Biological activity of complement fragment Ba

Ba是补体系统激活后产生的补体蛋白片段之一。Ba被属于补体系统替代途径的因子D从因子B裂解。近年来，已有文献报道了B因子B的Ba片段对淋巴细胞和巨噬细胞的生物学作用，但Ba的大量纯化存在困难，需要大量纯化的补体蛋白，纯化步骤非常复杂，阻碍了对Ba生物学功能的进一步研究。我们从人肝cDNA文库中获得了B因子的全长cDNA，并将其克隆到大肠杆菌中。由于Ba（234个氨基酸残基）对应于因子B的氨基末端三分之一，我们通过定点突变在第235个氨基酸残基处引入终止密码子。将编码Ba片段的突变型因子B cDNA转染CHO细胞并表达。用亲和层析柱纯化重组Ba，发现其与细菌/病毒感染和自身免疫性疾病有关。导致补体缺陷的分子缺陷尚未详细研究。我们研究了C6、C7、C8和C9缺陷的分子基础，这些缺陷在因子B的活化中很重要。我们首先确定了C6和C7缺陷的遗传异常。2例C6缺乏症患者存在两种1bp纯合性缺失，2例C7缺乏症患者分别存在一种无义突变和一种2bp缺失。我们已经证明在日本C9缺乏症中Arg-95的非义突变是主要的。

Ba is one of the fragments of complement proteins that is generated upon activation of complement system. Ba is cleaved from factor B by factor D that belongs to the altemative pathway of complement system. Recently, the biological effects on the lymphocytes and macrophages have been reported in the Ba fragment of factor B.However the further investigation of the biological functions of Ba has been hampered mainly by the difficulty in purifying Ba in a large amount that needs many Purified complement proteins and very much complicated purifilcation steps. We intended to circumvent this laborious procedures by making recombinant Ba fragment.A full-length cDNA for factor B was isolated from the human liver cDNA library. As Ba (234 amino acid residues) corresponds to the amino terminal one third of factor B,we introduced a stop codon at the 235th amino acid residue by using site-directed mutagenesis. The mutant factor B cDNA coding for Ba fragment was trnsfected and expressed in CHO cells. The culture media containing has been subjected to the purification of recombinant Ba by using an affinity column.The association of deficiencies in the complement proteins and bacterial/viral infections and autoimmune diseases has been implicated. The molecular defects leading to the complement deficiencies has not been studied in detail. We studied the molecular bases for C6, C7, C8 and C9 deficiencies that are important in the activation of factor B.We first identified the genetic abnormalities in the deficiencies of C6 and C7. A two kinds of homozygous lbp deletion were shown in two patients with C6 deficiency, while a nonsense mutation and a 2bp deletion were identified in two patients with C7 deficiency, respectively. We have shown a nonense mutation at Arg-95 was predominant in Japanese C9 deciciency.