Study On The Antigenicity Of Recombinant Protein OmpB Of R.japonica

[Backgrounds]SFGR is widely distributed in the world and causes tick borne zoonotic diseases in humans and animals.The clinical symptoms of SFGR includes common cold,fever,pain,nausea and vomiting,and often accompanied by a rash.Rickettsia japonica was discovered in 1984.Between May and July 1984,the Japanese doctors identified three patients with high fever and rash.All lived in the same rural area and had collected shoots from bamboo plantations on the same mountain.For two patients,an eschar was observed.Scrub typhus,caused by the mite-borne pathogen Orientia tsutsugamushi,was initially suspected because of the clinical similarity to the illnesses and because it is a well-known zoonotic disease in Japan.However,through a series of experiments and serology the disease was not scrub typhus but a spotted fever group rickettsiae.The causative agent was first isolated from patients in Shikoku in 1985 and was subsequently characterized as a new rickettsia of the spotted fever group and named Rickettsia japonica.Before 2000 years,China's research on Spotted fever group rickettsioses were mainly in the northeast,R.heilongjiang,R.sibirica and R.mongolia were detected in rodents from this area and at the same time,from the blood of patients with isolated R.heilongjiang strain.After 2000 years later,few studies on relevant Spotted fever group rickettsioses were reported in China.2013 years,our research group isolated Rickettsia japonica from a patient in the Dabie Mountains of Anhui Province called R.Japonica Anhui 120 strain.The targeted cells for Rickettsia in vitro cultivation is Vero cells and L929 cells,but studies from my research group showed that Rickettsia japonica not can grow inboth Vero and L929 cells.They were able to grow in THP-1(the macrophage like cells).Sequence analysis of spotted fever group rickettsiae genes such as omp A,omp B,gene D,16 Sr RNA and 17 k D showed deletion of 15 nucleotides bases at the position 3297~3311.The omp B gene is an outer membrane protein of Rickettsia japonica,which aids bacteria in invasion of host cells,so we we consider the lack of isolated cell tropism and omp B gene in 15 consecutive bases.Therefore,this paper constructs omp B prokaryotic expression plasmid,recombinant protein expression,purification and antigenicity.The research,conducted some research basis for the study of pathogenic mechanism of Rickettsia.[Methods](1)Rickettsia japonica whole cell preparation of polyclonal antibody: preserved by our laboratory strains to infect normal THP-1 cells,the cell under the microscope with diffquick staining was observed in the cells of the infection rate is about 100%,per week,a total of four times to 4 New Zealand white rabbits immune after the end of the acquisition.The immune rabbit heart blood,aseptic packaging,frozen in the refrigerator of-80?.(2)r Omp B recombinant protein expression and purification of omp B gene: a special connection to PET-30 a vector by T4 ligase,the induced expression of omp B gene by IPTG,using SDS-page and Western-blot(WB)were identified for reactogenicity of r Omp B recombinant protein,then through affinity chromatography and r Omp B the supernatant form inclusion body and purification of refolded technology.(3)Establishment of indirect ELISA system: using the chessboard titration method,in order to determine the optimal antigen concentration package,the best serum dilution concentration,the optimum enzyme concentration.And compared with the existing international standard test methods(American Focus company IF0100MG)to determine the specificity,sensitivity,coincidence rate and Kappa value.[Results](1)New Zealand rabbits were immunized with heart blood after aseptic preparation of Japanese dermacentroxenus polyclonal antibody.IF measured results strongly positive;ELISA method measured the titer of polyclonal antibody was 1:8000.(2)The enzyme digestion and sequencing,the recombinant plasmid containing omp B gene fragment,purified by Lowey was measured with the vector PET-30a(+)to construct the recombinant protein concentration was 4mg/ml.(3)The successful establishment of indirect ELISA method for identification of Rickettsia infection system,and this method has good specificity and sensitivity,and with the international standard test method(American Focus company IF0100MG),the coincidence rate was 99.2%.[Conclusion](1)r Omp B has good Immunorectivity;(2)R.Japonica Anhui 120 strain Omp B of indirect ELISA can be used as the detection of Rickettsia antibody Ig G levels and have has good specificity and sensitivity.