The Construction Of Variable Regions Of ScFv Library Against The Merozoite Of Eimeria Acervulian

Four strains Eimeria oocysts were obtained by the mothod of single oocyst isolation and were studied out to be Eimeria acervulian and Eimeria tenella at first. After purification and extraction genome DNA, by PCR amplification using species-specific primers, the four strains Eimeria were classified as monospecific Eimeria acervulian and Eimeria tenella.Eimeria acervulina merozoites were abstained from intestinal mucosa use a methed screen out from four methods for furification of Eimerria acervulina merozoites. total RNA was extracted from the spleen cells of BALB/C mice, which were immuned with merozoites soluble aetigen of E. acervulina by four times, and immune responses was measured by indirect ELISA.The frist chain of cDNA which derived from the total RNA with Oligo(dT)₁₅Primer by RT was used as template, VH and VL genes was amplified by PCR using gene specific primers. PCR pruduct be examined by agarose gel electrophoresis and purified by agarose gel DNA purificatin kit, the result shows the genes of VH and VL amplified were about 420bp and 390bp respectively. The purified pruduct was cloned PGM-T vector. The cloned plasmids were transformed into TOP 10. Colonies were selected on ampicillin-containing agar plates and plasmids were isolated from several independent clones. The clonies were sequenced by Sanger's dideoxy sequencing method. Nucleotide sequence of the VH and VL genes sequenced were compared with the published VHand VL sequence from the BALB/C mice. As expected, sequence difference between VH/VL and corresponding germline genes were identified, predominantly localized in complementary determining regions.