Prokaryotic Expression, Polyclonal Antibody Preparation And Idetification Of Goat △FosB Gene

ΔFosB, a naturally occurring truncated isform of FosB gene, existed in many tissues stability.ΔFosB may be related to the metabolism of calcium in bone and mammary gland and regulate the signal pathway of calcium transferred from bone to mammary gland. In this study, we expressed and purified target fusion protein in prokaryotic cells, then prepared high titer ofΔFosB polyclonal antibody that can be used for detecting the expression ofΔFosB protein. This polyclonal antibody was a basis for researching the molecular mechanism ofΔFosB regulated calcium transferred from bone to mammary gland. The main results of this research are as follows:(1)We constructed and identified the prokaryotic expression vector pET32a-ΔFosB using of the T vector has been cloned into the goatΔFosB Gene, then transformed pET32a-ΔFosB vector to E. coli lysogenic bacteria BL21 (DE3) pLysS for expression.(2) According to optimization of the expression conditions of prokaryotic vector pET32a-ΔFosB expressed in bacteria BL21 (DE3) pLysS, we obtained the optimal conditions: 37℃, 0.5mmol/L IPTG inducted to express 6h; the target protein will achieve the highest expression efficiency as the form of fusion protein expression in bacteria.(3) We purified from inclusion body lysates to obtain fusion protein His-ΔFosB using Ni-Agrose affinity purification and gel purification methods respectively. The purity of fusion protein His-ΔFosB purified from affinity column can reach 90%, but the recovery yield is less than 5%. Gel Extraction method can get relatively simple target band, and the recovery ratio is much higher than that of affinity column purification.(4) We injected two rabbits with the gel purified fusion protein His-ΔFosB, and obtained polyclonal antibodies with the titer of 1:51200 which can react specifically to the fusion protein His-ΔFosB. For a further research, we used of the prepared antibodies to perform a western blotting to detectΔFosB protein expressed in goats in vivo. The results showed thatΔFosB protein expressed in goat mammary gland, bone marrow, brain, muscle, heart, liver and lung tissue, but the pituitary and spleen did not express.