Studies On Differentially Expressed Genes Of Sporulated Oocysts And Sporozoites Of Eimeria Tenella

Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflict severe economic losses on the poultry industry. At present, conventional disease control strategies have relied on prophylactic medication and immunization with live vaccines. Due to the emergence of drug resistant parasite and the danger of live vaccines etc, Novel approaches are urgently need to study to control coccidiosis. At first, Identification important parasite antigen genes is crucial for the design of novel control approaches. Eimeria tenella is one of the seven species of parasites causing avian coccidiosis. So it was used to isolate and identify differential expressed genes of E. tenella sporozoy stages. The results may be provided important rationale for studying new vaccines and drugs against coccidiosis.1. Identification and analysis differentially expressed genes of sporulated oocysts and sporozoites of Eimeria tenella by Suppression subtractive hybridization and cDNA microarray techniquesIn order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of Eimeria tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated oocysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed . PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. The length of insert cDNAs was above 500bp in three subtractive cDNA libraries. 3004 clones were selected from three cDNA libraries to fabricate cDNA micrarrays. Microarray hybridization results suggested that 1371 clones were identified which showed differentially expression contained 582 clones from the sporulated oocysts subtractive cDNA library and 799 clones from the two sporozoites subtractive cDNA libraries. A total of 727 clones were selected and sequenced. 657 valid ESTs were obtained. The results of sequence analysis indicated that these clones represented 118 unigenes composed of 31 sporulated oocysts and 87 sporozoites unigenes. On homology searches of the public sequence databases, Among 31 sporulated oocysts unigenes, 12 genes had known protein functions, 19 genes had unknown protein functions, 7 genes of Eimeria were reported . Among 87 sporozoites unigenes, 23 genes had known protein functions, 64 genes had unknown protein functions, 6 genes of Eimeria were also reported. 5 differentially expression genes obtained were confirmed by Real-time PCR, which demonstrated that these genes were indeed differentially expressed. This results confirmed that SSH and microarray technology can be successfully applied to identify differentially expressed genes of the different development stages of E. tenella. Blast searches show that some proteins encoded by differentially expressed genes of sporulated oocysts shared significant identity with previously described proteins, including E. tenella microneme proteins, TA4 antigen protein, surface antigens , T.gondii heat shock protein. T. gondii PDI, C. hominis oocyst wall protein etc. Some proteins encoded by differentially expressed genes of sporozoites were homology with previously described proteins, including E. tenella SERPIN1 protein precursor, microneme proteins, reverse transcriptase, P. falciparum serine/threonine phosphatases, Calcium-dependent protein kinase 2 (PfCDPK2), P. yoelii sphingomyelin/lysocholinephospholipid-phospholipase C etc. These results suggested that these differentially expressed genes implicated in E. tenella oocysts sporulation, growth and development of E. tenella in host cell, invasion to the host cell, interaction between the parasite with host cell, cell metabolism , immunoloregulation, the regulation of cell cycle, signal transduction etc.2. Cloning and analysis differentially expressed novel genes of the sporoluated oocysts and sporozoites of E. tenellaBased on ESTs of differentially expressed genes of sporulated oocysts and sporozoites recerived by SSH and cDNA microarray , eight novel genes including a complete open reading frame were obtained with RACE technique. Among these novel genes, five new genes were up-regulated in sporulated oocysts of E. tenella, named as BW1-A05, BW1-E06, BW3-F04, BW3-D10, BW11-H07, respectively, three novel genes were up-regulated in sporozoites, named as ZB1-A02, ZB1-A08, ZB11-A04, respectively. With bioinfomatics analysis software, property and function of eight proteins encoded by these novel genes were predicted, including physical and chemical property, trans-membrane structure, antigen site, conserved domain, function site and homologous protein search. These results indicated that four proteins encoded by BW11-H07 , BW1-E06, ZB1-A02, ZB11-A04 were exhibited similarity to T.gondii hypothetical protein, PDI, toxomepsin and L. infantum protein kinase, respectively. These results provided important information to isolate useful genes of genetically engineering vaccines and novel drugs to control coccidiosis.3. Expression of novel genes of E. tenella in E.coli.Four differentially expressed novel genes were ligated to prokaryotic expression vector pGEX-4T-2 and constructed recombinant plasmids 4T-E06, 4T-F04, 4T-A02 and 4T-A08 successfully, respectively. These recombinant plasmids were transformed into BL21 (DE3 )for expression. After induction by IPTG, recombinant expressed proteins were identified by SDS-PAGE. Most of 4T-E06 recombinant proteins was soluble in cytoplasm, other three fusion proteins were expressed in the form of inclusion bodies. Four fusion proteins were purified successfully with GST resin, respectively. Western-blot revealed that four proteins were native antigens.4. Efficacy of recombinant proteins on induction of protection immunization against E. tenella challenge. Two purified recombinant proteins and four live E.colis containing recombinant proteins were used to immunize 1-weeks-old chickens, Several immunization protocols were utilized, Some chickens were immunized by intramuscularly injection with 25μg, 50μg, 100μg purified recombinant proteins, respectively. Other chickens were immunized orally with 150μg or 300μg live E.coli transformants expressing 4 recombinant proteins, respectively. After boosted on day 17 and day 27, chickens were challenged with 2×10~4 sporulated oocysts of E. tenella on day 31. Protective efficacy of recombinant proteins or live E.coli transformants against E. tenella infection was evaluated by body weight gain, mean oocyst output and mean cecal lesions. Partial protection was seen after immunization with different recombinant proteins or live E.coli. transformants . These results have provided the foundation for studying recombinant vaccines to control coccidiosis.