[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.

[Detection of anisakid nematodes by an SYBR green I real-time PCR].

OBJECTIVE: To establish an SYBR Green I real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait. METHODS: Anisakid larvae of six species (Anisakis simplex, A. physeteris, Raphidascaris trichiura, Contracaecum aduncum, C. muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait) were obtained from the guts of marine fishes and identified chiefly based on the morphological features. The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced. According to these sequences, six specific forward primers were designed and synthesized. Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E. coli DH5alpha. Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve. Sensitivity and reproducibility were determined. RESULTS: All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle (Ct) and template concentration. Melt curves were specific and all the 6 correlation coefficients were above 0.998. In the reproducibility test, the coefficients of variation (cv) of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%. The sensitivity of the real-time PCR was 1 x 10(2) copies/microl, about 100 times higher than the conventional PCR assays. The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report. CONCLUSION: An SYBR Green I fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.

[Cloning and expression of L-like cysteine protease of Anisakis simplex].

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 pET32a(+) 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 1211 bp of 3'-end of AsCP gene was amplified by 3'RACE, full length of the gene was 1462 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 plasmid pET32a(+)-AsCP showed that there was about 1150 bp fragment, the constructed recombinant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein (Mr 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.

[Expressed sequence tags (ESTs) analysis of Angiostrongylus cantonensis].

A total of 1,277 ESTs of Angiostrongylus cantonensis were downloaded from GenBank and analyzed with BlastX. SignalP V3.0 analysis was applied to predict potential putative antigen or allergen relative proteins with N-terminal secreted signal peptides or signal anchors. BlastX analysis showed that there were 614 ESTs scored more than 100, of which 14 were identical with A. cantonensis, 60 ESTs did not match any proteins in the databases. The identified 614 ESTs could be grouped into 10 categories, 80 ESTs expressed 22 antigen or allergen relative proteins, in which 12 had N-terminal secreted signal peptides and 3 had signal anchors.