Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli.</p><p><b>METHODS</b>SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition.</p><p><b>RESULTS</b>The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM).</p><p><b>CONCLUSION</b>The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.</p>

Screening and Cloning of Genes Encoding Schistosoma japonicum Antigens Related to the Serum Antibodies in Mirotus Fortis / 中国寄生虫学与寄生虫病杂志

Objective To understand and identify the molecules related to the natural resistance to Schistosoma japonicum infection in Mirotus fortis. Methods Sera from Mirotus fortis without schistosome infection were collected. The S.japonicum adult worm cDNA library was immunologically screened with the sera. The positive recombinants were identified, cloned, sequenced and analysed with software and internet. Results Seven genes encoding antigens relevant to sera antibodies in Mirotus fortis were cloned and sequenced. These antigens included glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine protease inhibitors(SERPIN), 70 kDa heat shock protein(HSP70), 22^6 kDa membrane-associated antigen, paramyosin (Sj97), cytochrome C and cathepsin B. Conclusion Many protein molecules might have been involved in natural resistance to \{S.japonicum\} infection in Mirotus fortis. The above 7 kinds of molecules may be identified as new candidates of vaccine against \{S.japonicum\} infection.