关键词: 简单异尖线虫, L-样半胱氨酸蛋白酶, 克隆, 原核表达

Abstract: Objective To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex （AsCP） . Methods According to L-like cysteine protease encoding gene of A. simplex from GenBank EST database, specific primers were designed to amplify 3′-end of AsCP gene using rapid-amplification of cDNA ends （RACE）, and the full lenth of the L-like cysteine protease gene was obtained. Specific primers were designed according to the full length of the gene. Using total RNA of A. simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT-PCR. The PCR product was digested by EcoR I and Sal I, and cloned into pET32а（+） vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21（DE3）. Expression of the protein induced by IPTG of gradient concentration （0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L） and by the same concentration （1 mmol/L） of IPTG at different time（0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h） was conducted. The expression situation of recombinant protein was analyzed by SDS-PAGE. Results A 1 211 bp of 3′-end of AsCP gene was amplified by 3′RACE, full length of the gene was 1 462 bp and coding 411 amino acids. It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans. Double enzyme digestion of the constructed recombinant plasimid pET32a（+）-AsCP showed that there was about 1 150 bp fragment, the constructed recombinant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein （M_r 60 000） was identical with the target. IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction. Conclusion The AsCP gene has been cloned and expressed.

Key words: Anisakis simplex, L-like cysteine protease, Cloning, Prokaryotic expression