Cloning And Prokaryotic Expression Of MSP5 Gene Of Anaplasma Marginale In E.coli And Development Of An ELISA Method

Bovine anaplasmosis, caused by Anaplasma marginale parasitic in bovine erythrocytes, is anarthropod-borne rickettsial disease. It is widely distributed in most of provinces in China, and hasresulted in a great deal of losses in cattle production.In the present study, a pair of primers was designed based on the MSP5 gene sequences of A.marginale available in GenBank. The MSP5 gene was amplified by PCR from genomic DNA purifiedfrom blood of a yak infected with A. marginale, and cloned into pGEM-T Easy vector to construct therecombinant plasmid "pGEM-T Easy-MSP5". The result showed that the identities of the cloned MSP5gene with the data published in GenBank in nucleotide and amino acid sequence were 98.6％and 99％,respectively. After then, Primers were designed according to the ORF sequence of the MSP5 gene, andthe pGEM-T-easy-MSP5 recombinant plasmid used as template in the PCR reaction, the amplifiedproducts were ligated to the pGEX-4T-1 vector, and transformed into DH5αhost cells. Expression of therecombinant plasmid was induced with IPTG, and resulted in a high level of protein expression. Theexpressed products were dissoluble, but most of them is inclusion body. SDS-PAGE experimentsrevealed that its molecular weight is approximately 45Ku. Western-blot analysis showed that theexpressed recombinant protein could be recognized by rabbit anti-A, marginale positive serum. TheMSP5 expressed level could reach up to 35.0％of total E. coli proteins.The recombination fusion protein was purified by lysis, washing, denaturation and renaturation. AnELISA method was developed based on this purified recombinant protein, and its coincidence of positiveand negative was determined to be 100％(100/100) and 98％(98/100), respectively. The antibodydynamics of the experimentally infected yaks was observed that the antibody could be detected on day 5post-infection (1:50), and the peak of the antibody (1:3200) appeared on day 90 post-infection. It isproved that 5 months after infection the antibody titration was still 1:1600. The method was applied toinvestigate the sera collected from cattle and yaks in six provinces in China, which are Xinjiang, InnerMongolia, Helongjiang, Jilin, Sichuan and Gansu province, and positive rates were 67.63％(700/1035),81.78％(184/225), 82.78％(149/180), 46.11％(139/180), 66.67％(83/180) and 77.22％(90/135),respectively. This result was in accordance with the previous data reported by the blood smear methods.It is concluded that the established method is suitable for sero-epidemiological survey of anaplasmosiscauses by A. marginale.