蚕素Bbx-b4在家蚕杆状病毒表达系统中的表达及功能研究

We found the Bbx-b4 gene from the cDNA library constructed by our laboratory. After Blasten based on NCBI database and alignment of deduced amino acid sequence homology，it was found to be the Bbx-b4 gene.Its open reading frame（ORF）is 273 bp and encod 90 amino acids . Bombyxin and human insulin are so similar that they have the same tertiary structure. Its molecular weight is about 4.0kDa. We cloned the fusion gene of Bbx-b4 and gene Polh(polyhedrin,Polh) into the vector pET-28a and pFastBac1 to construct recombinant plasmid, and the fusion protein was expressed by E.coil system and Bombyx mori NPV expressing system, respectively. The fusion protein expressed in the E.coil syste remained as inclusion body. According to the principle of the alkali fusion of Polh protein,we purified the fusion protein by dissolving inclusion protein in different PH buffer solution and metal-chelating affinity chromatography.The purified fusion protein was used to immunize male New Zealand rabbit. Western blot analysis revealed that the protein was expressed in siliworm cell and fresh silkworm pupa successfuliy.Flow cytometry cell cycle experiment proved the Bbx-b4 has the function of promoting the growth of cells. The expressed protein in fresh silkworm pupa could obviously reduce the tall fasting glucose in the mice whice were injected by ALX(Alloxan tetrahydrat)，but its effect is not obviously comared by the natural silkworm pupa.