Study On The Structure And Function Of Linear Epitopes Of PPV1 VP2 B Cells In PCV2 Loops CD And EF

Loop CD and Loop EF are important structures of porcine circovirus 2 capsid protein.PCV2 is a non-encapsulated virus,these loops play a key role in viral invasion,the interactions between virus and the receptor of host cells,and viral internalization.Our previous studies have shown that Loop CD could display PRRSV GP5 epitopes,the insertion of this epitope does not affect the normal assembly of VLPs in vitro,and can generate specific antibodies against both PCV2 Cap and GP5 in mice.Loop EF is the second largest loop structure,which plays an important role in the assembly process of PCV2 Cap.However,little is known about the function of Loop EF.Therefore,based on the previous study in our laboratory,the structure and function of PCV2 loops CD and EF which displayed PPV1 VP2 B cell linear epitopes were studied.?1?In this study,we first constructed a recombinant plasmid?Cap-M?which inserted the VP2 B cell linear epitope B5-E1(²²⁸QQITDA²³³)into Loop CD by using the conventional molecular cloning technique.Subsequently,the mutant protein was expressed in E.coli?BL21/DE3?and could be assembled into cVLPs in vitro confirmed by electron microscopy and IFA.The results of immunoassay showed that cVLPs can induce specific IgG antibodies against both PCV2 Cap and B5-E1 in the mice,which revealed that the Loop CD is not selective for the inserted foreign epitope?PPV1 VP2 and PRRSV GP5 epitopes?,and the inserted foreign epitope is displayed on the surface of the cVLPs.The results indicated that the ⁸⁵GS⁸66 amino acid sequence in the Loop CD is non-essential for PCV2 Cap protein assembly,and has the ability to display different foreign antigen epitopes.?2?We found ¹³⁰VTKATALT¹3737 was displayed on the outer surface of the capsid and highly variable by simulating the 3D mimic structure of PCV2 VLPs,comparing and analyzing the amino acid sequences of PCV1,PCV2 and PCV3.Therefore,we designed two mutants?Cap-M₁ and Cap-M₂?for this region,which were deleted this region?Cap-M₁?and the B5-E1 epitope was replaced by the region?Cap-M₂?,the results showed that both mutant proteins and 6×His tag could be expressed in E.coli?BL21/DE3?,but could not be purified using a nickel column,which indicated that ¹³⁰VTKATALT¹3737 may be an essential amino acid sequence for PCV2 Cap protein assembly.?3?Subsequently,the ¹³⁰VTKATALT¹³⁷ candidate mutation sites which may not participate in Cap protein assembly were screened by bioinformatics prediction and analysis,named the 132th lysine and the 133th alanine.And based on candidate mutation sites,we attempted to replace or insert a foreign epitope on the capsid surface.Firstly,two mutants ?Cap-M₃ and Cap-M₄?were designed for132th Lysine,and the site was mutated to uncharged alanine?A?and GSGS,in addition,another mutant was constructed using Cap-M recombinant plasmid as template,in which 132th Lysine was replaced by B5-E1.Three mutants based on 133th Alanine of Loop EF were designed,B5-E1 was inserted between132th and 133th amino acid,133th and 134th amino acid respectively,and the133th amino acid was replaced by this PPV epitope.All mutants were expressed in E.coli?BL21/DE3?,respectively.The proteins of mutants were purified by Nickel column affinity chromatography,respectively.The assembly of VLPs in vitro and the results of electron microscopy showed that the all mutant proteins could self-assemble into complete VLPs.These results confirmed that the mutation of the 132th and133th amino acid in Loop EF did not affect the assembly of VLPs,and the 132th and133th amino acid sites are non-essential for PCV2 Cap protein assembly,which indicated that it's feasible for PCV2 VLPs to introduce the exogenous epitope of Loop EF alone or with Loop CD simultaneously.The effects of Loops CD and EF mutations on the expression and assembly of PCV2VLPs were identified,which was helpful to reveal their structure and function,and laid the foundation for the study of PCV2 assembly mechanism.The results of this study lay the structural information basis for the molecular design of PCV2 cVLPs with multiple exogenous epitopes,and lay the foundation for the development and application of multivalent vaccines.