| BackgroudSince the ancient time, Mycobacterium tuberculosis(MTB) as a microorganism which has a strong ability of infectious,always to be a threat to humans and many other husbandry animals’ healthy. According to the latest statistics, there are about 9.6 million people / year infect the activity Mycobacterium tuberculosis, nearly 150 million / year people died of tuberculosis and its related complications. The rapid and accurate detection and right treatment of Mycobacterium tuberculosis in the begaining of the infection can greatly reduce the spread of tuberculosis in the crowd. As the new technology of specificity antigen in cellular immune response which has a rapid development in recent years, ELISPOT has been shown to can be used for diagnosis active and latent infection. But diagnostic reagents based on this principle rarely improve. The main reason that the main influence conditions of the test method is still not clear.Whole genome expression profiling chip technology that gradually developed mature in recent years can detect of a large number of genes expression at the same time.It has the characteristics of high throughput and high sensitivity. The main research that focus on ELISPOT is make sure the correction of the clinical diagnosis in different kind of patient.Only a little experiment to find how to improve the correction of ELISPOT. ObjectiveTo verifying the biological activity of specific recombinant protein ESAT-6 and CFP-10 by the clinicl samples. To establish the ELISPOT method with high sensitivity and specificity. To analysis the results mechanism of ESAT-6 and CFP-10 protein’ specific effection on specific T cells. through the of biological information result based on the gene chip. MethodTo construct the prokaryotic expression recombinant plasmid by CFP-10 and ESAT-6 gene fragmen through the polymerase chain reaction(PCR) amplification. After transforming the recombinant plasmid into the E.coil. Induced with IPTG.,then purifying the recombinant protein ESAT6 and CFP10 by affinity chromatography, analysis of the purity of the protein.Make the ELISPOT method can be applied to clinical by determining the suitable experimental conditionsBy magnetic activated cell sorting(MACS) pointed out the positive CD4 lymphocytes.By the suitable expression profiles gene chip, to find differentially significantly expressed gene loci by before and after stimulation protein.According to the go and KEGG analysis to predicte the corresponding pathway. To stablish the corresponding structure layout of the future experiments.Result 1.Our laboratory has been built a stable specific protein expression system, and a series of conditions for ELISOPT to detection of Mycobacterium tuberculosis infection has been established according to the experiental conditions. 2.Compared with T-SPOT kit, ELISPOT specificity and sensitivity were 82.35% and 88.50%(x 2 = 0.57, P > 0.05), the difference was not statistically significant.3.Through the chip detection before and after the stimulation of tuberculosis patient T cells’ different sites,CFP-10 stimulated expression increased relative genes in BP’s go entries is 1138, participate in the CC go entries of a total of 31, in MF go entries, a total of 59. Decreased expression of relative genes in BP entry 1273, participate in CC entries for a total of 154, MF entries to participate in a total of 181.ESAT-6 stimulated expression increased relative genes involved in BP’s go entries 1295, participate in the CC go entries for a total of 112, the participation of MF go item total of 71. Decreased expression of relative Genes involved in BP entry should be a total of 1089, a total of 112 entries in CC, a total of 139 entries in MF.4.Analysis the KEGG and differential gene expression with the rise and fall of the pathway,signaling pathway related with CFP-10 group,tuberculosis and IFN-γis following cytokine receptor interaction, toll like receptor, JAK stat, cell adhesion; signaling pathway related with ESAT-6 group, tuberculosis and IFN- γ is the JAK-STAT, toll like receptor, NF kappa B, PI3 K Akt.Conclusion: The research results show that the established in our laboratory assay has a good sensitivity and specificity,it has the basic screening of clinical specimens and reduce the detection cost. Through the analysis of gene chip results, screening out the higher specific loci and existing a certain correlation with IFN- γsecretion and tuberculosis infection, found a clear direction for the follow-up mechanism of the research and so on. |
