Shin Yong-Seung, Shin Gee-Wook, Kim Young-Rim, Lee Eun-Young, Yang Hyang-Hee, Palaksha K J, Youn Hee-Jeong, Kim Jae-Hoon, Kim Dae-Yong, Marsh A E, Lakritz J, Jung Tae-Sung
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Gyeong-nam 660-701, Republic of Korea.
Vet Parasitol. 2005 Nov 25;134(1-2):41-52. doi: 10.1016/j.vetpar.2005.06.021. Epub 2005 Aug 10.
This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.
本研究旨在利用蛋白质组学方法探究犬新孢子虫（N. caninum）的两个分离株（KBA - 2和VMDL - 1）之间的关系。为实现这一目标，通过二维凝胶电泳（2 - DE）分离KBA - 2和VMDL - 1的犬新孢子虫速殖子裂解物中的蛋白质，用硝酸银染色，并使用基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF MS）进行分析，以比较蛋白质谱。此外，将通过2 - DE分离的蛋白质转移到膜上，用牛抗犬新孢子虫KBA - 2免疫球蛋白G进行检测，并对两个分离株之间的反应性蛋白质进行可视化和比较。2 - DE图谱上的大多数斑点和2 - DE免疫印迹图谱上的抗原性斑点在等电点和分子量方面位于相似位置。两个分离株共有的蛋白质包括：热休克蛋白70、枯草杆菌蛋白酶样丝氨酸蛋白酶、核苷三磷酸酶、热休克蛋白60、丙酮酸激酶、微管蛋白α、微管蛋白β、烯醇化酶、假定的蛋白质二硫键异构酶、肌动蛋白、果糖 - 1,6 - 二磷酸酶、假定的核糖体蛋白S2、微线体蛋白Nc - P38、乳酸脱氢酶、果糖 - 1,6 - 二磷酸醛缩酶、丝氨酸苏氨酸磷酸酶2C、14 - 3 - 3蛋白同源物、犬新孢子虫致密颗粒 - 1和NcGRA2。因此，尽管犬新孢子虫KBA - 2和VMDL - 1分离株是从地理位置不同的地方分离得到的，但它们的蛋白质组和抗原蛋白质组图谱存在显著同源性。此外，蛋白质组学方法被证实是理解宿主针对原生动物不同分离株的免疫反应的有用工具。
