Construction Of MAGE-3 Prokaryotic Expression Plasmid PET30a (+)-MAGE-3 And Its Expression In Escherichia Coli

BACKGROUND: Melanoma antigen-encoding gene (MAGE) family was the firstreported example of the 'cancer-germline' genes. MAGE genes were first detected in 1991. Later, scientists recognize that they are a big family actually and this family comprises several related genes divided into 3 clusters, named MAGE-A, MAGE-B and MAGE-C. The gene MAGE-3 belongs to the MAGE-A cluster. Among the 12 members of the MAGE family, MAGE-3 is most frequently expressed in many kinds of carcinomas. The MAGE-3 protein is 314 amino acids long (34747Da). MAGE-3 was located in Xq28 of genomic. A number of MHC class I and class II restricted T-cell epitopes have been described for the MAGE-3 tumor antigen, including 12 MHC class I restricted epitopes and 8 MHC class II restricted epitopes. MAGE-3 tumor antigen is widely used in the study of tumor therapeutic vaccination. MAGE-3-based vaccines mainly include: vaccination with peptide, whole protein, the recombinant poxvirus encoding the peptide/the whole protein and the dendritic cells (DCs) pulsed by peptide/whole protein. All vaccinations above induce immune responses in vitro and have led to clinical benefits in vivo. Although the peptide-based vaccine is reported more than others, vaccination with peptides is restricted to patients with specific HLA haplotypes capable of interacting with those peptides. Using a whole protein as an immunogen makes it possible to circumvent this restriction and to obtain a broader repertoire of T-cell immune responses. The purpose of this study is to induce the expression of recombinant expression plasmid pET-30a(+)-MAGE-3 to express the MAGE-3 protein by IPTG. The recombinant plasmid was transformed intoBL21(DE3) E.coli and lay the groundwork for further research of the MAGE-3 whole protein-based vaccine and relative.METHODS: We extracted the whole RNA from the carcinomas tissues of small celllung carcinomas, and amplified the whole sequence of MAGE-3 by reverse transcriptase-polymerase chain reaction (RT-PCR). The gene was linked into pGEM-T Easy plasmid and the recombinant pGEM-T Easy-MAGE-3 was transformed into JM109. Using Amp fastness and blue/white color screening to select the positive colonies of transformed bacteria. The primer designed according to T7/SP6 sequence of pGEM-T Easy plasmid was applied to identify the linking correctness. Identified positive clone was sent to do the sequencing to identify the correctness of objective fragment cloned completely. Both recombinant plasmid pGEM-T Easy-MAGE-3 and prokaryotic plasmid pET-30a(+) underwent enzyme cutting with Xho I , and then purified by electrophoresis and gel extraction. After dephosphorylated by CIAP, the linearity plasmid pET-30a(+) was linked with the objective fragment to construct the recombinant expression plasmid pET-30a(+)-MAGE-3. Then, plasmid pET-30a(+)-MAGE-3 was transformed into BL21(DE3). The positive colonies were selected and the positive clone (the positive inserting recombinant) was identified by primer T7/MP2, and sent to do DNA sequence also. The expression of MAGE-3 protein was induced with IPTG and identified by 12% SDS-PAGE electrophoresis and Coomassie blue staining analysis and Western blot.RESULTS:1. Amplification of target gene and enzyme cutting identification: the whole MAGE-3 sequence at 954bp long was amplified by RT-PCR and was identified by the enzyme cutting with Hind IIIand EcoR I , the results was correct.2. Result of identification of recombinant of clone and subclone:Target gene linked with plasmid pGEM-T Easy. Then recombinant was transformed into JM109. After blue- white selection, 6 positive colonies were selected at random to undergo the PCR amplification with primer T7/SP6. The amplified fragment at 1118bp long was obtained from all samples.The recombinant of dephosphorylated pET-30a(+) linked with fragment MAGE-3 was transformed into BL21(DE3). 9 positive colonies were obtained and identified by PCR amplification with primer T7/MP2. 3 positive clones (the positive inserting recombinant) were identified for the correct ampli...