Prokaryotic Expression And Activity Of The Fusion Protein Append Determination

There are two key factors in tumour development: infinity increasing of the cells and the formation of the tumour blood vessel. If the anti-tumour druggery can aim at more than one targets, and attack tumour cells by multi-target manner, it will get the better effect.Apoptin, is a 13.6kDa basic protein of 121 amino acids which derived from the chicken anemia virus. It induces tumor specific apoptosis in human transformed and malignant cells but not in normal, diploid cells. Many study results have proved that the apoptin is safe when used as the anti-tumour druggery.Endostatin is one of the most effective factors which can inhibit the growth of blood vessels. Endostatin is 20kDa C-terminal fragment of collagen XVIII, which contains 184 amino acids.In this study, we have constructed a fusion gene, named Append, whose production is Apoptin-(Gly4Ser)3-Endostatin, we express the fusion protein in E. coli express system.The endostatin cDNA was amplified from total RNA of human embtyonic liver using RT-PCR.The apoptin gene was obtained from the recombinant plasmid pUC-Apoptin. After that, the Append gene was constructed by ligating apoptin gene and endostatin gene with a linker. Then the Append gene was cloned into pGEM-T easy vectors. Three expression plasmids were constructed by subcloned the target gene into three vectors pLLP-OmpA, pET-His and pET-DsbA. Three expression plasmids pLLP-OmpA-Append, pET-His-Append and pET-DsbA-Append were transformed into prokaryotic cells separately. The protein named Append was purified using Ni-NTA technology. The protein was identified by SDS-PAGE, Western blot and bioassays.Sequence analysis shows that we have obtained the 960bp gene fragment, and found the appropriate plasmid is pLLP-OmpA-Append, the souble protein is 1.43mg/L, and the purity is 91.7%. The bioactive Append is expressed in E. coli. The result has proved that the fusion protein has both function of apoptin and endostantin, that is, it can induce the apoptosis of the tumour cells specificly and inhibit the growth of the new blood vessels.The study has constructed the fusion gene append, and expressed the fusion protein Append in E.coli, which is helpful for creating the anti-tumour durggery...