Gene cloning, soluble expression and activity analysis of rSEA / 生物工程学报

The superantigen,such as staphylococcal enterotoxins, had been identified as possible anti-cancer molecules in many reports. In this paper, we cloned the entA gene encoding Staphylococcal enterotoxin A from the genomic DNA of Staphylococcus aureus(ATCC13565) by PCR, the sequence cloned was accordance with that reported in Genebank. The entA gene could be expressed effectively after inserted into plasmid pET-22b( + ), The rSEA was expressed as inclusion bodies when induced by IPTG at 37 degrees C and became soluble after induced at low temperature, the soluble part is about 55% of total rSEA products. Only one band was detected by western-blotting in expression product of BL-21 (DE3) with pET-SEA. The soluble rSEA was purified by Ni2+ chelating sepharose column. No other protein except rSEA was seen in SDS-PAGE gel stained by both Coomassie brilliant blue and silver salt, which showed that the rSEA was purified effectively. Homology modeling of rSEA determined the structure change was conducted, which indicated there was no apparent structure change between rSEA and native SEA. This result was also confirmed by proliferation assay of PBMC, for the rSEA could induced proliferation of PBMC as effectively as native SEA. The increasing anti-tumor activity of rSEA was also detected after the spleen cell activated in vivo by rSEA, which was accordance with others reports. This work paved the way for the further study of anti-cancer with rSEA.

Study on relationship between length of homologous sequences and chromosomic recombination rate in Saccharopolyspora erythraea / 生物工程学报

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.