Temperature control of prokaryotic expression and detection of a mutated SEAD227A fusion single chain antibody specific to HIV-1 gp41
ObjectiveTo obtain the recombinant protein comprised of anti-HIV-1 gp41 single chain antibody(ScFv41) and Staphylococcus aureus exotoxin A gene (SEA). MethodsScFv41 which can broadly recognize HIV-1 protein gp41 and SEA gene were obtained from GenBank. Codon GAT coding Asp at the site of 227 of SEA was mutated to codon GCT coding Ala. Both genes' codons were optimized and synthesized. The recombinant temperature control of prokaryotic expression vector pBV-41 and pBV-SL41 were respectively constructed. Recombinant protein was expressed by raising temperature. After gel filtration chromatographic protein refolding,ScFv41 binding activity with HIV-1 antigen strip and recombinant targeting toxins mediating CTL activity were detected. ResultsProduction of target proteins ScFv41 and SL41 were highest in the condition of introduced 6 h at 44℃ and 7 h at 42℃ in E.coli BL21(DE3)plys respectively and took 14.359% and 23.482% of the total cell proteins. After gel filtration chromatographic protein refolding,both target proteins can conjugate with HIV-1 antigen strip very well,SL41 can activate PBMCs and mediated CTLs to kill the target cells which steadily expressed HIV-1 gp160 protein. ConclusionThe recombinant protein SL41,was successfully obtained which laid a bases for the preparation of recombinant targeting toxin against HIV-1.