The Antigen Proteins Of MTB ESAT6 And RdESAT6 Cloned And Expressed In Pichia Pastoris To Evaluate The Potential Value In Early TB Diagnosis

6kD early secreted antigenic target, ESAT6 is a secreted protein presented in early culture filtrate of Mycobacterium tuberculosis with low molecular weight, which can induce intense cellular immunoreaction. It is only secreted by pathogenicity mycobacterium like Mycobacterium tuberculosis instead of nonpathogenicity ones, such as BCG, so it can be used in early diagnosis of tuberculosis as specific antigen. rdESAT6 is the recombinant dimer of ESAT6, with more reaction epitopes and stronger antigenicity than ESAT6. Because rdESAT6 was expressed in E.coli and M.smegmatis in the form of inclusion body, Pichia pastaris expression system was constructed to secrete rdESAT6 in order to get soluble antigen which is similar to the natural structure.Objective:Pichia pastoris expression system was constructed to express ESAT6 and rdESAT6 antigen secreted by Mycobacterium tuberculosis, in order to gain protein antigen in natural instructure and to evaluate the potential value as antigen stimulus in early TB diagnosis.Methods:The genes of esat6 and rdesat6 from Mycobacterium tuberculosis were amplified by polymerase chain reaction individually and then inserted into the vector pPic9k. After pPic9k-esat6 and pPic9k-rdesat6 transported into Pichia pastoris by electroporation, protein ESAT6 and rdESAT6 were expressed. Then Ni-NTA column was used to purify the object protein. Spleen lymphocytes of the mice immuned by rBCG were employed in ELISPOT (enzyme-linked immunospot assay) with the same antigen stimulus rdESAT6 expressed in E.coli, Mycobacteria smegmatis and Pichia pastoris to analysis the different ability on stimulating cells to product IFN-γ.Results:The antigen proteins ESAT6 and rdESAT6 were expressed successfully in Pichia pastaris. Expression level of ESAT6 was too low to purify. High-purity rdESAT6 was obtained through Ni-NTA. rdESAT6 was glycosylated in the process of secretion, leading to a higher molecular weight than normal. Digested by Endo H, the molecular weight of rdESAT6 protein was decreased to 24kD, which have specific immunoreaction with relevant antigen in Western Blot as well the specific immunospot in ELISPOT.Conclusion:rdESAT6 protein antigen can be expressed by Pichia pastoris, with a high express level at 5mg/L. During this process, the protein was glycosylated, which did not influence its antigenic specificity. On the other hand, memory-T-cell was stimulated to induce IFN-γ. A tentative study was presented to prove a new specific antigen on TB early diagnosis and to shed new insights for future research.