ABSTRACT
Objective To study the effects of arsenic compounds on metallothionein-3 gene expression in human normal hepatic cells. Methods cDNAs were cloned by SMART method. Bioinformatics was utilized to analyze the homologue, chromosomal localization, structure and encoding protein of the cloned gene and the trans-membrane information of the encoding protein. Metallothionein-3 gene expression level in L-02 cell line treated by arsenite was determined by cDNA microarrays. Results Metallothionein-3 cDNA gene was cloned and located in the chromosome 16q13. The results of bioinformatics analysis showed that the protein encoded by metallothionein-3 gene could cross the biologic membrane. Metallothionein-3 gene expression up-regulation in human normal hepatic L-02 cell line was found by cDNA microarrays in the early stage after the cells being exposed to arsenite. Conclusion The results of this study showed that the human metallothionein-3 gene was arsenic related gene and this gene might play a vital role in the detoxification metabolism of arsenic compounds at early stage.
ABSTRACT
Objective To screen a new schistosome vaccine candidate. \ Methods\ Schistosoma japonicum adult cDNA library was screened using sera from immune rabbits vaccinated with irradiated cercariae and monoclonal antibodies against membrane antigen of S.japonicum schistosomula. Three different fragments of S.japonicum cDNA genes were cloned into pGEM-T vector. The sequences of the inserts were determined using an automatic DNA sequencer and were analysed using Blast program. One of the unknown genes (B8) was selected and its ORF sequence (291 bp) was subcloned into eukaryotic expression vector. The recombinant plasmids were identified by restrictive enzymes and PCR amplification. The positive recombinant plasmids (pBK/SjB8) were transformed into host bacteria XL1-blue, and were then induced by IPTG for expression. SDS-PAGE and Western blotting analysis of total cellular protein from the bacteria were performed to detect the gene products. Results The results demonstrated that ORF of SjB8 gene was subcloned into the plasmid pBK-CMV and could express as fusion protein in XL1-blue. The results of SDS-PAGE and Western-blot also showed that the molecular weight of the fusion protein with 3 kDa ?-galactosidase was approximately 13^6 kDa and the actual molecular weights of the SjB8 was 10^6 kDa. The expressed fusion product of pBK/Sj-B8 could be recognized by immune serum and McAb. Conclusion A new gene of S.japonicum vaccine candidate (SjB8) was cloned into eukaryotic expression vector pBK-CMV and could express 10^6 kDa schistosome protein. The results provide foundation for further study of the protein for its posibility as candidate vaccine.