Cloning,Prokaryotic Expression And Identification Of Immunogenicity Of Trichomonas Gallinae G3 Protein

Trichomoniasis is caused by Trichomonas gallinae?T.gallinae?,which is belonging to flagellata,trichomonadidae,trichomonas,invase pigeon upper segments of digestive tract.All varieties and age of dove can be infected with T.gallinae,among which young pigeons are most susceptible,adults are more resistance and show latent infection.Trophozoite parasites in the intercellular gap of epithelial cells,characteristic symptoms of trichomoniasis is necrotic ulcer on the mucous membrane of oral,esophagus,crop and stomach,severe cases show rough yellow caseous exudate above the mucous membrane,leading to suffering from respiratory and feeding difficulties,oral secretions increasing,obvious symptoms is breast feathers messy,severe symptoms will lead to host death.At present,treatment of trichomoniasis usually uses chemical medicines,chemical medicines have a tendency that causing side effects on animals,and drug resistant trophozoite strains are easily induced by medicine selection pressure.It is nessesary to understand vaccine related protein molecules,explore a safe and effective novel way of prevention and treatment.The TgG3 protein has more than one open reading frame,has good immunogenicity.In this study,TgG3 protein was cloned and expressed for the first time,immunogenicity of protein was identificated,it will be helpful to value the possibility of developing novel vaccine,is conductive to the prevention and control of trichomoniasis.The main results are as follows:1.Cloning and sequencing of the T.gallinae G3 proteinPrimers were designed according to the partial ORF gene of T.vaginalis G3 gene in genebank,T.gallinae G3 protein base sequence were obtained by PCR amplification after extraction and of total RNA of T.gallinae and reverse transcript.T.vaginalis G3 gene was TA cloned into the pJET 1.2 clone plasmid and sequenced by the sangon biotech company.The ORF of TgG3 protein contains 1623 base pairs of nucleotide sequence,encoding 540 amino acids.The theorical molecular weight of the protein is about 63.7 kiloDalton.2.Base sequence and bioinformatics analysis of TgG3 proteinNucleotide sequence alignment between T.gallinae and T.vaginalis G3 protein showed that nucleotide homology between two protein was 78%,base sequence differencesdistributied uniformly every few bases from 5' to 3 'ends,G3 protein base sequence has no great homology with other proteins.Amino acids sequence alignment results showed that the homology between T.gallinae and T.vaginalis G3 protein was 98%,there were 9 amino acid sequence differences among them.The amino acid homology between T.gallinae G3 protein and Tritrichomonas foetus TRFO₁3881 protein was 83%.Bioinformatics softwares were used to analysis and predict protein characters base on the amino acid sequence of the TgG3 protein,the results showed that TgG3 protein had no pro-peptide and signal peptide,had hydrophobic,was basic amino acid,had no GPI,had SMC?structural maintenance of chromosomes?domain.3.Preparation of polyclonal antibody against recombinant TgG3 proteinRecombinant plasmid pET28a-G3 was constructed by CloneEZ PCR cloning kit using homologous recombination method,positive plasmid was transformed into E.coli transetta,recombinant TgG3 protein expressed inducing by IPTG,the bacteria cells were broken by supersonic then the inclusion body was dissolved in urea and purified by Ni-NTA.SDS-PAGE results showed that the molecular weight of prokaryotic expression recombinant protein was about 63.7kDa,which was equal to the predicted molecular weight of soft ware prediction.Purified TgG3 protein was used to immunize mice emulsified with freunds adjuvant,then polyclonal antibody was prepared,titer of the antibody was detected by indirect ELISA method.Antigenicity of TgG3 protein was identified and analyzed by westerm-blot,mice polyclonal antibody could recognize recombinant protein expressed by pET28a-G3,that meant prokaryotic expression recombinant protein had antigenicity.4.Identification of reaction of polyclonal antibody against natural TgG3 proteinT.gallinae under ogarithmic growth phase was disintegrated then sampled and electrophoresed,western-blot was conducted with mice polyclonal antibody.The western-blot results showed that 63 kDa protein of T.gallinae could be recognized by mice anti G3 protein antibody,it indicated that prokaryotic expression recombinant Tg G3 protein had good immunogenicity.TgG3 protein base sequence was obtained by PCR,prokaryotic expression plasmid pET28a-G3 was construced and 63.7kDa molecular weight of recombinant protein was expressed by E.coli transetta,mice polyclonal antibody against recombinant G3 protein couldidentify prokaryotic expression recombinant protein that meant the recombinant proteins showed good antigenicity.Polyclonal antibody had good reaction with natural whole trophozoite protein,indicating good immunogenicity of recombinant protein.