Expression of mastitis causing fibrinogen binding protein of gram positive bacteria in genetically engineered...

The current study is the first comprehensive report on the expression of fibrinogen binding protein (FIB) antigen in the genetically engineered switchgrass. Mammary tissue inflammation is one of the major infectious diseases caused by Staphylococcus aureus in the dairy animals. The aim of the present study is to develop an efficient and economical bioengineered immunogen for controlling mastitis in developing countries. Plant parts are served as bio-factories to produce antigens against infectious diseases. In this research, mastitis antigenic target (FIB) of S. aureus was expressed in switchgrass via Ag-nanoparticle mediated nuclear gene transformation to ease oral delivery of FIB antigen. FIB gene was cloned in expression vector through TOPO and Gateway cloning method. Transformation and integration of transgene was confirmed through PCR. The maximum concentration of total soluble fraction of FIB was calculated, and total soluble protein accumulated up to 0.5%. The recombinant FIB protein was purified and extract was prepared. FIB protein induced humoral immune response in mice and immunized orally. To administration oral immunogens against mastitis, FIB from S. aureus was commercially synthesized and PCR purified, the purified FIB gene was cloned into expression vector. Ag-NPs were encapsulated with pFIB and used as nanocarrier to target delivery of gene in forage grass. Forage seeds were successfully transformed through nuclear delivery and presence of transgene was confirmed through polymerase chain reaction. Transgenic lines of forage grass expressing FIB antigen is successfully developed. The transgenic lines expressing FIB gene were used for mouse study and in-vivo trials showed that switchgrass as transgenic immunogen developed antibodies in blood of animals upon orally delivering the FIB antigen. The expression of mastitis antigen in edible plants could contribute significantly to the development of cost effective and orally administered antigen-based subunit immunogen against dairy mastitis.