Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli.
OBJECTIVE
To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli.


METHODS
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.


RESULTS
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).


CONCLUSIONS
The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.
Cao
Jianping
J
Department of Immunology, Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, Shanghai 200025, China. caojp@yahoo.com
Liu
Shuxian
S
Song
Guangcheng
G
Xu
Yuxin
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012549:Schistosoma japonicum / Q000235:genetics*; D014335:Tropomyosin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014335:Tropomyosin