Molecular Mechanism Of Brucella Menlitensis Infecting Embryonic Trophoblast Cells

Brucellosis is a zoonotic disease caused by members of the genus Brucella. It is widely popular in the world, especially in developing countries. Recently, the rapid increasing cases in human and animals have caused widespread concern around the world. China released an emergent document "on the strengthening of prevention and treatment of Brucellosis notice". Brucella is a facultative intracellular parasitic bacterium. Due to its strong invasion and multiple approaches of transmission, patients show persistent infection, which is usually known as incurable disease. Infectious animal mainly caused abortion, which leads to serious loss for livestock industry. Animal placenta contains much Erythritol, which can promote the growth of Brucella. Embryonic trophoblast, knows as target cell of brucella, is a link between the pregnant animal and the fetus. Once infected, it will lead to abortion. However, its molecular mechanism of abortion is unclear. In this study, based on Brucella and embryonic trophoblast cells, the main research work carried out as the follows:1. Isolation and Identification of Brucella melitensis and ELISA method were constructed. According to bacterial morphology, PCR, biochemical examination, Brucella melitensis type 3, named as 027 strain, was obtained. After its SP41 gene was cloned, expressed, purified, indirect ELISA method was built. During our experiments, the best concentration of SP41 recombinant protein is 0.6μg/mL, the best dilution of serum is 1:50. The method has good reproducibility, specificity, positive rate of samples was higher than the rose Bengal plate test (RBPT) and standard tube agglutination test(SAT).2. Study on ery operon expression and regulation of Brucella melitensis during human embryonic trophoblast HPT-8 infected with pathogen. The real-time quantitative PCR method was used to detect expression differences of ery operon 4 genes in non-infected, infected 20min, 1h,2h,3h, and 4h. ery A, eryB and eryC in 2h has a significant over-expression. eryD in 3h has the highest expression compared to ery A, eryB and eryC. Interestingly, with the increase of eryD expression, ery A, eiyB and eryC decreased.3. The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain was constructed. The total RNA of infected cells at 20min, 1h,2h,3h and 4h Were extracted respectively. With cDNA synthesized by reverse transcriptase, homologous recombination construct infected HPT-8 cell cDNA library. The partial ESTs were sequenced. The results showed that cDNA library was successfully constructed. cDNA capacity was 1.43×106, the recombinant rate was 96.92 percent. The size of the inserted fragments is between 0.2-5kb.63 clones of cDNA library were random selected and sequenced. These gene were classified by BlastX and BlastN. The bait plasmid pGBKT7-omp25 was constructed into yeast Y187, which results show that the bait yeast is non-self-activated and non-toxic.4. The target protein were screened and verified. OMP25 interacted with 7 positive protein of HPT-8 cells by yeast two-hybrid. Ferritin and hypothetical LOC339123 were verified by immunoprecipitation.5. Study on the function of prey protein. According to nucleotide sequences of ferritin and hypothetical LOC339123, three specific interfering shRNA vectors transfecting HPT-8 cells, respectively, real-time quantitative PCR detected. Ferritin mixed group interference suppression rate of 76.18%, hypothetical LOC339123 mixed group interference suppression rate of 71.11%. After Ferritin was interfered, the relative number of Brucella in HPT-8 cells increased compared to hypothetical LOC339123 protein.The above results show that the isolate was Brucella melitensis type 3; indirect ELISA based on SP41 was successfully built; Brucella ery operon 4 genes expression differed at different times during infecting HPT-8 cells; The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain was successfully constructed; Brucella OMP25 interacted with HPT-8 Cell's ferritin and hypothetical LOC339123 protein; ferritin is related to Brucella infection while hypothetical LOC339123 protein is not. In this study, It is useful to demonstrate molecular mechanism of abortion, to screen specific candidate drugs to brucellosis, to give a upstream work for breeding of anti-brucellosis livestock.