Recombinant Filip1l Protein And Preparation Of Antibodies

BackgroundThe development and maintenance of a blood supply is critical for the growth and metastatic spread of tumors. Inhibiting the process of new blood vessel formation, as well as attacking established tumor vasculature, has been shown to be a viable strategy for treating cancer. A number o f antiangiogenic agents have been indentified, and several have entered clinical trials. These angiogenesis inhibitors that prevent endothelial cells from proliferating and migrating or result in the induction of apoptosis have been identified.Filamin A interacting protein 1-like(FILIP1L;previously known as down-regulated in ovarian cancer 1) was identified as one of the genes up-regulated in endothelial cells in response to these inhibitors. Some studies had suggest overexpression of FILIP1L resulted in inhibition of cell proliferation and migration and increased apoptosis. Overall, these findings suggest that the novel protein FILIP1L may be an important mediator of the effects of angiogenesis inhibitors and that FILIP1L has the potential to be an antivascular reagent for cancer therapy.In this study,the encoding extracellular of FILIP1L gene was fist cloned from 9706 cells line of esophageal squamous carcinoma. Then after verifying by DNA sequencing to construct Pet22b- FILIP1L eukaryotic expression vector,and transferred to the E.coli expression of feeling states. With affinity purification techniques of recombinant fusion protein, and preparing the FILIP1L protein polyclonal antibody. From that , we could study the interaction of FILIP1L expression.ObjectiveIn this study,we obeserved the components of each member of FILIP1L protein-binding protein complex and their functions, clarified the molecular mechanism of FILIP1L, the candidate tumor suppressor gene, in cancer how to play a suppressor role. It help us to further understand the molecular mechanisms for tumor development of multi-elements and multi-stages and to establish the basis work for the high-risk population screening and early diagnosis of biological indictors.MethodsWith mRNA, witch extracted from squamous cell carcinoma 9706 of esophageal epithelium, reversed transcription into cDNA and with RT-PCR to amplification the cDNA, which containing the full-length FILIP1L gene ( about 963bp). Then, the cDNA were directed into the prokaryotic expression vector pET22b to induce recombinant protein expression, after witch determined successfully construction by sequencing vector; With Western blotting to detection protein and get the induced-purified protein by affinity chromatography ways; Finally, with the induced protein to immune animals to get polyclonal antibodies, and detecting the difference of FILIP1L interacting protein expression with co-immunoprecipitation.Results1. The sequence of extracellular FILIP1L gene, which from squamous cell carcinoma 9706 of esophageal epithelium, was verified accuracy.2. We get the high purity protein with construction the prokaryotic expression vector of FILIP1L and inducing protein expressed.3. We get the high titer polyclonal antibodies of 1:1×106 from that purified protein.4. With co-immunoprecipitation technology, we found some difference of FILIP1L -related protein expression between normal esophageal epithelium NEC and squamous cell carcinoma 9706 by co-immunoprecipitation technology. ConclusionsThe extracellular segment of tumor suppressor gene FILIP1L was been extracted from the esophageal squamous cell line 9706, Construct a FILIP1L prokaryotic expression vector, use the plasmid expression the FILIP1L fusion protein ,and using the protein to prepare polyclonal antibodies. Detected the differential expression interacting proteins between normal tissue and tumor tissue ,and found six of the FILIP1L -related protein expression in esophageal cancer cells was significantly higher than normal esophageal epithelial cells, suggesting that in esophageal cancer cells, may be because the expression of these proteins abnormalities led to the FILIP1L tumor suppressor gene have no discrimination between in esophageal cancer tissues and normal tissues , but have not plays a key role in the tumor suppressor. This will help us to establish the basis by further clarified the molecular mechanism of FILIP1L protein in the tumor development process in the future and provide lots of candidate molecular markers for the high-risk population screening and early diagnosis of biological indictors.