Construction and Bioactivity Analysis of Fusion Protein of Polyvalent Anti-aflatoxin B1 Nanobody

KeyWord:aflatoxin B1  nanobody  tandem fusion  prokaryotic expression  enzyme linked immunosorbent assay

Abstract:

      Nanobody£¬derived from the variable domain of heavy chain only antibodies£¨VHHs£©£¬is the smallest antigen recognition unit£®It is an ideal candidate for the next generation of immunoassays due to its high structural stability and high expression yields compared with the conventional antibody£®This study aimed to investigate effects of the multivalency and gene fusion of anti-aflatoxin B1£¨anti-AFB1£© nanobody on the bioactivities of recombinant proteins£®The anti-AFB1 nanobody£¬namely G8£¬was screened from an immune VHH phage display library in a previous work£®The coding sequence of G8 was cloned into the vector pET30 between the restriction enzyme sites Nde¢ñ and Sfi¢ñ£¬generating vector pET30£­G8£®The overlap PCR was performed to assemble the tandem structures£¨DiG8 and TriG8£© with flexible linker£¨GGSGG£©£®The overlap PCR products encoding DiG8 and TriG8 were purified£¬and cloned into the vector pET30 at the same site as the G8£¬generating vector pET30£­DiG8 and pET30£­TriG8£¬respectively£®To construct expression vectors for the recombinant fusion proteins£¬the green fluorescent protein £¨GFP£© encoded gene was firstly amplified by PCR with specific primers F0 and R0£¬and then subcloned into the restriction sites between Sfi ¢ñ and Not ¢ñ of vectors pET30£­G8£¬pET30£­DiG8£¬and pET30£­TriG8£¬respectively. The resulting vectors£¨pET30£­G8£­GFP£¬pET30£­DiG8£­GFP£¬and pET30£­TriG8£­GFP£©£¬which fused GFPs at the C terminals of monomeric£¬dimer and trimer anti-AFB1 nanobody£¬were confirmed via colony PCR£¬restrictive digestion£¬and sequencing£®The vectors were transformed to Escherichia coli BL21 £¨DE3£©£¬respectively£®To induce the expression of the target proteins£¬isopropyl ¦Â-D-1-thiogalactopyranoside£¨IPTG£© was added to the culture£®Fusion proteins each contain six histidine tag at their C-terminals which were purified by affinity chromatography£¬respectively£®SDS£­PAGE analysis showed that the three fusion proteins£¨G8£­GFP£¬DiG8£­GFP and TriG8£­GFP£© were solubly expressed in E. coli yielding at least 6.0£¬7.5£¬4.0 mg/L£¬respectively£®Enzyme-linked immunosorbent assay£¨ELISA£© and fluorescence scanning were employed to estimate the antigen recognition bioactivity and fluorescence features of recombinant fusion proteins£®The half inhibitory concentrations£¨IC50£© of the recombinant proteins G8£¬G8£­GFP£¬and DiG8£­GFP were 12.10£¬14.10 and 2.19 ng/mL£¬respectively£®The results showed that the IC50 of the monomeric G8£­GFP was similar to the non-fused G8£¬wheras the IC50 of the divalent DiG8£­GFP was improved 5.5 times higher than that of G8£®Both G8£­GFP and DiG8£­GFP emitted fluorescence intensity above 3 000 at the concentration of 0.7 mg/mL£¬and the DiG8£­GFP showed better performance£®Indirect ELISA showed that the trimeric recombinant protein TriG8£­GFP could bound to AFB1£­BSA£®However£¬the IC50 cannot be calculated using the TriG8£­GFP as its fluorescence intensity was only 1 700£®This study lays a foundation for the subsequent establishment of immunological detection methods based on fluorescence signals£¬and also provides a reference for the optimization of affinity activity of nanobodies in immunological detection£®