Secreted Expression Of Human Basic Fibroblast Growth Factor (hu-bFGF) In Pichia Pastoris

Basic fibroblast growth factor (bFGF) is one important member of fibroblast growth factors, which is named as FGF-2. It can promote many cells growth and it's biological effection is very broad. At present, bFGF has not only set out new therapy way in many diseases, for example,nerve system diseases, coronary heart diseases, damnification and so on, but also show its active application foreground in hairdressing. It was also praised of"21st century cosmetics".The gene encoding hu-bFGF protein was amplified by PCR from the plasmid of pMD18-T/hu-bFGF. Then, the amplified fragment was cloned into pMD19-T Easy vector for sequence verification. In order to generate a secreting expression vector of pGAPZα-A/hu-bFGF, bFGF was insert into the pGAPZα-A vector with XhoI and XbaI . The location of bFGF was in the downstream of theα-factor signal sequence of pGAPZα-A vector. After linearized by BlnI restriction enzyme, the recombinant vector was transformed into Pichia pastoris GS115 competent cell by electroporation. Recombinant strains with high expression of recombinant hu-bFGF protein were identified by Zeocin resistance, PCR identification and SDS-PAGE, and a high expression strains were obtained and named as Yb5. By optimizing the cultural condition for recombinant strain of Yb5, the optimal ferment conditions were established as following: ferment culture medium composed of 3% yeast extract, 2% peptone, 5% maltose; the best cultured time was 96hrs,the original pH was 6.5; the inoculability rang was 1:500~1:50; the ratio of culture medium and volume container was not high of 10%. The yield of recombinant protein was 2.10 mg/mL show by data. After SDS-PAGE, the recombinant protein was further identified by Western blot. And the analysis indicated that the recombinant hu-bFGF protein had the antigenicity to rabbit anti-hu-bFGF polyclonal antibody.The results not only provide the basis of optimization in fermenter,but also lay the foundation of industrialization production and clinical practice.