Prokaryotic Expression And Activity Analysis Of Anti-IL-6R Nanobody

Objective: Anti-IL-6R nanobody VHH-0031 was expressed in recombinant engineering bacterium E.coli BL21(DE3)-pET28a-(VHH-0031)by IPTG induction.VHH-0031 was extracted and purified from recombinant engineering bacterium.The bioactivity of the target protein was analyzed.Methods: The recombinant plasmid pET28a-(VHH-0031)was previously constructed in our lab and following verified by sequencing.Then it was transformed into E.coli BL21(DE3)cells for protein overexpression.The expression of target proteins was induced by optimized temperature and IPTG,and analyzed by SDS-PAGE and Western-blot.His-tag magnet beads were used to separate and purify the target proteins which was washed and solubilized with optimized conditions,and the concentration of the purified proteins was analyzed by BCA method.The affinity and stability of the expression proteins was determined by ELISA method,and bioactivity was further investigated both in vitro and in vivo.LPS-activated RAW264.7 cell was selected to evaluate anti-inflammatory effects of VHH-0031.The RAW264.7 cell viability was evaluated by MTT assay,while the change of nitric oxide(NO)and inflammatory cytokines in culture supernatant were measured using Nitrate reductase assay and ELISA respectively.Adjuvant-induced arthritic(AA)rats were induced by complete Freund’s adjuvant(FCA)and divided randomly into certain groups.The apparent indicators including changes of body weights,paw swelling degrees and arthritis indexes,were analyzed to evaluate anti-arthritic effect of VHH-0031,while HE staining was adopted to observe pathologic morphology of the joint tissues.The levels of IL-6,IL-1β and TNF-α in serum and joint tissue were measured by ELISA and the status of related-proteins in tissues was examined by Western-blot and immunohistochemistry.Results: The expression of VHH-0031 nanobody was detected in the form of inclusion bodies.After washing,solubilizing and purifying processes,the purity of proteins can achieve over 90%.The expressed protein displayed high-affinity with IL-6R by ELISA,and exhibited great stability at high temperature and a broad range of p H.VHH-0031 reduced NO and inflammatory mediators’ accumulation in the culture medium of LPS-induced RAW264.7 though displaying no cytotoxic effect.VHH-0031 obviously suppressed the severity of AA rats by attenuating the indicators as mentioned above,and the levels of IL-6,IL-1β and TNF-α in serum and joint tissues were significantly decreased.Histopathological examinations indicated that VHH-0031 improved pathologic morphology of AA rats.Moreover,the level of JNK detected by Western blot and immunohistochemically in related tissues was down-regulated obviously.Conclusions: The nanobody VHH-0031 was highly expressed in E.coli BL21(DE3)-pET28a-(VHH-0031)in the form of inclusion bodies.After washing,dissolving and purifying,the nanobody VHH-0031 showed high purity over 90% and good thermal and p H stability.It also exhibited high affinity and biological activities in RAW264.7 cell and AA rats through the anti-inflammatory function assay.