Variation Characteristics Of Terminal Repeats In Outer Membrane Proteins MSP1a And Hypervariable Region In MSP2 During Persistent Infection Of Anaplasma Ovis

Anaplasma ovis(A.ovis)infects sheep,goats and wild small ruminants,causing anaplasmosis.Major Surface Proteins(MSPs)are outer membrane proteins and important antigens.To analyze the antigenic variation of MSPs during the persistent infection,we performed:1.Establishment of sheep model of A.ovis persistent infection.Three healthy sheep(No.106~#?134~#?174~#)were artificially infected by intravenous injection of A.ovis infected sheep blood(Haibei strain).Within one year after inoculation,blood samples were collected regularly for smear examination,PCR detection(target msp4 gene,869bp target fragment)and indirect ELISA detection,all these detections proved that the animal model of A.ovis persistent infection was successfully constructed.2.Analysis on antigenic variation of MSP1a protein.Artemis software was used to analyse the whole genome of A.ovis Haibei strain which was sequenced by our laboratory previously.The msp1a gene was found harboring tandem repeats.Primers AoMsp1a F3/R3 were designed based on the A.ovis strain Haibei genome sequence.The whole blood DNA was used as template from 10 different time points(During 1 year of persistent infection,the blood samples for DNA templates were collected at the inflection points of antibody curve detected by indirect ELISA).10 msp1a tandem repeats sequences were amplified by PCR with longth of 883bp and then cloned into pGEM-T Easy vector,transformed into E.coli DH5?competent cells.The plasmids were extracted and sequenced after the culture of single colony.Amino acid sequences alignment was performed after translation from gene sequences.The homology of 10 samples was>99%.The total length of tandem repeats was 197 amino acids,and its structure can be divided into 4 segments,3 completely identical regions with 55 amino acids in each,and 1 short region with 32 amino acids in length,which is consistent with the protein sequence of the original infected strain.Conclusion:the sequence and structure of A.ovis MSP1a protein remain stable in experimental sheep during persistent infection.3.Analysis of antigenic variation of MSP2 protein.The whole genome data of A.ovis Haibei strain sequenced in our laboratory was analysed.We found that the msp2 gene family contained one full-length coding sequence and five homologous genes.All the homologous genes contained HVR sequence.To amplify the HVR sequence,DNA templates were extracted as above from blood samples at 10 time points.Nested PCR primers lzn111(F/R)and lzn22(F/R)were designed based on the A.ovis strain Haibei genome sequence to target hypervariable region within the msp2 expression gene.Product longth 457bp.The amplified fragments were cloned into pGEM-T Easy vector,then transformed into E.coli(JM109)competent cells.The plasmids were extracted and sequenced after the culture of single colony.For each sample,10-15 HVR sequences were amplified and sequenced,a total of 130 sequences were obtainded.Then analysis based on amino acid sequence were performed after translation showed that the HVR sequence contains 2 microregions,separated by 3 conserved regions.During 1 year persistent infection,the HVR produced antigenic variations through gene recombination between msp2 coding region and homologous pseudogenes,increasing the sequence diversity.Conclusion:the encoding sequences of the MSP2 HVR is a continuously changing sequence during the persistent infection of A.ovis.