Comparison Of Three Prokaryotic Expression Systems And Research On Heat Resisitance Of Nanobody

Nanobody (Nb) is a single-domain antibody that only has variable domain of heavychain of heavy chain antibody (VHH),which was found in camelids and particularcartilaginous fish. Nbs can be easily produced in eukaryotic and prokaryotic expressionsystems by the means of gene engineering technology. Although Nbs showed higher solubilitythan traditional antibodies, there are differences in the spatial structures of Nbs,Theyield,solubility and activity of various Nbs in prokaryotic expression system are quitedifferent.In order to avoid those problems,the fusion tag technology was used for,fusion oneor more tags in the N-terminal or C-terminal of the interest protein. In order to find theoptimal prokaryotic expression system for expression anti-porcine circovirus type II (PCV2)Cap protein Nbs.Two kinds of Nbs of anti-porcine circovirus type II (PCV2) Cap protein havedifferent spatical structures as models, Three prokaryotic expression vectors pMAL-c2x,pE-SUMO, pCOLD I with MBP-,SUMO-and His-tags were used for expressingNbs,respectively.Then comparison of the yields,solubility,antigen-binding activity of thesetwo kinds of nanobodies,The results showed that expression of Nbs in the pE-SUMO vectorwas higher yield,solubility and antigen-binding activity than that in other two vectors.Theantigen-binding activity of natural Nbs was not affected after SUMO-tag was removed fromfusion Nbs.The pE-SUMO vector would be a better prokaryotic expression system for Nbs.The indrect ELISA is the main method for detection of PCV2specific antibodies ininfected animals. However, indrect ELISA could not accurately detect PCV2infection beforePCV2antibodies are produced in animals.Therefore, establishment of a double antibodysandwich ELISA for rapid detection PCV2antigen is urgently necessary. The key to establishthe double antibody sandwich ELISA is to find an antibody with high stability and affinity forantigen binding. Base on this point, pE-SUMO vector was used for expression the six kinds ofCap Nbs which were screened in our laboratory. We found that these recombinant Nbs havehigh heat resistance property after different temperature treatment,Those Nbs with highstability and affinity would provide important tools for PCV2detection using double antibodysandwich ELISA.The contents and results are as follows:1. Construction of Nbs in three prokaryotic vectorThe sequences of Nbs against Cap protein of PCV2were determined by yeasttwo-hybird system in our laboratory.The pCOLD-Nbs and pMAL-c2x-Nbs were constructedthrough BamH I and Sal I restriction enzymes sites.The His-SUMO-Nbs fusions wereconstructed through Bsa I and BamH I restriction enzymes sites.2. Expression and comparison of recombinant NbsThe pMAL-c2x-Nbs were transformed into E.coli TB1.The optimized expression conditions was0.3mM IPTG and induction for3h at37℃. The pE-SUMO-Nbs weretransformed into E.coli Rosetta. The optimized condition for expression was0.4mM IPTGand induction for4h at25℃. The pCOLD-Nbs were transformed into E.coli BL21. Theoptimized conditions for expression was0.4mM IPTG and induction for6h at15℃. Theexpression Nbs were analyzed by12%SDS-PAGE. The results showed that MBP-tagexpression was improved and the solubility was enhanced. The fusion proteins were analyzedby BCA assay. The yields of His-SUMO fusion Nbs were highest among these three fusionNbs. Indirect ELISA results showed that all fusion Nbs recognized the specific antigens.However, the affinity of these fusion Nbs was significant difference (P<0.05).His-SUMO-Nbs was the highest comparing to other two fusion Nbs (P<0.05). MBP-Nbsfusions showed the lowest affinitiy of the cognate antigen than that of His-SUMO-Nbs andHis-Nbs. In summary, we demonstrated that SUMO was a relatively better expression tag forNbs expression.3. SUMO tag removal and the antigen-binding activity of natrual NbThe SUMO tag was cleaved out from the recombinant Nbs by SUMO protease1(Ulp1),and natural Nbs were purified by TALON Metal Affinity Resin. The antigen binding activityof the purified natural Nb was analyzed by double antibody sandwich ELISA,His-SUMO-Nb2(Nb2, Nb clone2) was used as a control. The results revealed that thebinding-antigen activity of natural Nb2was similar with the His-SUMO-Nb2fusions proteins.Thus, further demonstrating that His-SUMO tag didn’t affect the affinity of Nbs.4. Heat-resistance of NbsSix His-SUMO-Nbs were successfully expressed. Indirect ELISA was conducted toanalyze the antigen binding acticity of six fusion Nbs under different temperature conditions.The results showed that although six fusion Nbs showed lower binding activity thantemperature untreated Nbs, they still have higher affinity to the antigen after high temperaturetreatment comparing to polyclonal antibody. These results indicated that they are heatresisitant. Using indrect ELISA analysis, we found that the temperature stability of Nb2washigher than other Nbs, and the original antigen-binding activity of Nb2was also good. Hence,the Nb2would be an ideal candidate antibody for dection of PCV2in double antibodysandwich ELISA method.