Construction And Expression Of Recombinant Mouse Stnf-α Protein And Its Purification

Tumor necrosis factor alpha (TNF-α) is a very important cytokine with a wide array of biological activities, which is chiefly produced by activated monocytes/macrophages and lymphocytes. There are two forms of TNF-α, namely a 17kD secreted TNF-α(sTNF-α) and a 26kD transmembrane TNF-α(mTNF-α). The both types of TNF-αhave biological functions.It was reported that solid tumor comes from chronic inflammation, and there were so many myeloid derived suppressor cells (MDSC) in chronic inflammation and carcinoma. MDSC are heterogeneous family of myeloid cells, including immature macrophages, dendritic cells and granulocytes, and they use a diversity of mechamisms to suppress multiple immune effects. It was reported that MDSC developed to tumor-associated macrophages (TAM) and vascular endotheliocyte, which were critical matrix cell in carcinoma tissue, and TAM could participate in the apoptosis of activated CD8+ T cells through direct and indirect contaction. These finding indicates that TNF-αmay be involved in accumulation and immune suppression of MDSC.To study the relationship between TNF-αand MDSC, mouse sTNF-αcDNA inserted into pTriex4hisC expression vector was first constructed and expressed effectively in E.coli. Mouse sTNF-αfusion protein was then purified by Ni-NTA agrose. The results of this study are as follows:1. Construction, cloning and identification of mutant WT-TNF-pcDNA3.1C recombinant plasmidsExtract the total RNA of mouse peritoneal macrophages, reverse transcription samples were taken to get cDNA.Using this cDNA as a template, the gene WT-TNF-αwas amplified with recombinant ploymerase chain reaction (PCR). After digestion with endonucleases, the WT-TNF fragment was inserted into pcDNA3.1c plasmid at BamHI and EcoR I by T4 DNA ligase. Then the mutant WT-TNF-pcDNA3.1C recombinant plasmid was transformed into E. coli DH5αand the positive clones were screened by ampicillin resistance.Identification of positive clones: The positive clones were confirmed firstly by colony PCR, followed by further identification with endonucleases digestion, showing the cleaved fragment from the recombinant plasmid with the molecule weights of 708bp, DNA sequence analysis also proved that the WT-TNF-pcDNA3.1C recombinant plasmid was successfully constructed.2. Construction, cloning and identification of mutant sTNF-pTriex4hisC recombinant plasmidsUsing the plasmid WT-TNF-pcDNA3.1C as a template, the gene sTNF-αwas amplified with recombinant ploymerase chain reaction (PCR). After digestion with endonucleases, the sTNF gene fragment was inserted into pTriex4hisC plasmid at BamHI and EcoR I by T4 DNA ligase. Then mutant sTNF-pTriex4hisC recombinant plasmid was transformed into E. coli DH5αand the positive clones were screened by ampicillin resistance.Identification of positive clones: The positive clones were confirmed firstly by colony PCR, followed by further identification with endonucleases digestion, showing the cleaved fragment from the recombinant plasmid with the molecule weights of 471bp, DNA sequence analysis also proved that the sTNF-α-pTriex4hisC recombinant plasmid was successfully constructed.3. Expression and purification of Recombinant Mouse sTNF-αProtein and Its identificationsTNF-αDNA inserted into pTriex4hisC expression vector was first constructed and expressed effectively in E.coli. The sTNF-αfusion protein was then purified by Ni-NTA agrose.The results of the identification of this fusion protein show that a highly purified and precise protein was gotten.