Cloning, Expression And Function Research Of Gametocyte Antigen Genes Of Eimeria Necatrix

Chicken coccidiosis is an intestinal disease caused by protozoan parasites which belong to the genus Eimeria, and is a disease of major economic importance to the poultry industry. In China, annual costs for the control of Eimeria are estimated to30-60million US dollars. Among seven species of Eimeiria in chickens, E. tenella and E. necatrix are the most pathogenic; they caused acute caecal and intestinal coccidiosis, respectively. Severe intestinal coccidiosis caused by E. necatrix mainly occurs in8to18weeks old chickens. During the last century, the cases of acute intestinal coccidiosis caused by E. necatrix are rare clinically in our country because the life span of broilers, mostly AA broilers, was short, whereas laying hens were raised in cages during the later period. In recent years, yellow-feathered broilers are widely raised for the special requirements of consumers to chicken flavor. They should be raised over a relatively long period and they are mostly raised on the ground or net in the company plus peasant household mode; resulting the cases of intestinal coccidiosis caused by E. necatrix gradually increase.Prophylactic medication has been successfully used to control avian coccidiosis. However, the increasing incidence of drug-resistant strains, and the escalating public anxiety over chemical residues in meat and eggs request the development of alternative control methods. Moreover, there are increasing government regulations on the use of anticoccidial substances as feed additives have been banned in the European Union, and designed to inform the decision on the phasing out of these coccidiostats by December2012. And there was a similar reports in U.S. Food and Drug Administration (FDA)(FDA-2011-D-0784). Therefore, the controlling of coccidiosis is being from chemoprophylaxis to immunoprophylaxis. The vaccine used in controlling coccidiosis are mainly came from live vaccine and subunit vaccine. And the live vaccine is composed with the mixture of parasites, while the subunit vaccine is constructed with proteins coming from purified gametocytes of E. maxima and had been confirmed with high immunogenic. Gametocyte protein is the oocyst wall precursor protein, which localizes in the wall forming bodies of macrogametocyte. To date, only a small number of genes encoding gametocyte proteins have been cloned and sequenced from avain Eimeria such as Emgam56, Emgam82and Emgam230(a partial sequence) in E. maxima, Etgam56(Etgam56tmp1), Etgam59(Etgam56tmp2) and Etgam22in E. tenella, and Eagam56(a partial sequence) in E. acervulina. To the best of our knowledge, there are no previous reports regarding gametocyte antigens of E. necatrix and their genes. In order to understand the molecular mechanism of the formation of oocyst wall formation and find the candidate genes for controlling coccidiosis, we focused on the researches as following.Firstly, total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification. The cDNA encoding gametocyte protein was cloned into pGEM T easy vector and sequenced. A731-nucleotide sequence of cDNA of E. necatrix had97.7%identity to that of Etgam22of E. tenella was obtained, named Engam22. It had a opening read frame with a length of597bp which encoding186amino acids and has a proline-histidine rich domain. A PCR product of-522bp was isolated from pGEM-T-Engam22and subcloned into a pET28a(+) bacterial expression vector prior to transformation into chemically competent E. coli BL21cells. The recombiant proteins expressed in the inclusion body and revealed a protein band of-29kDa, named rEnGAM22. The recombinant rEnGAM22can be recognized by mouse anti-6×HIS tag monoclonal antibody, mouse anti-rEnGAM22polyclonal antibody or the convalescent sera from chickens immunized with oocysts of E. necatrix or E. tenella or E. maxima. Acording to immunohistochemisty localization, EnGAM22was localized in wall forming bodies in gametocytes and the walls of oocyst and sporocyst, but not in the second generation meronts. And Real time PCR verified the results of immunolocalization that the Engam22gene was not expressed in second generation meronts and other merozoite stages but it starts to transcript and express in the stage of gametocyte development and participate the formation of oocyst wall.And then, two genes encoding gametocyte proteins of E. necatrix were obtained used the methods similar to Engam22, named Engam59and Engam56. Engam59was1473bp encoding a total of490-amino acids and has a tyrosine-serine rich domain. It had93.4%identity to that of Exgam22of E. tenella and had61.4%identity to that of Emgam56of E. maxima. Engam56was918bp containing a tyrosine-serine rich domain. It had73.2%identity to that of Emgam56of E. maxima, had63.2%identity to that of Engam59, and had60.9%identity to that of Etgam59. Two PCR products,687bp and522bp, were obtained from Engam59and Engam56using specific primers respectively and were subcloned into a pET28a(+) bacterial expression vector prior to transformation into chemically competent E. coli BL21cells, named pET28a(+)-Engam59and pET28a(+)-Engam56. The recombinant proteins both expressed in the inclusion body, and revealed a protein band of-28kDa of rEnGAM59and-24kDa of rEnGAM56. And rEnGAM59and rEnGAM56were recognized by mouse anti-6×HIS tag monoclonal antibody, mouse anti-rEnGAM59or anti-EnGAM56polyclonal antibody, or convalescent sera from chickens immunized with oocysts of E. necatrix. After immunohistochemisty localization, EnGAM59and EnGAM56mostly localized in wall forming body which played a important role in wall formation.At last, Chickens vaccinated with rEnGAM22at different doses and challenged with live parasites after the booster immunity except negative group. The effects of immune protection of rEnGAM22was evaluated on parasitological criteria (oocyst excretion, intestinal lesion scores and growth performance) and immunological response. There were two animal experiments. The results showed that the groups immuned with50μg of rEnGAM22had the best immune protective effect. And the groups immuned with rEnGAM22had lower fecal oocyst shedding, reduced lesion score and higher weight gain compared with non-vaccinated and infected control. Chickens immunized with rEnGAM22also stimulated higher production of anti-rEnGAM22serum antibodies and highter cellullar immunologic response.