Expression And Purification Of His-Porcine Growth Hormone Fusion Gene In E.coli And Baculovirus Expression Vector System

Porcine growth hormone (pGH) is a single peptide which is produced and released by the anterior pituitary. The main results in this research showed as below: 2. The cDNA of pGH without signal peptide was reconstructed by PCR, and then recombined with the vector pET-28a(+) in E. coli and the transferred vector pBacPAK-His1. The recombined plasmid, pPGH020 and recombined transferred vector, pPGH030 could be directly purified by Ni-NTA Agarose. 2. The pPGH030 and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN), then the recombinant virus, Bm-BacPAK6-pgh was obtained successfully. 3. The optimum conditions of pPGH020 expression and purification were studied. IPTG inducing expression in BL21 strain as the host bacterium had been done. SDS-PAGE and Western blot analysis showed that the pGH gene product, 26.5 k fusion protein consisted of His·Taq, thombin cut site and pGH sequence. Gel scanning showed that the expression quantity of pGH in E. coli were about 17.3 % of the total proteins. 4. The recombinant baculovirus, Bm-BacPAK6-pgh constructed with His-Tag pgh fusion gene expression in BmN cell and the individual of Bombyx mori were studied. SDS-PAGE using the cells which were infected by Bm-BacPAK6-pgh showed that the fusion gene was expressed both in the BmN cell and the larvae of Bombyx mori, and the specific band of expression product is 25 k. Western blot analysis showed the fusion protein expressed in BmN cell and the individual of Bombyx mori was the same in antigenicity with that expressed in E. coli cells. Bm-BacPAK6-pgh expressed in BmN cells started at the 24h post infection, while in the individual of Bombyx mori, 72h. The expression summit of BmN cell and the individual of Bombyx mori appeared at 96h and 120h, respectively. 5. The recombinant pGH fusion proteins can be attained by two-step purification method, that was 40% ammonium sulphate salt out and Ni-NTA Agarose affinity chomatography respectively. The obtain fusion protein has the electrophoresis purity and also has immunogenicity.