Establishment Of QPCR Diagnostic Method And Prokaryotic Expression Of Theileria Annulata Cargo Protein Gene

Tropical theileriosis is a tick-bone protozoan disease caused by Theileira annulata.It mainly infects domestic animals,which can lead to anemia,fever and loss of appetite.It is distributed in 13 provinces of northern China,which has brought huge economic losses to the cattle industry.At present,diagnostic methods such as PCR and indirect ELISA have been established for the diagnos is of T.annulata.However,those methods still have problems that antigens were not good enough for reactive and immunogenic and longtime needed for diagnostic.Therefore,it is necessary to explore new antigens for the development of diagnostic methods.In this study,bioinformatics analysis was used to analyze and predict the nucleotide sequence and amino acid sequence of Theileria annulata cargo protein.The fluorescent quantitative PCR diagnostic method was established,and the recombinant protein was expressed in prokaryotic expression system and purified to prepare polyclonal antibodiess.The reactivity of the recombinant protein and antibodiedwere analyzed with westernblotting.The present study provides a rapid and effective diagnostic method for the detection of tropical theileriosis,and also provides a new idea for the future research to establish a new serological diagnostic method.1、Cloning and bioinformatics analysis of Theileria annulata cargo proteinThe gene sequence was amplified by PCR.And the tmp21-pGEM-T Easy cloning vector was constructed and sequenced.The sequencing results were analyzed and predicted through bioinformatics.The obtained gen contained 213 amino acids and was a stable hydrophobic nonsecretory transmembrane protein.It contains eight modification sites and one transmembrane region.The highest transcription level is at the stage of piroplasma.It contains EMP24_GP25L domain.2、Establishment and application of the real-time PCR method based on Theileria annulata cargo proteinAim to establish a real-time PCR method for detection,a pair of specific primers was designed according to the gene sequence derived from the database of GenBank.The results showed that the established method was specific,and has no cross reactions with Theileria sinensis,Theileria orentails,Babesia bovis and Babesia bigemina.The minimum detection limit of this method is 10.4 copies.The variable coefficient between inter-and intra-groups of this method were less than 5%,which indicated the method has a good stability.Total 766 field samples collected from 10 provinces were used to test the applicable of this method,the results shown that the positive rate of established real-time PCR was higher than that of conventional PCR.In conclusion,the real-time PCR method established in this study has goodl specificity and stability.According to the results of field sample detection,the fluorescence quantitative PCR has better detection effect than ordinary PCR.3、Expression of Theileria annulata cargo protein and preparation its polyclonal antibody and reactivity analysisThe recombinant plasmid vector pET-32a-tmp21 was constructed and expression.The optimal induction expression conditions were optimized by exploring the concentration of inducer and induction time.The purified protein was mixed with Freund’s adjuvant to immunize mouse by subcutaneous injection.The reactivity of purified protein and mouse serum was analyzed by SDS-PAGE and Western-blot.The results showed that the recombinant protein was successfully expressed,and the protein size was 38kDa,which existed in the form of inclusion bodies,and had good reactivity and immunogenicity.