The Construction Of CDNA Library Of Brain Microvascular Endothelial Cell Infected By LM90

Listeria monocytogenes is an important intracellular Gram-positive bacterium which isresponsible for food-borne disease characterized by meningitis, meningo-encephalitis,materno-fetal and perinatal infections. The disease is thus due to the original property of L.monocytogenes to be able to cross three host barriers: the intestinal barrier, the bloodebrainbarrier and the materno-fetal barrier. So far,it is unknown that the mechanism which LM crossthe host bloodebrain barrier,many people doubt there may be various ligands on the LMsuface which can interact with different receptors of host cell.The interaction can mediate theentry of LM and the interactin with the proteins of host cell. In order to screen the hostreceptors which interact with the suface proteins of LM. The cultured Human/Mouse BrainMicrovascular Endothelial Cells served as a modle of blood brain barrier in vitro, The cellsinfected by LM90were used to construct cDNA library of LM90-MBMEC，LM90-HBMECto lay a foundation of the following yeast two-hybrid experiment. The main research contentsand results are as follows：1.The identification of listerial species by PCR:Accoreding to the published conservedsequence of Iap and lmo0038, the specific primers was synthesised which is used todifferentiate listerial species of the saved listerial strains in our lab. The final results showedlisterial strains1,2,3were Listeria.seeligeri, listerial strains5and freeze-dried powder3,4wereListeria monocytogenes.2.Differences of adhesion,invasion,intracellure growth of Listeria monocytogenes toHuman/Mouse Brain Microvascular Endothelial Cells:LM90,LM3(freeze-driedpowder3),LM4(freeze-dried powder4),L.seeligeri（listerial strains2）,L.innocua were used toinfect Brain Microvascular Endothelial Cells, the adhesion rate, invasion rate, and number ofintracellular growth were determined by plate count method. The results showed that theadhesion rate of LM90,LM3,LM4ranged from3.14%to7.83%, but there was no significantstatistical differences.The invasion rate of LM90,LM3,LM4varied from0.036%to0.128%,which was higher than L.seeligeri（0.0315%,0.00148%）, L.innocua was unable to invadeBMEC. There was significant differences between HBMEC and MBMEC of LM90、LM3、LM4(P<0.05).The maximum quantity of bacteria and the peak time in BMEC were differentwith LM strains,the clinical pathogenic strains LM90was higher than other strains.Discrepancies in adhesion,invasion and the intracellular growth in Human/Mouse BrainMicrovascular Endothelial Cells of LM strains suggested that there might be differencevirulence among LM strains.3.The construction of cDNA library of brain microvascular endothelial cell infected byLM90: The total RNA extracted from the cells that inoculated with100:1multiplicity ofinfection of LM90for different time. cDNAs were synthesized and amplified by RT-PCR. ThePCR products and prey vector were ligated after purification and digestion with sfiI，and thenthe ligated cDNAs with prey vector were transformed into E.col to construct the cDNA library.The clones randomly selected from cDNA library were sequenced and analyzed. A cDNA library of LM90-MBMEC was constructed that contained1.2×105clones with97.92%recombination rate, the percent of insert size from1kb to2kb was22.9%,from0.5kb to1kbwas70.83%,above0.5kb was93.75%, Calciumion binding protein, receptor signaling proteinand so on were involved in analysization of genes; The constructed cDNA library ofLM90-HBMEC contained1.2×105clones with87.5%recombination rate, the percent ofinsert size from1kb to2kb was7.14%,from0.5kb to1kb was71.43%,above0.5kb was78.57%, mRNA binding protein,skeletal system development protein and so on were involvedin analysization of genes.