[Cloning and expression of D-like aspartic protease of Anisakis simplex].

OBJECTIVE: To clone and express the full length of D-like aspartic protease gene (AsAP) of the third stage larvae of Anisakis simplex. METHODS: According to the partial information of D-like aspartic protease encoding gene of A. simplex from GenBank, specific primers were designed to amplify 3'end and 5' end of AsAP gene using rapid amplification of cDNA ends (RACE), and the full length of the D-like aspartic protease gene was obtained. Using total RNA of the third-stage larvae of A. simplex, coding sequence of the AsAP gene was amplified by reverse transcription-PCR (RT-PCR). The PCR product was digested by EcoR I and Sal I, and cloned into pET32 vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into E. coli BL21 (DE3). Expression of the protein induced by IPTG under gradient concentration and different time was conducted. RESULT: A 1 753 bp full length of AsAP was obtained, which contained 30 bp 5'UTR, 361 bp 3'UTR and a 1 362 bp open reading frame (ORF) encoding 453 amino acids with a predicted molecular mass of M(r) 50 726. It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum. The predicted amino acid sequence contains two conserved catalytic motif, an active site flap, an S2 subsite and an S3 subsite. A 20 amino acids signal peptide was found in the N-terminus, with significant hydrophobic property. Different concentration of the IPTG (0.2-1.6 mmol/L) showed little effect on the expression, and the production of the protein was up to maximum after 2 hours induction. CONCLUSION: The AsAP gene has been cloned and expressed.

PCR assay for the cell-free copro-DNA detection of Angiostrongylus cantonensis in rat faeces.

To facilitate improved detection of the first stage larvae (L1) of Angiostrongylus cantonensis from rat faeces, a TaqMan(®) probe real-time PCR method for the detection in situ was developed targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. cantonensis. The assay was capable of detecting a single L1 in a grain of fresh faeces (weight 320 ± 125 mg) from the experimental infected Sprague-Dawley rats, and the method can also detect cell-free copro-DNA from positive faeces placed for up to 12 months at ambient environment. The present study exhibited a high level of specificity for A. cantonensis, with no fluorescence signals were observed in reference control consisting of four parasite species commonly found in the intestine of rat. This approach can overcome the limitations of DNA-based identification that faecal materials should be stored in 70% ethanol or kept as frozen samples for further tests, and thus it might be suitable and feasible for the detection of target DNA in faecal materials preserved at ambient temperature, but the detecting efficiency will depend on the amount of DNA in the samples and the time placed for the samples due to DNA degradation.