Construction of hybrid gene of hepatitis B surface antigen carrying heat-stable enterotoxin of escherichia coli and its expression in mammalian cell line

Hepatitis B surface antigen is the first genetically engineered vaccine licensed for human use. Various strategies have been proposed to obtain a vaccine that would bypass the need for injection. In this study, a non-toxic portion of heat-stable enterotoxin of Escherichia coli that is capable of adhering to epithelial cells was inserted at amino acid position 112 of hepatitis surface antigen. The construct was used for transfection of human embryonic kidney cells in order to assess the expression of the hybrid protein. The data obtained showed a very low level of expression. In vivo antibody production and cytotoxic T lymphocyte response in B6 mice were assessed using DNA immunization. Three out of five injected mice responded with titers 10 mIU/ml anti-HBsAg and cytotoxic T-lymphocyte response was much higher with construct encoding the chimeric protein. Although this study proves that the chimeric protein is capable of eliciting both humoral and cellular responses, but further work is required to fully explore the feasibility of combining the properties of the two proteins

Synthesis and expression of modified bFGF gene in Escherichia coli cells

A new strategy for construction of synthetic gene encoding human basic fibroblast growth factor comprising DNA annealing-Iigation and augmentation by polymerase chain reaction was introduced. The sequence of the corresponding amino acid chain were modified in order to increase stability of the protein, First, 300 bp 160 bp fragments of the gene were assembled from 18 oligonucleotides and ligated separately. Then, the shorter fragment was completed by using PCR and combined with the longer one in a proper orientation in pUC 19, One extra nucleotide that had been found in the gene after DNA sequencing and resulted in frame shift, was rectified through the use of PCR directed mutagenesis. Finally, 5' -terminal region of the gene was augmented by means of PCR in order to restore the N-terminal part of the protein and to introduce the Nde1 recognition site The gene was subcloned into the inducible pET-3a expression vector under control of T7 promoter and expressed in Escherichia coli The identity of the recombinant protein and level of expression were detected by using Western blot analysis immunoassay. The proposed method has provided a useful strategy for synthesizing modified proteins that might be applied for protein engineering