Construction And Expression Of Recombinant Anti NI-35 Single Chain Antibody

PrefaceCNS myelin and oligodendrocyte membranes contain two minor proteins with strong inhibitory effects on growing neuritis (neurite growth inhibitors NI-35 and NI-250 ). Monoclonal antibody ( anti-NI-35 ) was obtained that neutralize this activity in CNS. We construct and express a recombinant cloning vector bearing the chemically synthesized cDNA of the recombinant anti-NI-35 single-chain antibody.Method35 segments with the length ranging from 40 ~ 50 bp were assembled in only one step by a PCR approach. The entire gene was cloned into pUC18 plasmid . Determined by auto-sequencing ,the target gene was cloned into prokaryotic expression vector pET28a( + ) in fusion form and transformed into E. coli BL21. Then, we used the IPTG to induce the E. coli BL21- pET28a( + ). At last, we used NI-NTA technology to purify the expression product and got the recombinant anti-NI-35 single-chain antibody protein.ResultSequence analysis showed that the splicing order, the direction and the sequence in the gene were almost correct . SDS-PAGE analysis showed that a new protein band with molecule weight of 31kD appeared as the expected size. After the purifing process, we got the anti-NI-35-scfv protein on the purification of 86%.ConclusionThe successful construction and expression of the recombinant vector pET-28 a ( + ) bearing the cDNA might provide material for the further research about the anti-NI-35 single-chain antibody.