Expression The Neutralizing Antibodies Of HIV-1in Baculovirus Exrpession System(BES)

Acquired immunodeficiency syndrome（AIDS）is caused by human immunodeficiencyvirus（HIV），which is one of the most serious and dangerous virosis to human health. AIDShas been reported for31years to date, but human still no effective vaccines to control thedisease. Fortunately, scientists have already found several broadly neutralizing monoclonalantibodies (mAbs), such examples as B12,VRC01,2G12,2F5and4E10could be used as oneeffective approach instead of vaccines. Among them, B12and VRC01have been confirmed toinhibit40%and90%pandemic HIV replication, respectively. Alone or joint use thesemonoclonal antibodies, can effectively block the HIV infection. Thus, we use de novo genesynthesis and overlap PCR method to gain the whole gene sequences of neutralizing antibodyB12and VRC01and express in the BES.According to the sequence of VRC01high variant region and our lab published B12sequence, we synthesized the light and heavy chains of B12and VRC01mAb genes, thefragments are710bp、1452bp、709bp and1434bp respectively. Meanwhile the nucleic acidsequences of interest have been codon optimized to permit high expression in insect cells.Then, the fragments of B12and VRC01light and heavy chain were cloned into pTriEx1.1shuttle vector. The result plasmids B12L-pTriEx1.1,B12H-pTriEx1.1,VRC01L-pTriEx1.1andVRC01H-pTriEx1.1were co-transfected with linear ACMNPV Bacmid transferred into sf9cells to generate recombinant ACMNPVs(rACs) rAC-B12L, rAC-B12H, rAC-VRC01L andrAC-VRC01H, respectively. Through infecting sf9cells with above rACs alone, we couldobserve the25kDa light chain and55kDa heavy chain expression by Western blot analysis.Then through infecting sf9cells with rAC-B12L/rAC-B12H (“/” represents infection together)or rAC-VRC01L/rAC-VRC01H, we could observe the160kDa full-length mAb expressionby non-reductive Western blot (no DTT in sample loading buffer) or both25kDa light chainand55kDa heavy chain by reductive Western blot (with DTT in sample loading buffer),respectively. Furthermore, the ELISA results showed that both recombinant antibodies(B12and VRC01) have the binding activity with the HIV gp120.In summary, we firstly cloned and expressed the full-length B12and VRC01mAbs genes with baculovirus-insect cells system. The Western blot and ELISA results confirmedthat full-length double chain antibodies could be right expressed and show specific bindingactivity with gp120. Our throughout mAbs baculovirus-insect cells expression platform willfurther push forward the study of novel HIV diagnosis methods and HIV therapeutic vaccines.