Preparation And Function Of SA-hTNF-α Bifunctional Fusion Protein

BackgroundMalignant tumors are serious harm to human health,and the major treatments for malignant tumors are surgery,radiation therapy,chemotherapy and combine biotherapy.The common defects of these treatments are lack of specificity,damage normal tissue and toxicity.These treatments are not only poor effect but also expensive.Therefore,we need to investigate new therapies and new drugs that are efficiency,non-toxic side effects and induce long-term immune protection.Tumor necrosis factor alpha（TNF alpha） is a cytokine with antitumor effect that can directly kill cancer cells and activate the immune system.Currently,recombinant human TNF alpha has successfully show antitumor activity in vivo and vitro.TNF alpha could direct damage the tumor and inhibit proliferation of tumor in vitro,and could make the tumor necrosis,hemorrhage,smaller and even disappear in vivo.But, it need a great quantity TNF alpha all over the body could to achieve the concentration of effective treatment in tumor local.Therefore,the methods of local administration were adopted to reduce the toxicity of TNF alpha in clinic.Because of the organization diffusion,the cytokines can not maintain long-term efficacy and have toxicity.The application of TNF alpha was greatly restricted in clinical antitumor.Because it has these disadvantages,we were developed a SA-TNF alpha fusion protein to improve TNF alpha effect of tumor therapy and avoid its side effects. We use the two properties that membrane surface is easily biotin and biotin combine streptavidin-（SA） with high efficiency,forcefulness and almost irreversible.The first,we made the biotin chemical crosslinking with the membrane protein on the surface of tumor cell or tissue,and then,the target protein SA-hTNF-αwas anchored at the surface of cells or tissues through streptavidin specific binding with biotin. Biotin reaction has no obvious adverse effects for cells,tissues and organisms.This technology can make SA-TNF alpha fusion protein quickly modify tumor tissues in home position and was fixed on tumor tissues and surrounding,and the quantity of fixation can accurately be controlled.It could avoid serious side effects which caused by largely TNF get into circulation system.This article constructed a SA-TNF alpha plasmid which had high efficient expression in Escherichia coli,and studied the purification,renaturation and activity of the SA-hTNF-αfusion protein,which provide a substantial foundation for TNF alpha apply in anti-tumor in the clinic.ObjectiveWe constructed a SA-TNF alpha recombinant plasmid,prepared the fusion protein（SA-TNF alpha） and studied its biological functions.Methods1.Construct the SA-hTNF alpha expression plasmidwe obtained a pET24a-SA-hTNF alpha prokaryotic expression plasmid by PCR identify.2.The SA-hTNF alpha fusion protein expression and the optimal conditions of fermentationWe cloned a pET24a-SA-hTNF alpha to BL21（DE3） cells and studied the conditions of its expression.3.The extraction and purification of SA-hTNF-αinclusion bodies Inclusion bodies were washed by five-step method and add to lysozyme ultrasound-breaking strain,high-speed.We collected and purified them by Nickel-metal -chelated（Ni-NTA）.4.The renaturation of SA-hTNF-αfusion proteinWe used different systems to study the renaturation of fusion protein and to identify by West blotting.5.To identify biological activity of SA-hTNF-αbifunctional fusion proteinWe detected SA-hTNF-αactivity of cytotoxicity in vitro by L929 cell,and analysised the membrane anchoring efficiency that SA-hTNF-αbiotinylated-modified tumor cell by flow cytometry.We detected the activity of SA-hTNF-αonce again after broke the membrane.Results1.The identification of pET24a-SA-hTNF-αplasmidWe constructed a plasmid of pET24a-SA-hTNF-αand identified it by PCR and restriction enzyme digestion.We obtained the fragment with hTNF-α（498） and SA-hTNF-α（986） which carried out DNA sequencing,and the results was same to GenBank.2.The expression of SA-hTNF-αfusion proteinThe recombinant plasmid was transformed into E.coli BL21/DE3.The purpose protein was induced by 37℃,0.5mmol/LIPTG,7h.Its expression was 30%in the total protein and its form was inclusion body.3.Washing and purification inclusion body of SA-hTNF-αTarget protein was adsorbed and eluted by 100mmol/L imidazole buffer.The purity of purpose protein was get up to 95.7%by SDS-PAGE electrophoresis and TLC analysis.4.The renaturation of SA-hTNF-αfusion protein We used two methods to study renaturation of the SA-hTNF-αfusion protein. The first method was urea gradient refolding dilution and the result was that the fusion protein mainly existed in form of dimer;The second method was precipitation of fusion protein that was dissolved in 7mol/L guanidine hydrochloride,and the protein in form of polymer.The mechanism of this result need further study.5.To identify the biological activity of SA-hTNF-αfusion proteinWe used L929 cell to detect SA-hTNF-αactivity of cytotoxicity in Vitro.There were no significant difference among standard protein(ED₅₀ for 0.1ng/ml),polymer’s ED₅₀ is 0.21ng/ml and the dimer’s ED₅₀ is 0.28ng/ml（P＞0.05）.The result of flow analysis was that both of dimer and polymer can anchor biotinylated MB49 cell membrane.The anchor rate of the dimer was 92.07%,and the polymer was 98.15%.6.To detect activity of fusion protein after anchoredSA-hTNF-αcombined with biotinylated MB49 tumor cell membrane,and broken the cell membrane.Then,we detected activity of SA-hTNF-α.The results showed that SA-hTNF-α’s dimer and polymer are both can be anchored on the surface of tumor cell membrane and can kill L929,but the standard protein can not anchor on the tumor cell membrane.Conclusions1.we was successfully constructed the plasmid of pET24a-SA-hTNF-α.2.we obtained the high expression of SA-hTNF-αfusion protein in E.coil and accounting for 30%of total protein.3.we successfully purified fusion protein and accounting for 95.7%total protein.4.We have successfully developed the SA-hTNF-αbifunctional fusion protein. These two proteins have a bifunctional activity that is anti-L929 cell activity and the anchor-modified biotinylated MB49 cell activity,and the functions of these two proteins was no significant difference.We successful developed the SA-hTNF-αfusion protein which may laid the foundation for carry out animal experiment and applicated possible for clinical anti-tumor.