| Gastric cancer is one of the most common human malignancies, which isthe second leading cause of worldwide cancer mortality. The prognosis is poorin China, where the5-year survival rate of gastric cancer is less than20%-25%. Early gastric cancer can be cured by surgically removal, supplementedby radiotherapy and chemotherapy. Those greatly improve the patient’s5-yearsurvival. However, in advanced gastric cancer those don’t work effectively.With the depth of the tumor study, people realized that the tumor growthmicroenvironment, especially in tumor angiogenesis, is very important totumor growth and metastasis. Therefore, blocking tumor angiogenesis hasbecome a new direction for cancer treatment.GX1-rmhTNFα is a neovasculature targeting drug for tumor treatment,which was prepared by genetic engineering technology. rmhTNFα has beenapproved for marketing in China for the treatment of small cell lungcancer.GX1is a small cyclic peptide targeting gastric cancer vascularlaboratory by phage display peptide library screening. Therefore we fusedGX1to rmhTNFα. The pre-test proved that the drug could selectively targeton the neovascularization in gastric cancer and would effectively inhibitangiogenesis in gastric carcinoma which played a great role in the treatment ofgastric cancer.Objective To further speed up the GX1-rmhTNFα used in the clinical treatment, this subject is designed to comprehensively test engineeringbacteria (DH5α/pBV-GX1-rmhTNFα)which expresses GX1-rmhTNFαprepared by recombinant DNA technology. The aim of our project is toestablish a stable engineering bacteria strain suitable for mass production andto establish a stable process of fermentation and purification. Methods First,we established a stable engineering bacteria strain library by comprehensivetest. Then we developed a stable production technology of fermentation byconsecutive three batches of fermentations in5L fermenter under the optimalcondition. At last, a purifiction procedure was developed according to thephysical and chemical properties of target protein, and then the protein wasidentified by SDS-PAGE, Western blot and HPLC. Results The test resultsshowed that the recombinant engineering bacteria(DH5α/pBV-GX1-rmhTNFα)was suitable for engineering bacteria strain library. Consecutivethree batches of fermentations in5L fermenter harvested about200g bacteiralrespectively and the protein expression level reached10%. The purity of thepurified GX1-rmhTNFα by HPLC was over95%. Conclusion The developedprocedures for fermentation and purification were simple and rapid, whicnlaid a foundation of large scale production of GX1-rmhTNFα. |
