【作者】 方绍庆；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2002， 硕士

【摘要】 猪链球菌2型是猪的重要病原菌之一，能产生多种毒力因子，溶血素是其中重要的一种，几乎所有的强毒株均产生溶血素，且其具有良好的免疫原性。本文研究了猪链球菌2型江苏分离株溶血素的纯化、化学修饰，并对其基因进行PCR检测和序列分析。 本试验采用硫酸铵沉淀，阴离子交换层析及凝胶过滤层析纯化猪链球菌2型江苏分离株溶血素，并在此基础上采用二硫苏糖醇等九种化学修饰剂对其氨基酸侧链基团进行化学修饰，以期探明其溶血活性基团。结果表明色氨酸、组氨酸、精氨酸残基是猪链球菌2型江苏分离株溶血素的活性必需基团，二硫键断裂可引起其活性增强。 根据GeneBank已公布的猪链球菌2型溶血素基因序列，设计和合成了一对可扩增其完整阅读框的引物，对HA9801等6株猪链球菌2型江苏分离株核酸进行PCR扩增，结果显示6株均呈阳性，对HA9801株PCR产物纯化后测序，序列分析表明HA9801株溶血素基因与1933株的同源性为99％，表明猪链球菌2型江苏分离株普遍存在溶血素基因。更多还原

【Abstract】 Streptcoccus suis(S.suis) type 2 is an important swine pathogen and produces varieties of virulence factors.Hemolysin of S.suis type 2 had been identified as a major virulence factor and a good protective antigen. In the present paper,purification,chemical modification of hemolysin and the gene encoding hemolysin was detected,sequenced and analysed.The hemolysin of Streptococcus suis type 2 strain HA9801 isolated in Jiangsu province was purified from the culture supernatant,by using different methods,including selective (NH4)SO4 precipitation,desalting,anion-exchange chromatography,and sephaceryl chromatography,and showed the activity of 128,2048,4096,1024 hemolytic units respectively.The specific activity of the purified hemolysin was enhanced 200 folds.The purified hemolysin appeared a single band with molecular mass of 54,OOODa by SDS-PAGE.It sugguested the purified hemolysin from Jiangsu isolate was analogue of suilysin purified from U.S.A. isolate previously reported.Effects of protein modification reagents on the Streptococcus suis type 2 hemoysin activity were detected. The hemolysin was not affected by PCMB, succinic anhydride( SA), EDC, and N-AI modification, and it indicated that sulfhudryl groups, Amino groups, Carboxyl groups and tyrosine residues were not essential to hemolysin activity. The hemolysin activity was significantly decreased after NBS, DEPC, 2,3-Diacetyl, H2O2 modification and was greatly increased after DTT modification.The results indicated that tryptophane residues, histidine residues, arginine residues and disulfides groups seemed to be essential to the hemolysin activity.A hemolysin gene from six Streptcoccus suis type 2 strains isolated in Jiangsu was amplified by PCR using a pair of specific primers.The PCR product from strain HA9801 was sequenced and nucleotide sequence analysis revealed the hemolysin gene from strain HA9801 had a high homology to strains 1933(99%).更多还原