Construction Of Yeast Two-hybrid Cdna Library From 18day Schistosoma Japonicum And Screen The Factors Of Interactive With Sjgcp

Schistosoma japonicum is an important public health problem in China that can result in serious disease both humans being and in the domestic animal reservoir hosts. Eggs from paired mature schistosomes are responsible for most of the pathology caused by Schistosoma japonicum, and the eggs is the root too make the disease disseminates. The triggering and maintaining of female schistosomes maturation are dependent on Male schistosomes. When paired females are separated from male they stop laying eggs and regress to an immature state, and they maturate again if they are allowed to couple. The SjGCP(Schistosoma japonicum gynaecophoral canal protein) exhibits surface expression in adult parasites especially in male worms in which expression is limited to the gynaecophoric canal,the site of direct interaction between the mating pair. And interestingly, the transcription profile of SjGCP shows its peak expression at the time coincides with worm pairing. This suggested SjGCP may have the potential role in male-female interactions and the male-stimulated reproductive maturation of female schistosome. So the studies on SjGCP approach will be importment for reveal the mechanism of schistosome maturation. For this purpose, we constructed the Yeast Two-Hybrid cDNA library from 18 days worms of Schistosoma japonicum and preliminary screened the SjGCP interactiving proteins.Construction of Yeast Two-Hybrid cDNA Library from 18day Worms of Schistosoma japonicum:Total RNA was extracted from 18 days worms of S. japonicum by Trizol reagent, and the total RNA were puried by digestion of remain genomic DNA and use RNeasy Protect Mini Kit. The first strand cDNA was synthesized by reverse transcription using random primer. Ds cDNA were get by performing LD PCR using 5'amplimer and 3'amplimer the ds cDNA were clean up by using CHROMA SPIN TE-400 Column to purify ds cDNA. Transform AH109 Yeast competence cells with PGADT7-Rec and ds cDNA according to the screening method of Yeast Mating, PGADT7-Rec and ds cDNA can homology compose the plasmid of cDNA library with ring shaped and have active of duplication. The clones grown on SD/-Leu harvested is Yeast Two-Hybrid cDNA Library. The cell density of transformants>2×107cells/mL, the titer of library was 2.5×106 pfu/mL, the PCR identification result showed that recombination rate was 98%, in which inserted cDNA fragments's longth were between 0.25 bp and 2.0bp. The library possess satisfactory representation. All the six known objective genes of S.japonicum were amplified from the library. The Yeast Two-Hybrid cDNA library from 18days Schistosoma japonicum was constructed.Construction and confirmation of fused plasmid with SjGCP bait gene in yeast two-hybrid system:The intact ORF of SjGCP gene was PCR amplified the as the bait protein gene and cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-SjGCP, The pGBKT7-SjGCP were transformed into the competent Y187 yeast cells. The transcriptional activation, the expression and toxicity. the results shows the SjGCP bait protein is suitable for two-hybrid library screening.Screen of SjGCP interactive proteins:The screen were performed by Screening the factor that can interactive with SjGCP from the Yeast Two-Hybrid cDNA library used the method of Yeast mating., reduplicate streak the clones on selective medium to eliminate the false positives. We got 330 clones and we isolate the AD plasmids of 5 positive yeast clons then transform them into E.coli and sequencing. We got 5 EST sequence of S.japonicum:SJCHGC0566829,SJCHGC09129,SJCHGC 00694,SJCHGC06072,SJCHGC05265. Retest 3 interaction in yeast by cotransformatiom.