关键词: Klenow（exo-）蛋白制备, RAA技术, SNPs检测

Abstract: A preparation method of Klenow (exo-) protein was established and its application in RAA technology was preliminary studied. The target gene fragment was constructed by the Overlap point mutation method, and the Klenow(exo-) protein expression vector was constructed and transformed into Escherichia coli BL21 (DE3) strain to induce expression. The expressed product was subjected to preliminary purification by Ni-NTA chelate chromatography, followed by Heparine column chromatography for further purification, and the purified product was identified by SDS-PAGE. A protein of higher purity was obtained. The purified RAA reaction system was prepared using the purified target protein, and the mutation site was designed for the actual sample of the meat source. The experimental results showed that the system has strong specificity and dont amplified when there is a double base difference at the 3′ end of the template gene sequence, compared with the conventional PCR detection method, the system could effectively distinguish double base mutation. The system was expected to applied in detection of SNPs in the future.

Key words: Klenow（exo-） protein preparation, RAA, SNPs detection