Purification and characterization of superantigenic factors produced by viridans streptococci isolated from acute phase Kawasaki disease (KD) patients

翠绿菌群链球菌（VGS）通常主要存在于急性期RD患者的口腔咽拭子培养中。如果这些细菌产生有丝分裂因子，那么考虑到KD病因学的超抗原假说，它们将被认为是一个候选病原体。本文报道了从8例急性期KD患者中的4例患者中分离出的4株菌株产生强效有丝分裂因子，暂定名为SS4、M15、K-9和R2-6。通过API Strep20等检测，鉴定SS4和M15为血链球菌，K-9和R2-6为血链球菌。兔和人外周血淋巴细胞培养检测有丝分裂活性。这些有丝分裂菌株在胰蛋白胨（Mikuni蛋白胨）-酵母浸膏透析液培养基中培养，然后用Diaflo YM10或YM05膜过滤器浓缩培养上清，硫酸铵饱和度75%沉淀，凝胶渗透和离子交换高效柱层析纯化。在分子大小范围内发现有丝分裂活性，约为。10 - 17kda。然后，这些因子是一种基本蛋白质，因为它们不粘附在阴离子交换凝胶（如DEAE-Sephacel）上。然而，采取拒绝，这样做激活是不稳定的，净化过程向前推进。实验中使用了蛋白酶抑制剂，从蛋白酶存在的可能性来看，对活性下降没有影响。这些结果可能提示了KD病因学研究的困难。进一步纯化和鉴定VGS有丝分裂原对于了解它们是否真的是超级抗原以及是否在KD中起致病作用具有重要意义。

Viridans group streptococci (VGS) are often found predominantly in oral throat swab cultures from acute phase RD patients. If these bacteria produce mitogenic factors, they will be assumed to be a candidate as the pathogen in view of the superantigen hypothesis on the etiology of KD. Here report the production of potent mitogenic factors by four strains, tentatively named SS4, M15, K-9 and R2-6, isolated from four patients among eight acute phase KD patients studied. SS4 and M15 were identified as S.mitis, K-9 and R2-6 were as S.sanguis by use of API Strep20 and some other tests. Rabbit and human peripheral blood lymphocyte cultures were used to detect mitogenic activity. These mitogenic strains were cultured in a tryptic peptone (Mikuni peptone) -yeast extract dialysate medium, and then the culture supernatants were concentrated with a Diaflo YM10 or YM05 membrane filter, precipitated at 75% saturation of ammonium sulfate, and purified by gel permeation and ion-exchange high performance column chromatography. The mitogenic activity was found in the range of molecular size, approx. 10 to 17 kDa. Then, these factors are a basic protein because they don't adhere to an anion-exchange gel (such as DEAE-Sephacel). However, the taking declined that to do this activated was unstable and for the process of the purification to move ahead. It did the experiment used the protease inhibitors from the possibility of the existence of the proteases but there was not an affect in the activated decline. These results may suggest the difficulty on the etiology of KD. Further purification and characterization of the VGS mitogens are important in addition to know whether they are really superantigens and whether play pathogenic role in KD.