Full-length Cloning And Expression Of Arginase And Serine Protease Gene In Hyriopsis Cumingii

Arginase (Arginase, Arg) is a sign enzyme among organisms of urea cycle. It is not only related to many diseases in organisms, but also used to treat tumors and cancer as an important tool enzyme. Serine protease (Serine protease, SP) is a special super-gene family enzyme. It includes a variety of proteases and has different functions. Present study finds that many serine proteases involved in the immune response, particularly occurred in the lower invertebrates.According to the Hyriopsis cumingii (H. cumingii) expressed sequence tags (EST) obtained by constructing subtractive hybridization cDNA library, the gene full-length cDNA sequences of arginase and serine protease in H. cumingii were cloned by RACE-PCR technique. The structural characteristics of the two genes' nucleotide and amino acid sequences were analysed. At the transcriptional level, semi-quantitative RT-PCR was used to analyse the expression of two genes in different tissues. The results show that the length of arginase gene cDNA sequence is 1720bp, and its open reading frame (65-1072)has 1008 bp, encoding 335 amino acids, flanked by a 64 bp of 5'untranslated region(UTR)and a 648 bp of 3'-UTR. The relative molecular weight of arginase is about 36.81KDa. The length of serine protease gene cDNA sequence is 1046bp, and its open reading frame (74-937) is 864 bp, encoding 287 amino acids, flanked by a 73 bp of 5'untranslated region (UTR) and a 109 bp of 3'-UTR. The relative molecular weight of serine protease is about 32.08KDa. Differential expressions in different tissues indicated that the two genes were expressed widely, and the two genes were expressed in various tissues.By means of constructing H. cumingii serine protease prokaryotic expression vector and transforming it to expression system BL21 (DE3), induced with IPTG, the serine protease was expressed. Then the product was analysed by SDS-PAGE electrophoresis, a fusion protein with molecular weight about 50 kDa was obtained which coincided with the expected result. The results provide the basis for the function study of serine protease.