| The chicken coccidiosis is a protozoan parasitic disease caused by one or more virulent Eimeria species parasitize in cecum which lead to serious damage to the poultry industry. The E.tenella microneme-2 protein(Et Mic2) is secreted from the microneme organelles, and it involved in the process of the invasion, and also it can cause the chicken body immunity reaction. Interleu kin-2(IL-2) is one of immune regulation cytokines which can enhance immune protection and host immune response. In this study, we constructed recombinant DNA vaccine of Hebei strain Et MIC2 and IL-2, the recombinant plasmids were identified by restriction enzyme digestion and transfected into 293 T cells, the target genes were verified expression by RT-PCR method. The study was divided into the following sections:1. Cloning and bioinformatics analysis of IL-2 gene in chickenThe part of the research we designed a pair of specific primers according to Gen Bank in the IL-2 m RNA sequences, then used the RT-PCP method for cloning the IL-2 gene from chicken. The sequence analysis showed that the full length of IL-2gene was 432 bp, including a 432 bp open reading frame, and encoding 132 amino acids. And we found the sequences which we built was different from the one from NCBI, in the sites of 28、30、32 and120 amino acids happened mutation. The prediction of protein structure showed that the molecular weight of encoded protein is 16.429 KD, the theoretical isoelectric point is 5.66, the molecular formula is C728H1178N184O225S10. The protein has a strong hydrophilic, contain protein transmembrane region, its position before the signal peptide of 22 amino acids, the protein deduced transmembrane protein secretion; secondary structure to α-helix and random coil-based extension chain and β corner just appeared in the local; subcellular localization showed that the primary role of protein in the cell membrane.2. Cloning and bioinformatics analysis of EtMIC-2 gene in Eimeria tenella hebei strainThe EtMIC-2 gene of E.tenella Heibei strain was amplitied by RT-PCRtechnology, then the gene was inserted into p MD-19 T and sequenced, the sequence analysis and protein structure prediction were carried on. The sequence analysis showed that the full-length of Et MIC-2gene was 1029 bp, including a 1029 bp open reading frame, and encoding 342 amino acids, the Gen Bank(AF111839.1) and published Et MIC-2 nucleotide and amino acid sequences encoding the similarities were 99.8% and 99.4%, with two-base mutation,(655 A to the base G, 808 bit is set by the base T into C). Sequence analysis showed that: Hebei strain Et MIC-2 gene and E.tenella hybrid strains between Beijing strains isolated from Guangdong Province, Howden strain Et MIC-2 gene is relatively conservative, while Brandt Eimeria MIC- 2 genes species differences. The prediction of protein structure showed that the molecular weight of encoded protein is 35 KD, the theoretical isoelectric point is 5.66, the molecular formula is C1500H2429N431O528S5. The protein hydrophobicity is not strong, no transmembrane region, location of its signal peptide may be for the first 22 amino acids of the protein deduced transmembrane protein secretion. The signal peptide is located in the first 22 amino acids. The secondary structure consists of 16 alpha-helices, 20 beta angles, 17 corners, 25 random coils.3. Construction of recombinant eukaryotic vector pcDNA3-EtMIC-2-IL-2Pc DNA3-Et MIC-2-IL-2、pc DNA3-Et MIC-2、pc DNA3-IL-2 DNA vaccines were constructed by the means of DNA recombinant. The recombinant plasmids were Identified by restriction enzyme digestion and transfected into 293 T cells,The target gene were verified expression by RT-PCR method. The results showed the The resu Lts showed the pc DNA3-Et MIC-2-IL-2 、 pc DNA3-Et MIC-2 、 pc DNA3-IL-2 DNA vaccines were expressed in 293 T cells. |
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