Objective A specific candidate antigen of stage V larvae of Angiostrongylus cantonensis was cloned,expressed,and identified.Methods Based on the results of screening the stage V larvae of A.cantonensis cDNA library using immuno-probes,the Ac11 gene was immunoreactive.The Ac11 gene was amplified and submitted for BLAST analysis online.Then,it was subcloned into the expression vector pET28a(+) and expressed in E.coli.The recombinant protein Ac11 was purified and identified immunologically.Results The Ac11 gene was predicted to encode sulfite oxidase.The recombinant plasmid pET28a(+)-Ac11 was successfully constructed.The recombinant protein Ac11 with a molecular weight of about 30 kd was highly expressed in the form of inclusion bodies according to SDS-PAGE analysis.It was also highly immunoreactive in Western blot.Conclusion The recombinant protein Ac11,which is highly immunoreactive,was obtained in vitro and may provide a basis for further immunodiagnostic study of angiostrongyliasis.

Objective To construct and screen a cDNA library of Ⅴ stage larvae of Angiostrongylus cantonensis to ob-tain specific antigen genes for diagnosis. Methods Total RNA was extracted from Ⅴ stage larvae of A. Cantonensis.After that, an unamplified library was constructed the using SMART~(TM) cDNA Library Construction Kit from Clontech Laboratories. The titer and the recombination rate of the unamplified library were detected and then amplified. Positive clones were obtained by using antisera of rats infected with A. Cantonensis as an immuno-probe. Some of the inserts of positive clones were specifically amplified by PCR and sequenced. The nucleotides and the prediction of open reading frames (ORFs) and physico-chemical properties of deduced amino acids were analyzed using bioinformatics. Results This study constructed a cDNA library of Ⅴ stage larvae of A. Cantonensis using SMART LD-PCR, and eleven positive clones were obtained through preliminary immunoscreening. The inserts of nine positive clones were sequenced and ana-lyzed for their homology in the GenBank database. Results showed that one EST of Ⅴ stage larvae of A. Cantonensis had homology of 99% and that one was not homologous to genes in GenBank. The maximum homology of the other six se-quences, two of which were the same, was 91% in comparison to nucleotides from Caenorhabditis elegans and Cae-norhabditis briggsae. There were total seven positive clones with complete ORFs. Conclusion Constructing and immu-noscreening a cDNA library of Ⅴ stage larvae of A. Cantonensis yielded seven significant clones, providing a basis for fu-ture research on these genes.

To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium,the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly.The positive clones were identified by EcoR I,Xho I digestion and sequenced.The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares.The P30 gene was recombined with plamid pET-28 a(+) to construct the prokaryotic expression vector and was expressed in E.coli with IPTG induction.Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting.It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified,and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively.Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes.The expressed plasmid was identified by EcoR I/Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay.It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.

Objective To clone the galactose/N-acetylgalactosamine-specific lectin P30 gene of Cryptosporidium parvum into the plasmid pMD18-T vector for nucleotide sequencing and bioinformatics analysis. Methods The P30 gene was amplified from the genomic DNA of C. parvum by polymerase chain reaction (PCR) and cloned into the pMD18-T vector directly. Positive clones were screened and identified by EcoRⅠ and XhoⅠ digestion and sequenced. Then the DNA sequence was analyzed by relevant bioinformatics software. Results The P30 gene was specifically amplified, and comparison analysis of the nucleotide and deduced amino acid sequences were 98%-100% and 99%-100%, respectively. Conclusion The P30 gene was successfully cloned, and there were nine predicted potential antigenic determinants for the deduced P30. These results lay the foundation for future research.