Expression,Purification And Activity Analysis Of Several Broadly Neutralizing Antibodies To HIV-1

In recent years,several broadly neutralizing antibodies,such as b12,2F5,4E10,2G12,PG9,PG16,VRC01 etc.,which can recognize the conserved epitopes on envelope proteins of HIV,have been found.These broadly neutralizing antibodies can neutralize many stains of HIV-1 in vitro.The findings of these antibodies provide positive evidence of the possibility to design vaccines which can induce neutralizing antibodies.These antibodies are lucubrated to illuminate virus neutralizing epitopes,which can provide theoretical basis for designing epitopes to induce protective antibody reactions.The amino acid sequences of VRC01 and b12 have been published by NCBI,and the gene sequences were designed based on Escherichia coli codon preference.In this study,the primers were synthesized according to the gene sequences.The variable regions of the heavy and light chain of broadly neutralization antibody b12 and VRC01 were amplified by polymerase chain reaction.The two regions were linked together as scFv-VL-linker-VH by over-lap PCR and cloned into a prokaryotic expression vector pET28a to make the construct of pET28a-b12-scFv and pET28a-VRC01-scFv,which was then transformed into Escherichia coli BL21(DE3)cells.Induced by IPTG,the b12-scFv protein expressed was mainly soluble with an insoluble part aggregated in inclusion body.The protein was purified by affinity chromatography using Ni-NTA His Bind Resin,producing a yield of about 2 mg/L purified soluble scFv.By enzyme-linked immuno sorbent assay(ELISA)and flow cytometry(FCM),b12-scFv was shown to give specific and good reactivity to both secreted and membrane anchored HIV-1 gp120,suggesting its potential application in probing the receptor binding site on HIV-1 gp120.However,VRCO1-scFv protein was insoluble and aggregated in inclusion body.After being dissolved by urea,the inclusion body was purified using Ni-NTA His Bind Resin,and VRCO1-scFv protein was eluted between 50 mmol/L and 500 mmol/L imidazole.VRC01-scFv protein eluted by 500 mmol/L imidazole was dialyzed in PBS,and then refolded VRC01-scFv protein was detected in supernatant.Nevertheless,the refolded protein was not sufficiect for subsequent reactivity detection because of low renaturation rate,and the optimal condition of renaturation needs to be determined by further investigations.pET28a-b12-scFv-Linker-4E10-scFv and pET28a-VRC01-scFv-Linker-4E10-scFv were constructed based on pET28a-b12-scFv and pET28a-VRC01-scFv,using the method of restriction enzyme digestion and fragment insertion.Then the plasmids were respectively transformed into Escherichia coli BL21(DE3)cells.Induced by IPTG,the b12-scFv-4E10-scFv and VRC01-scFv-4E10-scFv proteins expressed were insoluble and aggregated in inclusion body.The inclusion body proteins were denaturated by urea and purified using Ni-NTA His Bind Resin,and b12-scFv-4E10-scFv and VRC01-scFv-4E10-scFv proteins were eluted between 50 mmol/L and 500 mmol/L imidazole.The denatured proteins were dialyzed by the decreased concentration of urea,and centrifuged to spin down precipitated proteins.After dialysis,only a small amount of soluble proteins were obtained.The optimal condition of renaturation also needs to be down by further studies.Using genetic engineering technology,single-chain and bispecific antibodies can be prepared in vitro for passive immunity prophylaxis of AIDS.The engineered antibodies can also be used for epitopes probing of antigen in vaccines design to reveal the interactions between neutralizing antibodies and antigen of HIV,which can provide theoretical basis for new generation vaccines design.