Reconstruction Of Fusion Protein Tumstatin-TNF Secreted Eukaryotic Expression Vectro And Stable Expression On Chinese Hamster Ovary Cells

Objective:Tumstatin is a novel anti-angiogenic protein domain derived from theα3 chain of typeⅣcollagen,which consists of 244 amino acids.The NC1 domain of theα3 chain(tumstatin) was most potent in inhibiting the proliferation of blood vessel endothelial cells and causing apoptosis when compared with the otherα(Ⅳ) chain NC1 domain.It can inhibit tumor growth.Human tumor necrosis factorα(hTNFα) is one of the most important cytokines that acts as a mediator in immunoregulation and plays a key role in anti-tumor defence mechanism.The aim of this study is to Cloning and construction of fusion protein tumstatin-TNF gene.tumstatin was amplified from 293 embryonic kidney cells using RT-PCR.tumstatin and TNFαwere linked by a flexible hinge in order to make tumstatin-TNF as a dimer.Then fusing expression of an antiagiogenic inhibitor(tumstatin) with a cytotoxic factor(TNFα) yielded a novel secreted fusion protein,tumstatin-TNF,in order to improve its anti-tumor activity.Methods:The cDNA fragment of tumstatin was obtained by a reverse transcriptase-polymerase chain reaction(RT-PCR) with total RNA extracted from 293 embryonic kidney cells.The RT-PCR product was cloned into pGEM-T vector,then the pGEM-T/tumstatin plasmid was obtained.The typeⅣcollagen signal peptide that takes overlapping district with tumstatin part was obtained by a polymerase chain reaction(PCR) with pGEM-T/tumstatin.Sig-tumstatin-linker fragment and linker-TNF fragment were obtained by PCR from Sig-tumstatin fragment and PBV220-TNF-a vector.Then the sig-tumstatin-linker-TNF fusion gene was obtained by a splicing by overlap extension polymerase chain reaction(OSEing-PCR) with the previous products. The fusion gene that digested with NheⅠ/BamHⅠwas cloned into pIRESneo3 expression vector with the same restriction enzyme digestion and positive clones were screened.We obtained the positive recombinant pIRESneo3/sig-tumstatin-linker-TNF plasmids.Colony PCR,restriction enzyme digestion and gene sequencing were used to verify the obtained sig-tumstatin-linker-TNF fusion gene.The recombinant pIRESneo3/sig-tumstatin-linker-TNF plasmids were transfected into Chinese hamster ovary-K1(CHO-K1) cells by the lipofection method.After transfection,the cells were cultured in fresh medium containing G418(700 mg/L) for 2 weeks,and then stable transfectants were obtained by G418 selection.We detected the expression of sig-tumstatin-linker-TNF out of these cells.Through cell-based assays,such as endothelial cells proliferation and inhibition tests,endothelial cells tubular structure formation test,the apoptosis of endothelial cells test and L929 cells restraint active test, by which we can know whether tumstatin-TNF fusion protein maintained both tumstatin and tumor necrosis factor-alpha activities.Results:The obtained sig-tumstatin-linker-TNF fusion gene(1.32 kb) was identical to the sequence of designed human fusion protein tumstatin-TNF.Double enzyme digestion demonstrated that the recombinant pIRESneo3/sig-tumstatin-linker-TNF secreted eukaryotic expression vector was successfully constructed.The transfected CHO-K1 cells successfully secreted expression tumstatin-TNF(45 kD) fusion protein by western blot. Tumstatin-TNF fusion protein inhibited apparently the proliferation of endothelial cells in a dose-dependence manner through endothelial cells proliferation and inhibition tests (ED50,2μg/ml).Tumstatin inhibited the proliferation of endothelial cells also (ED50,7.6μg/ml).Tumstatin-TNF obviously restrained endothelial cells pipe structure form.The formatting percentage of endothelial cells pipe structure was 4.1±1.4%with 2μg/ml tumstatin-TNF.Tumstatin-TNF(100 ng/ml) induced the apoptosis of endothelial cells aspect was higher than tumstatin(5μg/ml).Tumstatin-TNF fusion protein in for L929 cells restraint active aspect was no difference than TNF.In addition, the CHO-K1 cells transfected with pIRESneo3 vector and the CHO-K1 cells transfected with recombinant pIRESneo3/sig-tumstatin-linker-TNF were slower than the non-transfected CHO-K1 cells in the growth speed.In a word,Tumstatin-TNF fusion protein maintained both tumstatin and tumor necrosis factor-αactivities as demonstrated by cell-based assays.Conclusion:Study to construct the bifunctional secreted fusion protein tumstatin-TNF pass through gene recombination technology. We have successfully constructed fusion protein tumstatin-TNF secreted prokaryotic expression vector.Recombination expression vector pIRESneo3/sig-tumstatin-linker-TNF successfully secreted express in eukaryotic cell CHO-K1.Tumstatin-TNF fusion protein inhibited apparently the proliferation of endothelial cells in a dose-dependence manner,Tumstatin-TNF fusion protein inhibited the proliferation of endothelial cells active was higher than tumstatin.Tumstatin-TNF fusion protein in for L929 cells restraint active aspect was no difference than TNF. Tumstatin-TNF(100 ng/ml)induced the apoptosis of endothelial cells aspect was higher than tumstatin(5μg/ml).tumstatin that restrain ability endothelial cells pipe structure form is not as good as tumstatin-TNF.We have successfully constructed the recombinant tumstatin-TNF fusion protein prokaryotic expression vector and obtained CHO-K1 cells that can stable express tumstatin-TNF fusion protein.