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首页> 外文期刊>Infection and immunity >Purification of Immunogenically Active Ribonucleic Acid Preparations of Salmonella typhimurium: Molecular-Sieve and Anion-Exchange Chromatography
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Purification of Immunogenically Active Ribonucleic Acid Preparations of Salmonella typhimurium: Molecular-Sieve and Anion-Exchange Chromatography

机译：鼠伤寒沙门氏菌免疫原活性核糖核酸制剂的纯化：分子筛和阴离子交换色谱

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Immunogenic Salmonella typhimurium ribonucleic acid (RNA) preparations, prepared by differential centrifugation, phenol extraction at 65 C, and ethanol precipitation from 0.5% sodium dodecyl sulfate solution, maintained their immunogenicity through lyophilization. As measured by survival, differential pathogen counts 5 days postchallenge, or clearance of the infecting organism from the tissues, immunization with 50 μg (dry weight) of the lyophilized preparation proved as effective as immunization with 0.1 LD50 of attenuated S. typhimurium cells. Chromatography of the immunogenic fraction through Biogel P-6 (exclusion limit > 4,600) or through Biogel P-300 (exclusion limit > 300,000) resulted in only one immunogenically active protein of the eluate found in the void volume of the columns. Diethylaminoethyl (DEAE) cellulose anion-exchange chromatography of the RNA preparations showed that the immunogenic activity was eluted from the column at 0.8 to 1.0 m NaCl in a linear 0.1 to 2.0 m NaCl gradient. Nonimmunogenic, protein-containing minor peaks were eluted at 0.1 to 0.5 m NaCl. Serial fractionation of the crude RNA preparations over Biogel P-6 to DEAE cellulose to Biogel P-300 molecular-sieve or anion-exchange columns did not alter the immunogenicity of the RNA preparation. Incorporation of the column fractions into Freund's incomplete adjuvant did not increase their relative effectiveness in eliciting anti-salmonella resistance. Chemical analysis of the immunogenic preparations indicated that they were lacking in detectable protein, lipid, and deoxyribonucleic acid. These results suggest that the immunogenic moiety of the crude nucleic acid fraction is either RNA or an as yet undefined polysaccharide of greater than 300,000 molecular weight.
机译：具有免疫原性的鼠伤寒沙门氏菌核糖核酸（RNA）制剂是通过离心，65°C苯酚提取以及乙醇从0.5％十二烷基硫酸钠溶液中沉淀而制得的，通过冻干可以保持其免疫原性。根据存活率，攻击后5天或从组织中清除感染生物后的差异病原体计数，用50μg（干重）冻干制剂进行免疫接种与用0.1 LD 50 免疫接种一样有效衰减的 S。鼠伤寒细胞。通过Biogel P-6（排除限> 4,600）或通过Biogel P-300（排除限> 300,000）对免疫原性级分进行色谱分离，仅在色谱柱的空隙体积中发现了洗脱液的一种具有免疫活性的蛋白质。 RNA制剂的二乙氨基乙基（DEAE）纤维素阴离子交换色谱表明，免疫原性从柱中以0.8至1.0 m NaCl线性0.1至2.0 m NaCl梯度洗脱。非免疫原性，含蛋白质的次要峰在0.1至0.5 m NaCl中洗脱。在Biogel P-6上将粗制RNA制备物在DEAE纤维素上进行连续分馏，在Biogel P-300分子筛或阴离子交换柱上进行分级分离不会改变RNA制备物的免疫原性。将列级分并入弗氏不完全佐剂中并没有增加它们引起沙门氏菌抗药性的相对有效性。免疫原性制剂的化学分析表明，它们缺乏可检测的蛋白质，脂质和脱氧核糖核酸。这些结果表明，粗核酸级分的免疫原性部分是RNA或分子量大于300,000的尚未确定的多糖。

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