Construction Of CDNA Expression Library And Screening Gene Of Eimeria Necatrix

The Phylum Apicomplexa is composed entirely of parasitic protists,including many pathogens of veterinary and medical importance (e.g. Eimeria, Plasmodium, Babesia, Toxoplasma, Cryptosporidium and Sarcocystis). Among various parasitic infections, coccidiosis caused by obligate intracellular protozoan parasite of the genus Eimeria is the major constraint for modern poultry production. Coccidiosis, caused by various Eimeria spp., not only threatens the health and welfare of poultries, but also causes significant economic losses in poultry industries around the world. Among various Eimeria spp., Eimeria necatrix, which causes caecal coccidiosis, is highly pathogenic. In spite of advances in immunological, biotechnological and genetical methods, control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drugresistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Among the several alternative Eimeria control measures only host vaccination against Eimeria appears promising. In this research, our strategy was constructing the cDNA expression library of the sporozoites by Eimeria necatrix sporulated oocysts, and screening, cloning the immunogen genes, as well as expressing the interesting genes, analysing the protective immunity. Results of studies displayed as following.1. The isolated-strain was propagated and identified according to classical indexes such as the prepatent period, location of coccidia in the intestine, the shortest sporulation time, size and shape of oocysts and sporocysts. The results showed that the isolated-strain was pure strain. In order to confirm whether the isolation was monospecific, another method was used based on PCR amplification by species- specific primers which were designed according to E. acervulina,E. tenella,E. maxima genome. The results proved that the isolated-strain of E.necatrix (Guangdong strain) was monospecific.2. Chickens were orally infected with sporulated oocysts of E. necatrix for preparations of anti-E. necatrix positive serum. Rabbit was infected with sporulated oocysts of E. necatrix for preparations of anti-E. necatrix positive serum. The results showed that rabbit anti- E. necatrix positive serum could be use for immunoscreening from cDNA expression library.3. We first constructed successfully cDNA expression library of sporulated oocysts of E. necatrix with SMARTTM cDNA library construction kit in China. The result showed that the titer of primary cDNA library was 4.72×106 pfu/mL, the titer of amplified cDNA library was 2.62×1010 pfu/mL, the recombination rate was 97.5℅, the average insert fragments was 500～1 100 bp. These showed that constructed the cDNA library of E. necatrix had good quality.4. cDNA expression library of E. necatrix was immunoscreened with positive serum of rabbit anti-E. necatrix. Thus 3 positive clones were obtained.After identificated the positive clones were same.It has a ORF of consisted of 344 nucleotides encoding 133 amino acids. Compared with Eimeria necatrix small subunit ribosomal RNA gene and Eimeria tenella small subunit ribosomal RNA of the gene data the homology of nucleotides acide sequence of the gene data were 98% and 97%. All those confirmed that the gene was small subunit ribosomal RNA gene of E. necatrix. After that the cDNA library was changing into plasmid and obtained ESTs, which were same. Compared with Eimeria tenella small subunit ribosomal RNA the homology of nucleotides acide sequence of the gene data were 97%.