Purification and characterization of protective antigen (PAG) against Leptospira interrogans

采用氯仿-甲醇-水（1:2:8 .8,V/V）法从钩端螺旋体血清lai菌株017中提取保护抗原（PAG），采用硅胶柱层析法进行部分纯化。在SDS-PAGE中，考马斯亮蓝染色未检测到PAG，银染色观察到PAG呈单一的涂片状条带。该条带对应于标准蛋白的24 ~ 30kd。由于外包膜（OE）组分呈同一波段，表明PAG是OE的组分之一。比较了PAG与OE的免疫原性和保护活性。PAG能特异性诱导对钩端螺旋体病具有重要保护作用的凝集抗体，与OE和全细胞相比，PAG不能诱导除凝集素外的任何针对钩端螺旋体细胞抗原的抗体。此外，通过测量由活性氧产生的化学发光，PAG免疫小鼠血清对钩端螺旋体表现出拮抗活性。PAG对仓鼠钩端螺旋体致命感染具有保护作用，可作为钩端螺旋体感染的成分疫苗。

Protective antigen (PAG), glycolipid nature substance, was extracted from Leptospira interrogans serovar lai strain 017 with chloroform-methanol-water (1:2:0.8, V/V) and was partially purified by silica gel column chromatography. In SDS-PAGE, the PAG was not detected by Coomassie brilliant blue staining, but was observed as single smear-like band by silver staining. The band corresponded to 24-30 Kd of standard protein. Since outer envelope (OE) fraction showed the same band, suggesting that the PAG is one of components of OE. Immunogenicity and protective activity of the PAG were compared with those of OE. the PAG was able to induce agglutinating antibody specifically, which is important for protection in leptospirosis, and did not induce any antibodies against cellular antigens of Leptospira except for agglutinogen in comparison with OE and whole cells. Furthermore, sera of mice immunized with the PAG exhibited opsonic activity against Leptospira by measurement of chemical luminescence derived from reactive oxygen. The PAG possessed protective activity against lethal infection of Leptospira in hamsters and seemed to be useful as component vaccine against leptospiral infection.