S. D. Audarya, A. Sanyal, L.K. Pandey, J. K. Mohapatra and B. Pattnaik
TNF-α is one of the critical cytokine involved in important processes of immune defense system. In the present investigation total RNA from peripheral blood mononuclear cells (PBMCs) from healthy Holstein-Friesian cattle (Bos taurus x Bos indicus) was extracted. Using extracted total RNA complementary DNA (cDNA) was prepared through reverse transcription technique. This cDNA was used in polymerase chain reaction (PCR) for partial amplification of TNF-a cytokine gene with the help of commercially available readymade primers. In the gel electrophoresis, desired TNF-a DNA product of 360 bp was documented. After purifying the resulting PCR product it was inserted into the cloning vector, pGEMÂ®-T Easy Vector System I by T-A cloning technique. Successful transformation of desired amplified DNA product of TNF-a was accomplished in Escherichia coli (JM 109) bacterial cells. Further confirmation of actual success in transformation was achieved on amplifying specific product of TNFa in PCR when extracted plasmids from transformed Escherichia coli (JM109) cells were used as a template. After treating the plasmids obtained from transformed Escherichia coli (JM109) cells with restriction enzyme, release of the specific inserts in the plasmid was also documented. Finally to reconfirm the results extracted plasmids were sequenced successfully and found to be of TNF-a. The transformed Escherichia coli (JM 109) cells containing TNFa insert were further preserved. This clone of TNF-a cytokine gene can be used as a positive control to compare and for quantitative analysis of the cytokine TNF-a for healthy, diseased and vaccinated bovines in real time polymerase chain reaction.
Keyword: Cattle; Cloning; mRNA, RT-PCR; Sequencing; TNF--α;