Genomic DNA probe and purification of Theileria sergenti merozoites in Korean cattle

Chae, Joon-seok; Lee, Joo-mook; Kwon, Oh-deog; Chae, Keon-sang

Korean Journal of Veterinary Research 1994;34(2):387-394.

Joon-seok Chae¹, Joo-mook Lee¹, Oh-deog Kwon¹, Keon-sang Chae²

¹College of Veterinary Medicine, Chonbuk National University
²Department of Molecular Biology, Chonbuk National University

한우에 감염된 Theileria sergenti merozoite의 순수분리와 genomic DNA probe에 관한 연구

채준석¹, 이주묵¹, 권오덕¹, 채건상²

¹전북대학교 수의과대학
²전북대학교 자연과학대학 분자생물학과

Abstract

To make the genomic DNA probe of Theileria sergenti, the merozoites were purified from bovine erythrocytes. The infected erythrocytes were lysed by Aeromonas hydrophila(Ah-1) hemolysin, and the parasites were isolated by ultracentrifugation on a Percoll discontinuous density gradient. For construction of a T sergenti genomic DNA library, T sergenti DNA was digested with Pstl and the fragments were ligated into the PstI site of pUC19 before transformation of Escherichia coli JM83. Out of thousands of transformants obtained by transformation of E coli JM83 with the genomic library, three plasmids were chosen. The sizes of the inserted DNAs were 2.9kb(2.4kb and 0.5kb) in pKTS1, 4.3kb in pKTS2 and 1.5kb in pKTS3, respectively. The DNA fragments used as probe KTS1(2.4kb), KTS2(4.3kb) and KTS3(1.5kb) were labeled digoxigenin-11-dUTP for the Southern hybridization. In Southern hybridization, all of the probes(KTS1, KTS2 and KTS3) reacted specifically to T sergenti DNA, but not to bovine leucocyte DNA. In order to find out the sensitivities of the digoxigenin-11-dUTP-labeled KTS1 and KTS3 as the probes, purified merozoite DNA and bovine DNA (control) were checked by dot blot hybridization with the probes. Both of the probes, KTS1 and KTS3, detected as minimum amount of 975pg of the T sergenti DNA, but not bovine DNA even to 500ng.

Key Words: bovine, Theileria sergenti, DNA probe, Southern hybridization, dot blot hybridization