Alamuri Anusha, Kumar K Vinod, SowjanyaKumari S, Linshamol L, Sridevi R, Nagalingam M, Roy Parimal, Balamurugan V
Indian Council of Agricultural Research -National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Yelahanka, Post Box No. 6450, Bengaluru, Karnataka, 560 064, India.
Jain University, Bengaluru, Karnataka, India.
Mol Biotechnol. 2020 Dec;62(11-12):598-610. doi: 10.1007/s12033-020-00278-4. Epub 2020 Oct 2.
The expressed recombinant leptospiral surface adhesion lipoprotein (Lsa27) of pathogenic Leptospira in E. coli was evaluated for the detection of Leptospira antibodies in cattle sera by latex agglutination test (LAT). The Lsa27 lacking signal peptide coding gene sequences from L. interrogans serovar Pomona was amplified (~ 660 bp) by PCR and the amplicon was cloned into pETiteN-HisKan vector. The expressed recombinant Lsa27 histidine-tagged fusion protein (rLsa27) was Ni-NTA affinity purified under denaturation followed by renaturation methods. The purified rLsa27 was characterized by SDS-PAGE and immunoblot, which confirmed the leptospiral protein with a MW of ~ 25 kDa. Further, the prepared sensitized latex beads coated with rLsa27 were evaluated as a diagnostic antigen for detection of pathogenic Leptospira antibodies by using known microscopic agglutination test (MAT) positive (n = 74) and negative (n = 62) sera for Leptospira antibodies in LAT, which revealed the relative diagnostic sensitivity of 91.89% and specificity of 87.10% against the gold standard serological test, MAT. Furthermore, on evaluation of developed rLsa27 LAT using serum samples from cattle associated with the history of abortions and reproductive disorder (n = 309), the relative sensitivity of 96.15%, and specificity of 89.11% were observed. Therefore, this rapid field test using the rLsa27 is first of its kind and it could be used as a screening test for the detection of Leptospira antibodies or it can be complemented by other diagnostics for the diagnosis /surveillance of bovine leptospirosis.
通过乳胶凝集试验（LAT）评估了大肠杆菌中致病性钩端螺旋体表达的重组钩端螺旋体表面粘附脂蛋白（Lsa27）用于检测牛血清中的钩端螺旋体抗体。通过PCR扩增问号钩端螺旋体波摩那血清型中缺乏信号肽编码基因序列的Lsa27（约660 bp），并将扩增产物克隆到pETiteN-HisKan载体中。表达的重组Lsa27组氨酸标签融合蛋白（rLsa27）先经变性后用Ni-NTA亲和纯化，再进行复性。通过SDS-PAGE和免疫印迹对纯化的rLsa27进行表征，证实其为分子量约25 kDa的钩端螺旋体蛋白。此外，使用已知的显微镜凝集试验（MAT）阳性（n = 74）和阴性（n = 62）血清检测钩端螺旋体抗体，评估了包被有rLsa27的致敏乳胶珠作为诊断抗原在LAT中的应用，结果显示相对于金标准血清学试验MAT，其相对诊断敏感性为91.89%，特异性为87.10%。此外，使用来自有流产和生殖障碍病史的牛的血清样本（n = 309）评估所开发的rLsa27 LAT，观察到相对敏感性为96.15%，特异性为89.11%。因此，这种使用rLsa27的快速现场检测尚属首次，可作为检测钩端螺旋体抗体的筛查试验，或者可与其他诊断方法互补用于牛钩端螺旋体病的诊断/监测。
