Cloning, Expression And Immunogenicity Of Orientia Tsutsugamushi 56-kDa Membrane Protein

Scrub typhus is a natural foes disease caused by Orientia tsutsugamushi(Ot), prevalent in the tropical and subtropical areas of Asia-Pacific regions, widespread severly in our contry. Mites are the transmission vector and murine is main infection source of the disease. Human being is infected after the introduction of O. tsutsugamushi through the skin by the bite of a larval-stage (chigger) trombiculid mite. The major clinical features include febrile, eschar or anabrosis, lymphadenectasis and rash.In China, misdiagnosis of scrub typhus is common because of lacking in diagnostic reagents, clinician not understanding the importance of scrub typhus and non typical clinical symptoms. Diagnosis of scrub typhus depended on serological assayes such as indirect immunofluoresence assay (IFA), which need special equipment like fluorescence microscope and the patients'convalescent serum. Isolation and cultivation of O. tsutsugamushi are reliable for diagnosis but difficult for general laboratory and time consuming. The molecular biological technology based on PCR targeted for the special genes of O. tsutsugamushi also require equipments. Therefore, a simple, rapid, sensitive, special,economic and sutable for application in rural areas diagnostic methods is urgently needed. A great quantity of researches domestic and abroad indicated O. tsutsugamushi 56-kD membrane protein was a type-specific antigen which accounts for 10-15% of the overall bacterium protein. The protein is proved to have effective Immunogenicity and nearly 95% person could produce antibody against it after infecting with the bacteria. It is confirmed that this protein also play a significant role during invasion of its host cells.The purpose of the study is to clone and expression of 56-kD membrane protein. The dominant epidemiological strain in China - O. tsutsugamushi strain Karp was selected for bioinformatics analysis including secondary structure, hydrophilicity and B-cell epitope and so on. The polypeptide fragments (from 100 to 468 amino acids),which contain major antigenic determinants except signal peptide was chosed for expression as goal protein.The construction of pET30a(+) expression vector were conducted by PCR amplying the 1104bp fragments of 56-kD membrane protein gene and then ligated to the pET30a(+) expression vector.The combinant vector was transformed into the Rosetta compatant cells. An expected molecular size of protein was observed when pET30a(+) expression vector was induced by IPTG. The mass-spectrum analysis demonstred that he amino acid sequences of the expressed polypeptide was 100% homologous with the selected fragments of the original 56-kD membrane protein.The ELISA test and western blot as recombinant protein as antigen while anti-Karp rabbit serum as positive reference antibody(IFA:1:512) showed the sensitivity of the recombinant protein(ELISA:the titre of antibody 1:1600 and western blot:the titre of antibody 1:1600) were higher than the traditional assays-IFA.Cross reaction between 16 rabbit serum immunined with common member of order Rickettsiae and the recombinant protein was conducted by ELISA using the recombinant protein as antigen and the results demonstrated that all serum except anti serum against Ot strain TA763,TA817 and B.quintana,A.phagocytophilum were nagitive. Here we concluded that the recombinant protein in the study exhibited excellent antigenicity and specificity and could be a candidate of developing a simple and rapid diagnostic reagent and vaccine of O. tsutsugamushi.