The Primary Research Of EtCBS And EtCHP317 From Eimeria Tenella

Coccidiosis is a very harmful parasitic disease which leads to high economical losses to the poultry industry every year. The disease is caused by Eimeria spp which belong to the Apicomplexa phylum. Among several Eimeria spp, Eimeria tenella is now known as the strongest one in virulence, it can cause fatal damage to the chickens. Most Apicomplexa are obligate intracellular parasites and the cell invasion mechanism is highly conserved. A ring-shaped moving junction(MJ) is formed between the anterior end of the parasite and the host cell plasma membrane during the parasite invade to the host cell, The MJ is a highly conserved structure and plays an important role during parasite invasion. Recent works in Toxoplasm and Plasmodium have inforced that the apical membrance protein-1(AMA1) which secreted from micronemes is the key component of MJ and plays a central role in host cell invasion. In Eimeira, the further research on AMA1 is very important for understanding the mechanism of the parasite invasion the host cells.In our previous studies, a yeast two-hybrid c DNA library of E.tenella sporozoites and the bait recombinant plasmid p GBKT7-EtAMA1 were constructed. The bait protein was used to screen the interacting proteins of EtAMA1 by used the yeast two-hybrid system. Some ESTs which maybe the interacting proteins of EtAMA1 were obtained. In this study, we selected two ESTs(clone No. 23, Genebank No. JZ905767 and clone No. 317, Genebank No. JZ905773), then verified the interaction relationship with EtAMA1 and we also analysed the function of these two proteins.1. Cloning and bioinformatics analysis of EtCBS and EtCHP317 geneSince the two selected EST sequences contain a ploy(A) in the 3’end, so the full-length 5’-ends of the c DNA for the two genes were obtained by 5’ RACE technique. The 2423 bp full-length c DNA was obtained with the EST No. 23 sequence, including an ORF of 1887 bp, encoding 629 amino acids with predicted protein molecular weight of 68 k Da. The deduced amino acid sequence had no predicted transmembrane region and signal peptide, but had 22 B cell antigenic sites, the protein encoded by the gene is 100 % homologous to cystathionine β synthase, so the gene was named as E. tenella cystathionine β synthase(EtCBS). The full-length of the gene 317 is 1113 bp including an ORF of 501 bp, encoding 167 amino acids with predicted molecular weight of 18 k Da. The deduced amino acid sequence had no transmembrane and signal peptide, had 8 B cell antigenic sites, the protein encoded by the gene is 100 % homologous to protein conserved(hypothetical conserved protein, Etm102F05), therefore this gene was named EtCHP317. QRT-PCR was used to detect the transcription level of the two genes. Among these four development stages(sporulated oocysts、sporozoites、 unsporulated oocysts and second-generation merozoites), the m RNA level of EtCBS was higher in sporozoites than the other three stages. The m RNA transcript level of EtCHP317 was highest in the second-generation merozoites.2. Prokaryotic protein expression and preliminary function Study of EtCBS and EtCHP317The ORF fragment of EtCBS and EtCHP317 was respectively cloned into the prokaryotic expression vector p ET-28 a and transformed into E. coli BL21(DE3). Then recombinant proteins His-EtCBS(rEtCBS) and His-EtCHP317(rEtCHP317) were expressed successfully by induced with IPTG at low temperature, respectively. Moreover, two recombinant proteins are soluble proteins and purified by His Bind resin. Two purified proteins were used to immunize the rabbits, respectively. Then the polyclonal sera were obtained. Using antibody against rEtCBS or rEtCHP317, the localization of these two proteins in sporozoites, second-generation merozoites and during first schizogony were investigated in vitro by immunofluorescence. The results showed that EtCBS was mainly located on the surface of sporozoites and merozoites, during the parasite invasion host cells, the protein was on the anterior region of sporozoites. Following the development of parasites in cells, EtCBS protein was uniformly dispersed in parasites and increased. So we postulated that EtCBS might be involved in invasion and intracellular growth of the parasites in the host. The positioning of EtCHP317 showed that the protein was mainly located on the surface of sporozoites and merozoites, when the parasites invade the host cells, the protein was distributed on the surface and the head of the parasites. So the protein maybe involved in invasion. Inhibition experiments were performed using sporozoites preincubated with anti-rEtCBS or anti-rEtCHP317 respectively. We found that the two antibodies pretreatment significantly reduced the capacity of sporozoites to invade the DF-1 cells, and that the inhibitory effects of these two antibodies were dose-dependent. Compared with the same dose of Ig G from naive rabbit serum(negative control), when the antibodies concentration increased to 400μg/ m L, the two antibodies can prevent about 70% the parasite invasion cells, respectively. These results suggested that two proteins may play an important role during the invasion of host cells.3. Verification of the interaction of the EtCBS and EtCHP317 with the EtAMA1In order to verify the interaction between EtCBS with EtAMA1 and EtCHP317 with EtAMA1, EtCBS and EtCHP317 were cloned into the eukaryotic expression vector pcDNA3.1(+) and pBiFC-VN155, respectively. Four eukaryotic recombinant expression plasmids were constructed successfully, including pcDNA3.1-EtCHP317, pcDNA3.1-EtCBS, pBiFC-VN155-EtCBS and pBiFC-VN155-EtCHP317. pcDNA3.1-EtCHP317 and pcDNA3.1-EtCBS were co-transfected into DF-1 cells with the pcDNA3.1-flag-EtAMA1 which had been obtained in our lab, respectively. The interactions of these two proteins with EtAMA1 were identified by Co-IP with the anti-flag tag antibody, the results indicate that EtCHP317 is an interacting protein of EtAMA1, but EtCBS is not an interacting protein. At the same time, pBiFC-VN155-EtCBS and pBiFC-VN155-EtCHP317 were co-transfected into DF-1 cells with pBiFC-VC155-EtAMA1 which had been obtained in our lab, respectively. Bi FC was used to identify the interactions. The results of Bi FC show that there were no green fluorescencein the DF-1 which was co-transfected by pBiFC-VN155-EtCBS and pBiFC-VC155-EtAMA1, while the specific green fluorescence was observed in the the DF-1 which was co-transfected by pBiFC-VN155-EtCHP317 and pBiFC-VC155-EtAMA1. Additional, these interactions were verified by GST Pull-Down experiments. These results show that there is no interaction between EtCBS and EtAMA1, while EtCHP317 and EtAMA1 interact with each other.4. Protective effect of rEtCBS and rEtCHP317 on chickens against E. tenellaA total of 120 one-week old chickens were randomly divided into 6 groups of 20 chickens with equal mean body weights, including unchallenged control, infection control, rEtCHP317(50 μg/bird),rEtCHP317(100 μg/bird), rEtCBS(50 μg/bird), rEtCBS(100 μg/bird). The experimental groups were immunized with different recombinant protein emulsified in Montanide ISA 71 adjuvant, the chickens of the infected control group and unchallenged groups were injected with PBS. A booster immunization was carried out 8 days later with the same dose as the primary immunization. 8 days after the second immunization, all the chickens except the unchallenged control group, were challenged orally with 1×104 sporulated oocysts of E. tenella. While chickens of unchallenged control were given equal volume PBS orally. Nine days after the challenge, all chickens were weighed and slaughtered. Average body weight gain, oocyst decrease ratio, lesion score were calculated. The results showed that, compared with the control group, the experimental group have a protectivce effect because they could increase weight gain, reduce both cecum lesions and oocysts shedding. These results showed that the recombinant protein had partial protection against E.tenella challenge.