series. In the latter instance, 0.4 st ammonium formate containing 0.003 st NaNa was employed as eluting buffer at a flow rate of 20 mlfur. Fractions containing choleragen antigen were again identified by Ouchterlony precipitation, pooled, and concentrated by ultrafiltration. Gel filtration columns were calibrated periodically by determination of elution volume of known molecular weight markers, according to methodology adapted from Andrews (13).