Establishment Of T Cell Response To Mycobacterium Tuberculosis Elispot IL-12 Detection Technology

Objective:To establish a detection technique, which was used for evaluate the level of cellular immunity after infection with Mycobacterium tuberculosis(MTB), thus provide a new method for the diagnosis, treatment and therapeutic effect of tuberculosis.Methods:1.The primers of the PPE36,TB10.4 gene fragment of Mycobacterium tuberculosis were designed and synthesized. Using the DNA of MTB standard strain(H37Rv)as a template, the polymerase chain reaction was performed.The amplified product were connected with the cloning vector, transformed into E.coli strain DH5 a and cultured, and the plasmids were extracted for identifying and saving.2. The plasmid were connected with the expression vector and the ligation products were transformed to E.coli BL21(DE3) for expression under induction of IPTG, and the expressed product were purified and identified.3. Using mouse anti human IL-12 antibody coated PVDF film, the ELISPOT IL-12 detection technique was established by biotin streptavidin method.4. The TB10.4, PPE36 proteins were used for stimulate the peripheral blood mononuclear cells of normal people and patients infected with Mycobacterium tuberculosis, for verifying the feasibility of clinical application of Elispot IL-12 detection technology.Results:1. The sequences of PPE36,TB10.4 gene obtained from experiment were in accord with the sequences on GenBank, and the corresponding proteins were successfully expressed.2. The results of SDS-PAGE electrophoresis showed that the PPE36,TB10.4 proteins there were a clear band each in molecular weight 30 kD and 10 kD. It consistent with the expected size.3. The results of Elispot showed that there were no spots in the blank control group and the normal group, while the positive control group and infection with mycobacterium tuberculosis patients group was significantly.Conclusion:1. The proteins PPE36 and TB10.4 of Mycobacterium tuberculosis were acquired by genetic engineering methods.2. After identification, the molecular weight of target proteins were consistent with the literature.3. The Elispot IL-12 detection method of reactive T cells of Mycobacterium tuberculosis has been preliminarily established.