The Construction Of GST Fusion 15-peptide Library And The Detection Of Its Anti-tumor Activity

The classical construction of the peptide library is the phage-display technology, but with this method, every peptide in the peptide library can not be expressed solely and screened on basis of its function and structure. Further value of the peptide library can not be implemented in the extensive application and comparison. Purpose of our research is to construct a peptide library with the method of the gene-engineering technology which is distinguishing to the phage-display technology. In this peptide library, each peptide should have the integrity of the function, and its activity can be detected, its number can be counted, and its applied value can be estimated.On the premise of these requirements, we design this project in aim to construct a peptide library answer for the request. First, design the sequence of the random polypeptides, ligate it into plasmid pGEX4T-1, then transform the plasmid into DH5a, spread the cells onto the LB(Amp) agar platesj examine the efficiency of the ligation and the transformation, we can get the capability of this peptide library, it is about 2.5× 10~4. After the establishment of the peptide library, we chose 100 recombined plasmids which were ligated with foreign DNA sequence and induced the fusion proteins expression by EPTG The fusion proteins were purificated with the Glutathione Sepharose 4B.We measured the approximate concentration of the fusion proteins compare to the BSA which has certain concentration. We make use of MTT assay to measure its anti-tumor activity, the result showed that a majority of the fusion proteins represented the function of inhibiting to the HeLa tumor cells. We discussed the feasibility of building the peptide library in use of the gene-engineering technology and prospected the works which should be done in future.