问号钩端螺旋体lipL21基因改建及其表达产物免疫原性变化和定位

Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product

目的：改建问号钩端螺旋体（简称钩体）lipL21基因核苷酸序列以提高表达产量并了解基因改建前后表达产物免疫原性变化，确定外膜脂蛋白LipL21在钩体表面的定位。方法：参照大肠杆菌偏爱密码子设计，合成lipL21基因核苷酸序列并构建其原核表达系统。采用SDS—PAGE和BioRad凝胶图象分析系统检测改建前后lipL21基因表达量变化。采用钩体TR／PatocI抗血清为一抗的Westernblot鉴定改建前后两种目的重组蛋白rLipL2ls的免疫反应性，显微镜凝集试验（MAT）比较改建前后rLipL21s抗血清对不同钩体血清群的交叉免疫凝集效价变化。应用免疫电镜技术对LipL21进行定位。结果：改建前后lipL21基因表达量分别为细菌总蛋白的8．5％和46．5％。两种rLipL21均能与TR／PatocI抗血清发生免疫结合反应，免疫家兔后均能产生特异性抗体。两种rLipL21兔抗血清对我国15群15型钩体参考标准株MAT效价相近，均为1：80～1：320。LipL21位于钩体外膜表面。结论：LipL21是钩体表面抗原。改建后的lipL21基因可明显提高原核表达产量，其产物可保持良好的抗原性和免疫反应性，其抗体具有广泛的交叉免疫凝集活性。

Objective. To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction,and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. Methods： According to the preferred codons of E. coli, the nucleotide sequence of lipL21 gene was designed and synthesized,and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor,the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test （ MAT ）. Immuno- electronmicroscopy was applied to confirm the location of LipL21s. Results： The expression outputs of original and reconstructed lipL21 genes were 8.5% and 46.55 of the total bacterial proteins,respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits,the animals could produce specific antibody. Similar MAT titers with 1 ： 80-1 ： 320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. Conclusions： LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed /ipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.