Aim: To prokaryotically express and purify fusion protein containing extracellular region of human BTLA and prepare the antiserum of it.
Methods: Human BTLA (hBTLA) gene was amplified by PCR, digested with enzymes, ligated and subcloned into a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET28a-hBTLA. Then pET28a-hBTLA was transformed into E.coli BL21 (DE3). The hBTLA fusion protein was obtained upon IPTG induction, purified by Ni-NTA Purification System, and analyzed by SDS-PAGE. Rabbit anti-hBTLA antiserum was prepared and identified.
Results: The pET28a-hBTLA plasmid was confirmed to carry the correct hBTLA gene by sequencing. It expressed a 15.7kD protein that could be purified by Ni-NTA purification system. The titer of the multiclonal antibody was 1:16 detected by double diffusion test, and 1:20 000 by ELISA, respectively. The anti-hBTLA antibodies can bind to hBTLA specifically shown by Western blot.
Conclusion: The prokaryotic expression vector has been constructed successfully, leading to highly purified hBTLA protein. The antiserum of hBTLA has been prepared, with high titer and specificity.