Authié E, Muteti D K, Mbawa Z R, Lonsdale-Eccles J D, Webster P, Wells C W
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
Mol Biochem Parasitol. 1992 Nov;56(1):103-16. doi: 10.1016/0166-6851(92)90158-g.
A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.
刚果锥虫的一种33 kDa蛋白是感染牛体内的主要抗原，针对该抗原产生的抗体似乎与对锥虫病抵抗力的增强相关[4]。使用针对33 kDa抗原产生的单克隆抗体（mAb 4C5）进行免疫电子显微镜观察显示，其定位于溶酶体，类似于先前描述的刚果锥虫32 kDa半胱氨酸蛋白酶的定位。mAb 4C5和感染牛产生的抗33 kDa抗体在蛋白质免疫印迹中均与通过胱抑素-琼脂糖亲和层析纯化的半胱氨酸蛋白酶结合。用琼脂糖偶联的mAb 4C5从刚果锥虫血流形式中亲和纯化抗原。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，亲和纯化的抗原在非还原条件下分子量为33 kDa，在还原条件下为40 kDa。感染牛产生的抗33 kDa抗体在蛋白质免疫印迹中与非还原和还原的亲和纯化抗原均结合。用生物化学方法纯化的酶免疫的兔血清也与亲和纯化的抗原结合。亲和纯化的抗原在含纤维蛋白原的SDS-PAGE中以及对偶氮酪蛋白显示出蛋白水解活性。它水解苄氧羰基-苯丙氨酸-精氨酸-7-氨基甲基香豆素（Z-苯丙氨酸-精氨酸-NHMec），其米氏常数与生物化学纯化的酶相似。抗原的蛋白水解和肽水解活性被半胱氨酸蛋白酶抑制剂、胱抑素和反式环氧琥珀酰-L-亮氨酰-酰胺基（4-胍基）丁烷（E-64）抑制。在二维凝胶电泳中，该抗原显示出与生物化学纯化的酶相似的特征。我们得出结论，刚果锥虫的33 kDa抗原和半胱氨酸蛋白酶是同一分子。
