Objective To understand the immunological characteristics of Rv0081, a dormancy-associated antigen of Mycobacterium tuberculosis.

Methods The Rv0081 gene of M. tuberculosis was amplified by polymerase chain reaction (PCR), and the prokaryotic expression recombinant plasmid pET28a-Rv0081 was constructed. The target protein expression was induced and confirmed by Western blot. The soluble protein was purified by affinity chromatography. Mice were immunized subcutaneously and intramuscularly with Rv0081 without adjuvant, and the levels of serum antibody and cytokines secreted by splenocytes were detected by enzyme-linked immunosorbent assay (ELISA). Splenocyte proliferation was detected by cell proliferation aassay kit. Recombinant protein was used as coating antigen to detect the serum antibody levels to Rv0081 of guinea pigs and mice infected with M. tuberculosis, as well as tuberculosis (TB) patients, and receiver operating characteristic (ROC) curves were drawn.

Results The target Rv0081 gene was amplified by PCR, and a prokaryotic expression vector for Rv0081 was successfully constructed. This vector expressed the target protein with an approximate molecular weight of 15 kDa, and purified native recombinant Rv0081 protein was obtained. The immunization of mice with purified recombinant Rv0081 protein via subcutaneous or intramuscular routes induced specific antibody, with significantly higher antibody level in the intramuscular immunization group. Intramuscular immunization with Rv0081 promoted splenic lymphocyte proliferation and induction of increased secretion of helper T (Th) cell cytokines, especially the increased production of Th1-type cytokines. Subcutaneous immunization with Rv0081 mainly induced increased secretion of Th1-type cytokines.The level of serum antibody to Rv0081 increased in the guinea pigs and mice infected with M. tuberculosis for at least 24 weeks , especially in the persistent infection phase. The level of serum antibody to Rv0081- of TB patients also increased significantly.

Conclusion This study obtained a soluble recombinant Rv0081 protein withstrong immunogenicity, which can induce humoral and cellular immune responses in mice and can be used to detect the specific antibody in individual with persistent M. tuberculosis infection. The findings provide a foundation and reference for the development ofRv0081-based subunit vaccines against TB and for the design of diagnostic reagents in combination with other antigens.