The Construction And Screening Of CDNA Library Of Eimeria Maxima Sporulated Oocysts

Eimeria maxima is one of nine species of fowl coccidians which belong to Eimeria genus, Eimeriidae family, Eucoccidiorida order, Sporozoasida class, Apicomplexa phylum. Eimeria maxima is a world-wide-spreading-species and has been discovered in 25 provinces of China. Eimeria maxima has a strong immunogenicity and is an important component of vaccine against coccidiosis. By present, cDNA libraries of E. tenella and E. acervulina have been constructed both in and abroad and important genes have been screened from these cDNA libraries. This paper is to construct a cDNA library with the Eimeria maxima strain isolated in our laboratory, and screen the cDNA library with two different methods to discover the valuable genes.The first part of the paper is to make sure the separated strain to be E. maxima. 2-week chickens were infected with sporulated oocysts and involved indexes were checked. The results are as follows: the prepatent period is 144 h, location of parasites is the middle piece of small intestine; the shortest sporulation time is 23 h; the average length and width of 100 oocysts is 21.25～37.25μm×16.50～28.00μm; the average length and width of 100 sporocysts is 15.00～17.50μm×7.50～10.50μm. The results prove that the separated strain is the pured Eimeria maxima.The second part of the paper is to construct a cDNA library of Eimeria maxima. First, oocysts were cleaned with gradient centrifugalization. Second, total RNA was extracted from the oocysts, with high quality for two reasons: OD260/OD280 of the total RNA was 1.9 and electrophoresis strips of 28 S and 18 S were clear. Third, a full-length cDNA library of E. maxima sporulated oocysts was constructed with the total RNA isolated from the sporulated oocysts of E. maxima as template andλTriplex2^TM as vector. The titer of unamplified library is 1×10⁶ pfu/mL and the titer of amplified library is 5×1010 pfu/mL. The percentage of recombinant clones is 95%. E. maxima gene for immunoglobulin heavy chain binding protein can be amplified from the constructed library. This library has high quality and can be qualified for new gene screening.The third part of the paper is to screen the valuable genes from the cDNA library. There were two different ways to obtain the ESTs of E. maxima. The first way was immunoscreening. Cony blood serum containing antibody was used as probe to screen the cDNA library and obtained one EST. The second way was changing the wholeλTr iplEx2 cDNA library into pTripIEx2 cDNA library and obtained 15 ESTs. The results proved the reliability of the constructed cDNA library.