Establishment Of PCR Method For Detecting CP8 Gene Of Tririchomonas Foetus From Cats And Its Prokaryotic Expression

Trichomoniasis in cats is a protozoan disease that has been caused by the infection of Trichomonas foetus(T.foetus).T.foetus can directly invade the ileum,cecum and colon of cats,causing acute enteritis.The most typical clinical manifestation after infection is chronic or recurrent diarrhea.This disease is prevalent worldwide,and poses a serious threat to the health of cats.For the diagnosis of this disease,microscopic examination is a commonly used method.However,due to the lower sensitivity of microscopic examination and subsequent the need for professional researchers to distinguish and diagnose,it is very difficult to diagnose clinical Trichomonas.Cysteine proteinase 8 plays an important role,in the process of Trichomonas,which infects host.Studies have shown that CP8 protein is related to the adhesion and toxicity of Trichomonas,so a further study of CP8 protein is necessary.Based on the above problems,this study has done the following four aspects:1.Establishment of PCR detection method for Trichomonas felis based on T.foetus CP8 geneSpecific primers were designed and synthesized according to the CP8 gene sequence registered in GenBank,and a PCR detection method for diagnosing T.foetus was established.Its specificity,sensitivity and stability were tested.The results showed that the detection method established in this experiment has no cross reaction with coccidia felis,Giardia felis,feline coronavirus(FCoV)and feline parvovirus(FPV).The minimum detectable template concentration is 2.37×106 copies/μL.The target bands of T.DNA could still be detected of cryopreservation at-20℃ after 12 months.This method was used to detect the clinical specimens about 20 cats.A total of 16 cases were detected this disease.Compared with the conventional microscope(13 cases),the results were more accurate and sensitive.2.Establishment of a real-time fluorescent quantitative PCR method for detection of Trichomonas felis based on T.foetus CP8 geneTo establish a rapid and accurate real-time fluorescent quantitative PCR method for detecting Trichomonas felis,a pair of primers were designed according to the CP8 gene sequence obtained by sequencing,and the plasmid standard CP8 pUCm-T vector was used as the template.By optimizing the reaction conditions,a real-time fluorescent quantitative PCR method for the diagnosis of T.foetus SYBR Green I was established.The results showed that the method could specifically detect the genome of T.foetus,and detection result of the genomes of coccidia felis,Giardia felis,FCoV and FPV were all negative;The lowest detection limit of plasmid standard can reach 2.37×102 copies/μL.The intra-assay and inter-assay coefficients of variation were 0.20%～0.47%and 0.27%～1.40%,respectively.The positive rate of 20 clinical samples was 85.00%(17/20),while the positive rates of ordinary PCR and traditional microscopy were 80.00%(16/20)and 65.00%(13/20).The positive samples detected by microscope and ordinary PCR were positive by fluorescent quantitative PCR,which proved that this method was more accurate and sensitive than ordinary PCR and traditional microscopy.3.Cloning and bioinformatics analysis of T.foetus CP8 proteinIn order to understand the relevant information of CP8 protein,this experiment extracted total RNA from Trichomonas,obtained cDNA through reverse transcription,amplified CP8 gene from cDNA,and then predicted and analyzed CP8 protein by using bioinformatics online software.The results showed that CP8 protein was composed of 315 amino acids.It was a hydrophilic protein with no transmembrane domain and no signal peptide.It has 24 serine phosphorylation sites,15 threonine phosphorylation sites and 20 tyrosine phosphorylation sites.There were two domains.The secondary structure prediction of the protein showed that there were 12 α-Spiral,19 β-Fold,19 β-Corner,12 irregular curl segments.The tertiary structure prediction showed that the protein had no ligand,and the oligomeric state was homotrimer.The subcellular localization prediction showed that the protein existed in the lysosome of eukaryotic cells,and there were 11 antigen sites of B cells.4.Prokaryotic expression and purification of T.foetus CP8 proteinIn order to express T.foetus CP8 in vitro,PET30a-CP8 expression vector was established and transformed into BL21 competent cells for preliminary expression.SDS-PAGE protein gel electrophoresis showed that the protein was an inclusion body protein with a size of 37 kDa,which was consistent with the expected size.After a large amount of purified protein,Western blot showed that the protein reacted with His tag antibody,indicating that the target protein was successfully expressed.To sum up,this study established two molecular diagnostic methods for detecting T.foetus in cat fetuses by cloning CP8 gene,which provided a new method for early diagnosis and epidemiological research of T.foetus in grass-roots veterinary institutions.The expression and purification of CP8 protein provided a reliable basis for the subsequent preparation of monoclonal antibodies and the development of colloidal gold.