Soluble Expression Of Ricin Toxin B Chain And Its Process Optimization And Purication

Objective:To optimize the expression conditions of Escherichia coli and to express a large amount of soluble expression of recombinant Ricin Toxin B chain protein and improve its purity.The aim is to use it as an antigen to prepare related antibodies in order to provide pre-technical support and theoretical basis for the study of anti-toxin vaccine.Methods:In this study,The gene of RTB was cloned into clone vector pMD18-T by the PCR method.The gene of interest was sequenced and then inserted into expression vector P300-TrxA-Sumo and pGEX-4T-1.The expression vector P300-TrxA-Sumo-RTB and pGEX-4T-1-RTB were constructed and transformed into competent cell E.coli BL21(DE3).The expression conditions were optimized by orthogonal test at different induction times,different IPTG concentrations,and different temperatures.The recombinant protein was expressed in a soluble form and then was purified with Ni-charged chelating sepharose by affinity chromatography method.Finally cut the sumo tag from the recombinant protein,then the target protein was ELISA and Western blot analysis were performed to confirm that the recombinant protein was successfully expressed in E.coli,and its high immunogenicities was detected as well.Level of Nitric Oxide was detected when RTB acted on RAW264.7 cells.Finally,MTT assay was used to detect the survival rate of RTB on mouse macrophage RAW264.7.Results:The recombinant expression vectors P300-Trxa-Sumo-RTB and pGEX-4T-1-RTB were successfully constructed.After the optimization of the induction conditions,a number of soluble expression of recombinant RTB was successfully achieved.The expression level was 11%and 8%respectively.Purified by Ni-NTA affinity chromatography column to obtain a relatively high purity of the target protein.Western blot and ELISA were used to verify that the target protein was immunogenic.Nitric Oxide test results showed that the value of RTB group is higher than the normal cell group.It proved that RTB can promote the release of NO,macrophage activation.RTB can be used as a treatment of macrophage-mediated inflammatory disease method.The MTT assay demonstrated that the purified RTB was not toxic to mouse macrophages RAW264.7.Conclusion:After the optimization of the induction conditions,a number of soluble expression of recombinant RTB was successfully achieved.The protein was biological activity after purification by Ni-NTA and GST affinity chromatography.Also,the recombinant protein was immunogenic and was not toxic.