Precocious Strain Selection Of Eimeria Nectrix And Expression And Immunoprotective Analysis Of REnGAM59 Of E.Necatrix

Avian coccidiosis is a severe parasitic protozoa disease causing considerable economic loss in the poultry industry.The chemotherapy and vaccine immunization are manly methods that are used to control the disease.The vaccines used to control coccidiosis currently include live vaccine and subunit vaccine.There are two major classes of vaccines,virulent vaccine and attenuated vaccine.Because the virulence of strains are low,the attenuated vaccines receive the favor of the farms.The production of subunit vaccine needs to isolate and purify the gametocytes from the infected chicken,and then gametochyte proteins are isolated and purified with the method of affinity column chromatography.Therefore the production of subunit vaccine is time-consuming and expensive.Use of recombination-mediated genetic engineering can solve this problem.E.necatrix is one of the most pathogenic coccidial species in chickens.It may cause very severe intestinal coccidiosis in 8-18 weeks old chickens.In recent years,yellow-feathered broilers are widely raised and they are mostly raised on the ground or net,resulting the cases of intestinal coccidiosis caused by E.necatrix gradually increase.In this study,an precocious line of E.necatrix were established with the precocious selection method.Meanwhile,the gam59 gene of E.necatrix was expressed.The immune protection of recombinant proteins and the precocious line of E.necatrix were evaluated.The research may lay a foundation for the development of attenuated vaccines and genetic engineering vaccines for chicken coccidiosis.1 Establishment of an E.necatrix single-oocyst strain and its PCR identificationThe single oocyst of E.necatrix was isolated to infect 3-day-old coccidial-free chicken and the oocysts were isolated from feces of the infected chicken.After sporulation,the oocysts were used to infected with coccidial-free chicken to expend the number of parasite.Then,the oocysts were used to infected with coccidial-free chicken to observe characteristics of intestinal lesions,parasites and oocyst morphology.The genome DNAs,extracted from oocysts of E.necatrix,E.maxima,E.tenella and E.acervulina,were used as templates for PCR with four sets of species-specific primers designed according to the previously published ITS-1 sequences of these four species of coccidian,respectively.The PCR products were analyzed using agarose gel electrophoresis.According to the morphological characteristics of intestinal lesions,parasites,and oocysts,the single oocyst isolate was identified to be E.necatrix with a potential period of 156 h.The PCR result showed that the species-specific DNA would be amplified when both the templates and primers used in PCR come from the same Eimeria species.It concluded that these four avian coccidia were the pure strains.2 Precocious strain selection of E.necatrixAn E.necatrix single oocyst isolated strain was used as a parent strain,and the precocious strains of E.necatrix were selected by precocious strain selection method.After 26 generations of precocious breeding,two successive generations of oocysts were expanded.14-day-old chicken were infected precocious strains at doses of 0.5 × 104,1 ×1 04,3 × 104,and 5 × 104 oo-cysts/feathers,and the parent strain was used as the control group.Average body weight,mor-tality and intestinal lesion were used to evaluate pathogenicity of the precocious strains.The results showed that potential period was shortened from 156 h to 140 h after 26 generation breeding,and pathogenicity of precocious strains decreased significantly compared with paren-tal strains.3 Prokaryotic expression of EnGAM59 of E.necatrixThe primer was designed according to the ORF sequence of the Engam59 gene and the enzyme cleavage site of plasmid pET28a(+).The recombinant plasmid pGEM-T-easy-gam59 was used as the template to amplify expression fragment with a size of 1419 bp(the signal pep-tide portion was removed).The gene was subcloned into a pET28a(+)bacterial expression vec-tor prior to transformation into E.coli BL21 cells,named pET28a(+)-Engam59,then the protein was induced expression.The fusion protein was shown to be about 57 kDa by SDS-PAGE analysis.The rEnGAM59 can be recognized by mouse anti-6 ×HIS tag monoclonal antibody.Soluble analysis showed that the recombinant protein exists in inclusion bodies.Western blot showed that the E.necatrix natural gametophyte protein can be recognized by mouse anti-r EnGAM59 polyclonal antibodies and a specific band with a size of approximately 56 kDa was observed.The results showed that the gametophyte protein EnGAM59 was successfully ex-pressed in vitro and had good immunogenicity.4 Immunoprotective analysis of rEnGAM59 of E.necatrixThe immunoprotective of recombinant protein rEnGAM59 was evaluated by oocysts re-duction,lesion score and average weight gain.The specific antibody level was detected after immunization.The results showed that,compared with the non-immunized group,the recom-binant protein rEnGAM59 could reduce the oocyst yield and reduced the lesion score and in-creased the average weight gain,but could not achieve the immune protection level of the oo-cyst immunization group.The immunoprotective of precocious strain immunization group and the parent strain immunization group were similar,and had no significant difference.