Srivastava Y, Rathaur S, Bhandari Y P, Reddy M V R, Harinath B C
Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, 221 005, India.
J Helminthol. 2004 Dec;78(4):347-52. doi: 10.1079/joh2004254.
A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.
采用DEAE - Sepharose CL6B和Sephadex G - 100连续柱层析步骤，从成年雌性猪蛔虫的体细胞提取物中分离并纯化出具有胶原酶活性的175 kDa抗原组分。发现175 kDa胶原酶的最适pH为7.0。对多种抑制剂和激活剂的敏感性表明，175 kDa胶原olytic酶本质上是金属丝氨酸酶。该酶能水解多种蛋白质底物，如血红蛋白、酪蛋白、偶氮酪蛋白（一般底物）和胶原蛋白、FALGPA（呋喃酰 - 丙烯酰 - 亮 - 甘 - 脯 - 丙氨酸），后者是胶原酶的特异性底物。该酶受到沙鼠抗体细胞胶原酶抗体57%的抑制，与正常沙鼠血清反应不明显。进一步研究了175 kDa胶原酶在体外和原位诱导沙鼠（长爪沙鼠）对马来布鲁线虫（一种人体丝虫寄生虫）免疫的免疫预防潜力。针对该抗原产生的沙鼠免疫血清在体外促进了腹腔渗出细胞对马来布鲁线虫微丝蚴（mf）和感染性幼虫（L3）的部分黏附，并在48小时内对寄生虫诱导了部分细胞毒性。抗猪蛔虫175 kDa抗原血清在诱导对马来布鲁线虫L3的细胞毒性方面比mf更有效。在植入免疫沙鼠体内的微室中，宿主细胞可迁移并黏附到mf和感染性幼虫上，从而在48小时内部分杀死它们。
