[Construction of the female subtractive cDNA library and screening of the specific expressing genes].
OBJECTIVE
To screen the Schistosoma japonicum female specific expressing genes.


METHODS
S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms.


RESULTS
Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB017097, AF519182, M32281, and S. japonicum unknown gene AY813556 respectively.


CONCLUSIONS
SSH is essential to screen the differentially expressed genes of S. japonicum female worms. A number of female specific genes have been found by this method.
Wang
Yan-hai
YH
Department of Parasitology, Southern Medical University, Guangzhou 510515, China.
Peng
Hong-juan
HJ
Chen
Xiao-guang
XG
Shen
Shu-man
SM
D000818:Animals; D001483:Base Sequence; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011817:Rabbits; D012549:Schistosoma japonicum / Q000235:genetics* / Q000302:isolation & purification; D012554:Schistosomiasis japonica / Q000469:parasitology*