The Construction Of A Yeast Two-hybrid CDNA Library Of Eimeria Tenella And Preliminary Screening Of Interaction Proteins With EtMA1

Coccidian parasite belongs to apicomplexan (Eimeria), it has a complex life cycleincluding stages of intracellular (schizogony, gametogony) and extracellular(sporogony). The sporozoite is the first developmental stage of the life cycle to incadeintestinal cells. Studies showed that the expression of Apical Membrane Antigen1(AMA1) gene was highest in the stage of sporozoite of E.tenella, and studies on otherapicomplexan (Toxoplasma, Plasmodium) indicated that AMA1was an importantprotein involved in invasion. So we selected AMA1of E. tenella (EtAMA1) as bait, andscreened the interaction proteins with EtAMA1from the constructed yeast twohybrid cDNA library of E. tenella sporozoites. The result may provided a molecularfoundation for studying invasion-related proteins of sporozoites of E. tenella.1.Construction of a yeast two-hybrid cDNA library of sporozoites of E. tenellaTo construct a yeast two-hybrid cDNA library from sporozoites of E. tenella,the chickens were infected with purified sporulated oocysts of E.tenella.Unsporulated oocysts were collected7d after inoculation and then the sporozoiteswere preparated and purified after sporulation. Total RNA was extracted fromsporozoites by using Trizol reagent and mRNAs were isolated from total RNA. Thefirst-strand cDNAs were synthesized by reverse transcription using MMLV. Thedouble-strand cDNAs acquired by Long-Distance Polymerase Chain Reaction (LD-PCR)and purified by Chroma Spin TE-400Column. The purified double-strand cDNAs weretransformed into AH109yeast competent cells with pGADT7-Rec, which is a yeastexpression vector, according to the screening method of yeast mating. All cloneswere harvested and the yeast two-hybrid cDNA library of the sporozoites of E. tenellawas constructed. The quality of cDNA library was identificated by counting thenumbers of clones on the plates of SD/-Leu. The results showed that the capacity andtiter of the library were1.1×1012cfu and2.68×109cfu/mL, respectively. PCRamplification revealed that the library contained approximately97%recombinantclones, and the inserted cDNA fragments of39clones were between300bp and2000bp. In addition, the supernatant of cDNA library after repeated freezing and thawing was used as template, seven specific genes of E. tenella (clone number:ZB11-A04、ZBZ-DO7、ZBZ-A03、AMA1、BW4-C03、RON2、ETLDH) were amplifiedfrom the constructed cDNA library in the presence of seven specific primers ofE.tenella. It could be concluded that a yeast two-hybrid cDNA library of thesporozoites of E. tenella was successfully constructed. Seven specific genes ofE.tenella were amplified from the constructed cDNA library. These results can providea foundation for screening invasion-related interaction proteins of E. tenellasporozoites by yeast two-hybrid method.2.Construction of a bait vector pGBKT7-EtAMA1In order to construct the bait vector applied in yeast two-hybrid system, anAMA1fragment of E.tenella was amplified and ligated to pGBKT7, the yeastexpression vector, and the bait vector pGBKT7-EtAMA1was constructed. Theidentification, transcriptional activation and toxicity were then tested. The1440bpfragment of target gene was obtained and identified by PCR and restriction enzyme.The pGBKT7-EtAMA1was transformed into Y187and AH109yeast competent cells,respectively. No white transformant clones grew in high selective medium(SD/-Trp/-His、SD/-Trp/-Ade、SD/-Trp/-His/-Ade/-Leu) but grew in low selectivemedium (SD/-Trp、SD/-Trp/X-α-gal) suggested that the bait vector was inactive. Onetransformant clone of pGBKT7-EtAMA1was cultured in SD/-Trp medium for22h andthe OD600was more than0.8, there was no significant difference in the growth ratebetween transformant of pGBKT7and pGBKT7-EtAMA1, these results showed thatthe protein was nontoxic. It could be concluded that the bait vector pGBKT7-EtAMA1was successfully constructed without activation and toxicity. The bait vectorpGBKT7-EtAMA1could be applied in screening the yeast two-hybrid cDNA libraryfrom sporozoites of E. tenella.3.Screening the interaction proteins with EtAMA1by yeast two-hybridIn order to screen the interaction proteins with EtAMA1in the stage ofsporozoites during invading the epithelial cells, the constructed yeast two-hybridcDNA library of sporozoites of E. tenella and the bait vector pGBKT7-EtAMA1wereapplied in yeast two-bride system, and the high selective medium was used to screen the interaction proteins. The result showed that710blue clones were acquired.42clones were selected randomly to extracted plasmids, and then transformed into E.coli DH5αcompentent cell, these selected positive clones were then sequenced afterPCR identification.14non-repeating ESTs of positive plasmids were obtainedincluding5unknown ESTs,7unnamed proteins and2reported proteins (EtZB1-A08and EtMIC2). The function of EtZB1-A08and EtMIC2is consistent with previouslyreported which revealed that they were invasion-related proteins of coccidian. Itcould be concluded that interaction proteins with EtAMA1were screened by yeasttwo-hybrid method from the constructed cDNA library, and these results providedthe candidate molecules for further research about some invasion-related proteins ofsporozoites of E. tenella.