The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and...The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and pET15b-NGF were transformed into P. pastoris host cell GS115 (Mut^+ , His^- ) and E. coli strain BL21 (DE3) respectively. Expression and secretion of hNGFB in P.pastoris was attempted under the direction of the AOX1 promoter and PHO1 signal sequence. The positive colonies growing on medium without histidine were further selected by PCR. The yield of rehNGFB in GS115 was about 14.4% of total cellular secretive protein. The secreted protein was immunological active on Western blotting with rabbit anti-mNGFB antibodies. The fusion protein yield of rehNGFB in E. coli BL21 (DE3) was about 10.3% of total cellular protein after IPTG induction. Western blot detection showed its immunological activity.展开更多
文摘The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and pET15b-NGF were transformed into P. pastoris host cell GS115 (Mut^+ , His^- ) and E. coli strain BL21 (DE3) respectively. Expression and secretion of hNGFB in P.pastoris was attempted under the direction of the AOX1 promoter and PHO1 signal sequence. The positive colonies growing on medium without histidine were further selected by PCR. The yield of rehNGFB in GS115 was about 14.4% of total cellular secretive protein. The secreted protein was immunological active on Western blotting with rabbit anti-mNGFB antibodies. The fusion protein yield of rehNGFB in E. coli BL21 (DE3) was about 10.3% of total cellular protein after IPTG induction. Western blot detection showed its immunological activity.
