Construction And Screening Of CDNA Library Of Eimeria Necatrix Sporozoite

Eimeria necatrix is one of the important pathogen of chicken coccidiosis. Acute small intestinal coccidiosis caused by E. necatrix mainly occurs in 8 to 18-week-old chickens. In recent years in china, as the number of the yellow feather chicken increasing, the acute small intestinal coccidiosis caused by E. necatrix are becoming common, and caused great economic losses to the poultry industry. The sporozoites is the primary stage of chicken coccidia invading intestinal cells and is also the first target for prevention and treatment of coccidiosis. For this reason, in the present study, a sporozoite cDNA library was constructed using the sporozoites of E. necatrix, and screened using immunological and conversion of λ phage library to plasmid library methods in order to obtain the candidate antigens or functional genes, respectively. The results may lay a foundation for the development of E. necatrix genetic engineering vaccine and finding the mechanisms of invasion of E. necatrix.Firstly, separation and purification of E. necatrix sporozoite and preparation of polyclonal antibody.25-day-age healthy chickens were inoculated 2×104 E. necatrix sporulated oocysts per birds. From 7-12 day after infection, the oocysts were isolated from feces using the conventional method and cultured to sporulated oocysts. After the sporulated oocysts were disinfected using 20% sodium hypochlorite solution for 20 minutes, the sporulated oocysts were purified with 1.1 M sucrose solution. The oocyst were vortexed with glass beads to break oocyst walls. Then the oocyst solution were filtrated through the 10 um membrane, and the filtrates were collected and centrifugaled to obtain sporocysts. The sporocysts were digested using 0.75% trypsin to release the sporozoites. The sporozoites were separated and purifiing using DEAE-52 cellulose column. Six-week-old BALB/c mice were immunized with 100 μg of sporozoite antigen for three times at 2-week intervals. Polyclonal mouse anti-purified sporozoite antigen was separated from the blood 7 days after the third booster dose. Indirect enzyme-linked immunosorbent assay (ELISA) was constructed using mouse anti-purified sporozoite antigen as the first antibody, horseradish peroxidase (HRP) conjugated rabbit anti-mouse IgG as the second antibody and tetramethylbenzidine (TMB) as chromogenic agent. As results, the recovery rate of purified sporozoite was 40%, and the optimal antigen coating concentration is 1 μg/mL in ELISA combined with the 1:200 dilution of mouse anti-purified sporozoite polyclonal antibody and 1:2000 dilution of horseradish peroxidase (HRP) conjugated rabbit anti-mouse IgGSecondly, construction of E. necatrix sporozoites cDNA library. Total RNA of E. necatrix sporozoites was extracted by Trizol. And reverse transcription and synthesis of first strand cDNA were conducted after the analysis of integrity and purity detection of the total RNA. The double-strand cDNA was synthesized by LD-PCR techniques. After digestion with proteinase K and Sfi I, the ds-cDNA was fractionated by CHROMA SPIN-400 Column. The ds-cDNA were ligated into the λTriplE×2 vector by T4 DNA ligase. After package in vitro, the cDNA library was constructed. The titer of the primary cDNA library and the amplification cDNA library and the recombination rate were determined according to the number of plaque formation in E. coli XL 1-Blue plate and the proportion of blue plaque to colorless plaque is the basis of. Finally, the sizes of the inserting fragments were determined by PCR. The result shows that the OD260/ OD280 value of total RNA is 1.9, and the 28 S and 18 S were clear in the electrophorogram. The titer was 3.1×106 pfu/mL in the primary cDNA library and 1.1 × 109pfu/mL in the amplification cDNA library. The recombination rate was 96.3%. The length of inserted fragments were concentrated in 300～1500 bp.Finally, screening of E. necatrix sporozoite cDNA Library using two ways and obtained EST sequence. (1) In immune screening, the library was screened using mouse polyclonal antibody as probe.35 positive clones were obtained in the initial screening, and 19 positive clones were obtained by rescreening. The rescreening positive phage λTriplEx2 were transformed into plasmid pTriplEx2, and then send to company to sequence. The sequences of ESTs were analysesed. A total of 8 contigs were obtained, which were 1027、820、900、1176、 645、987、719、858 bp in length. (2) In the screening methord of conversion of λ phage library to plasmid library, The λTriplEx2 phage library was transformed into pTripIEx2 library. A total of 50 clones were selected to be sequenced, and 11 clones were succeed. Comparison of ESTs obtained from two methods, five sequences are identical. The bioinformatics analysis of 8 contigs showed that these ESTs encode Eimeria regulatory proteins such as endonucleases, exonuclease and phosphatase, serine protease inhibitor domain protein and microneme protein which is related to sporozoite invasion, respectively.