The Construction And Screen Of CDNA Library For The Eimeria Necatrix Gametocytes And Expression Of Dynein Gene

Avian coccidiosis is a diseases caused by intracellular protozoa belonging to Eimeria,and has a major economic importance to poultry industry.E.necatrix is a pathogenic,and caused acute small intesnial coccidiosis among eight to eighteen week-old chickens.Nowadays coccidiosis is mainly controlled by the use of chemotherapeutic agents and live vaccines.However,the use of anti-coccidial drugs has been greatly restricted with the continual emergence of drug-resistance and the public concern of safety in animal foodstuff.Meanwhile,the use of live oocyst vaccine has some drawbacks including the high production expenses,the possibility of causing morbidity and mortality in vaccinates,and the risk of attenuated organisms reverting to a more pathogenic state,and so on.Therefore,cloning of protective antigen gene from coccidian and expression of recombinant proteins have been a research focus.For this reason,in the present study,cDNA library was constructed using the gametocyte of E.necatrix.The ENH₀0080190 gene was cloned and expressed using a prokaryotic expression system.This research established foundation to develop the subunit recombinant vaccines against avian coccidiosis.1.The identification and purification of the gametocytes of E.necatrixTwenty-four-day-old chickens were infected with 10 000 sporulated oocysts of E.necatrix.At 144 h post infection,the second generation merozoites were released from the mucosal tissues,the second generation of merozoite inject directly into the chicken cecum through the surgical method and make merozoite to gametophyte synchronous development.At 30h after inoculation,the chickens were killed,and the mucosal tissues in the cecum was removed.By means of filtration with 60 mesh copper sieve,260 mesh nylon mesh and 17 microns PET membrane,respectively,the gametophyts were isolated.Finaly after centrifuged at 5 000 rpm for 15 minutes with 30%and 50%percoll,the gametophyts were purified.The results showed that the purity and quantity of gametophyts obtained from the tissues were high,and can be used in the gene transcription omics,proteomics and immunology in thegametophyte stage of E.necatrix.2.The Construction of a cDNA library for of the gametocytes E.necatrixThe whole RNA from the gametophyts of E.necatrix was extracted using Trizol reagent.Then,the cDNA library was constructed by SMART technique.The result shows that the OD260/OD280 value of total RNA is 1.95,and the 28 S and 18 S were clear in the electrophorogram.The titer was 3.42×106 pfu/mL in the primary cDNA library and 2.6×1010 pfu/mL in the amplification cDNA library.The recombination rate was 90%.The length of inserted fragments were concentrated in 250?1000 bp.3.The immunological screening of a cDNA library for the gametocytes of E.necatrixFirstly,the positive sera of anti-E.necatrix and mice anti-gametocyte of E.necatrix.The sera titers were detected with ELISA,which showed that sera could be used for immunoscreening cDNA library.Secondly,the library was screened using mouse polyclonal antibody as probe.Five positive clones were obtained in the initial screening,and two positive clones were obtained by rescreening.Finally,the positive clones were identified using PCR,and the PCR products were sequenced.The results showed that the three ESTs were 734 bp?270 bp and 606 bp,respectively.The bioinformatics analysis showed that the ESTs were E.necatrix specific protein,related partial mRNA,E.necatrix dynein light chain,and E.necatrix hypothetical protein.4.The expression of E.necatrix dynein gene and immunogenicity analysis of recombinant proteinFirstly,the dynein gene?ENH₀0080190?of E.necatrix was synthetic and subcloned into express vector pET-28a?+?.The vector then was transformed into E.coil BL 21,and was induced with IPTG for expression.The expression products were analyzed with SDS-PAGE.Finaly,the recombinant proteins were analyzed by Western-blot using the positive sera of anti-E.necatrix oocysts.The results showed that gene was consisted of 270 nucleotides,which encoding 89 amino acids.The fusion protein was about 13 kDa,and expressed in incluble body.The recombinant proteins can be recognized by the positive sera of anti-E.necatrix oocysts,which suggested that the recombinant protein had good immunogenicity.