ABSTRACT
By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Subject(s)
Antibodies/genetics , Gangliosides/immunology , Immunoglobulin Fragments/biosynthesis , Interleukin-2/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Antibodies/metabolism , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Interleukin-2/genetics , Molecular Sequence Data , Recombinant Fusion Proteins/geneticsABSTRACT
Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.
Subject(s)
Enterotoxins/metabolism , Melanoma/immunology , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Single-Chain Antibodies/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enterotoxins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Melanoma/drug therapy , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/therapeutic use , Single-Chain Antibodies/geneticsABSTRACT
The rare codons of a fragment in staphylococcal enterotoxin A gene were turned into the most high usage frequency codons in E. coli by overlap PCR technique. Genes of sea and seam were cloned into 7ZTS expression vector and transformed into JM109(DE3), respectively. The result shows that expression level of sea gene was very low, but the expression level of seam was as high as 15% of total cell proteins. The expression product shows activity of antitumor in vivo.
Subject(s)
Codon/genetics , Enterotoxins/genetics , Animals , Base Sequence , Electrophoresis, Polyacrylamide Gel , Enterotoxins/metabolism , Enterotoxins/pharmacology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Male , Mice , Molecular Sequence Data , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Point Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacologyABSTRACT
An about 700 bp DNA fragment was amplified from genome DNA of S. aureus TSTw by PCR. This fragment was cloned into pGEM-7Zf(+) and the recombinant plasmid was transformed into E. coli DH5 alpha. The sequencing result of the recombinant plasmid demonstrated that it contains seb gene with 717 bp (without signal encoding region of 81 bp) which has the same nucleotide sequence as described in literature. The seb gene was cloned into expression vector 7ZTS and was transformed into E. coli JM109 (DE3). The expression level of SEB was as high as 33.3% of the cell total proteins.
