ABSTRACT
Introducing an effective vaccine for tuberculosis (TB) is an urgent need. Mycobacterium tuberculosis (Mtb) Ag85 complex is suggested for making protective immunodominant antigens for design and development of novel TB vaccine. In the present study, a pDR2EF1-Fcγ1 vector has been used to make Ag85B:hFcγ1 recombinant fusion protein. Briefly, specific XbaI and NotI site incorporated primers were used to amplify Mtb-fbpB gene by PCR, TA-cloned and amplified in E.coli DH5α. The resulting vector then subcloned into the pDR2EF1.Fcγ1 vector and transferred to Chinese hamster ovary (CHO) cell line. DNA sequencing was performed to confirm that Ag85B:hFcγ1 construct is precise and in-frame. Then, Ag85B:hFcγ1 protein was produced by CHO expression system and recombinant protein was purified using HiTrap rProtein A Sepharose Fast Flow column. The presence of recombinant fusion protein confirmed by immunofluorescence (IFA) and Western blotting (WB). This fusion protein containing Fc fragment of human IgG1, apart from stability and adjuvanticity potential, could bind to FcRγI (CD64) on the surface of antigen-presenting cells (APCs) and induce cross-presentation in favour of host immune response and can be used as a potential candidate along with other subunit vaccines against Mtb.
Subject(s)
Acyltransferases/metabolism , Antigen Presentation , Antigen-Presenting Cells/metabolism , Antigens, Bacterial/metabolism , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Recombinant Fusion Proteins/metabolism , Acyltransferases/genetics , Animals , Antigens, Bacterial/genetics , CHO Cells , Cricetulus , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Protein Binding , Receptors, Fc/metabolism , Recombinant Fusion Proteins/geneticsABSTRACT
Mycobacterium tuberculosis (Mtb) is one of the most life-threatening mycobacterial species which is increasing the death rate due to emerging multi-drug resistant (MDR) strains. Concerned health authorities worldwide are interested in developing an effective vaccine to prevent the spread of Mtb. After years of research, including successful identification of many Mtb immunogenic molecules, effective therapeutic agents or a vaccine have yet to be found. However, among the identified Mtb immunogenes, antigen 85 (Ag85) complex (Ag85A, Ag85B, and Ag85C) is receiving attention from scientists as it allows bacteria to evade the host immune response by preventing formation of phagolysosomes for eradication of infection. Due to their importance, A85 molecules are being utilized as tools in diagnostic methods and in the construction of new vaccines, such as recombinant attenuated vaccines, DNA vaccines, and subunit vaccines. This paper represents a comprehensive review of studies on Mtb molecules examining pathogenicity, biochemistry, immunology, and the role of Mtb in therapeutic or vaccine research.
