Construction Of LTBI-RD Multi-epitope Molecule HP16118P And Expression Of Its Fusion Protein In Vitro

Objective Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis.The most common one is pulmonary tuberculosis,which can invade multiple organs.Studies have found that about 90% of the world’s 1/3 infected population have Latent tuberculosis infection(LTBI).Due to the lack of effective differential diagnosis methods,the screening,prevention,and treatment of LTBI face significant challenges.In this study,the LTBI-RD associated antigens with potential advantages in the differential diagnosis of tuberculosis were screened from the Mycobacterium tuberculosis latent period antigen and the Mycobacterium tuberculosis Region of difference(RD)target antigen.The dominant Helper T lymphocyte(HTL),Cytotoxic T lymphocyte(CTL),and B cell epitopes of LTBI-RD related antigens were predicted and screened.LTBI-RD multi-epitope molecule HP16118 P was constructed.Bioinformatics and immunoinformatics technology analyzed the physicochemical properties,spatial structure,and simulation reaction of HP16118 P.The epitope molecule HP16118 P was further purified and expressed.ELISPOT and high-throughput liquid phase protein analysis were used to verify the immunological characteristics of the epitope molecules.This study provides a foundation for delivering new candidate target molecules for the diagnosis and differential diagnosis of LTBI.Methods 1.Construction of the LTBI-RD epitope molecule(1)LTBI-RD related antigen screening In this study,from 21 LTBI-RD associated antigens screened previously,15 antigens with LTBI differentiation potential or inducing significant differences in IFN-γ+T lymphocytes were selected for HTL,CTL,and B cell epitope prediction and screening.(2)HTL epitope prediction and screening of antigens HTL epitopes were screened and predicted by Allele Frequency Net Database.Vaxi Jen2.0 was used to predict and select the antigenicity of HTL epitopes.IFN epitope server Aller,TOP2.0,and Aller FP1.0 were used to predict and screen the non-allergenicity of HTL epitopes.(3)CTL epitope prediction and screening CTL epitopes were screened and predicted by Allele Frequency Net Database.The immunogenicity of CTL epitopes was expected using the IEDB database.Vaxi Jen 2.0 was used to predict the antigenicity of epitopes.Non-sensitization of epitopes was predicted using Aller TOP2.0 and Aller FP1.0.(4)Prediction and screening of B cell epitopes ABC pred Prediction Server and IEDB B Cell Epitope Prediction were selected to predict linear B cell epitopes.(5)Construction of LTBI-RD epitope diagnostic molecule Sixteen HTL cell epitopes,11 CTL cell epitopes,and 8 B cell epitopes were selected.GPGPG,AAY,and KK were used as the linkers of HTL,CTL,and B cell epitopes,PSMα4 as the adjuvant,EAAAK as the adjuvant linker,HBD-3 and PADRE as the adjuvant peptides.All epitopes were connected to construct LTBI-RD multi-epitope diagnostic molecules.(6)Prediction of physicochemical properties and immunological properties of LTBI-RD multi-epitope molecules The physicochemical properties of the epitope molecules were predicted using Expasy Prot Param.IEDB Immunogenicity was used to predict the immunogenicity of epitope molecules.Vaxi Jen v2.0 and ANTIGENpro were used to predict antigenicity,Aller TOP v.2.0 and Allergen FP v.1.0 were used to predict sensitization,and Toxin Pred was used to predict toxicity.(7)Spatial structure prediction and immune response simulation of LTBI-RD multi-epitope molecules PSIPRED was used to predict the secondary structure of the epitope molecules,and I-TASSER was used to predict the epitope molecules’ three-dimensional(3D)spatial structure.C-immsim was used to predict the ability of epitope molecules to induce immune cells to produce specific antibodies and various cytokines.(8)Expression and purification of LTBI-RD epitope diagnostic molecule HP16118 P in vitro The fragment between Bam HI and Xho I recognition sites of the p ET-28a(+)vector was replaced with the HP16118 P nucleotide sequence to obtain the recombinant expression vector p ET-28a(+)-HP16118 P.Recombinant vector p ET-28a(+)-HP16118 P was introduced into Escherichia coli BL21(DE3)receptive cells to obtain recombinant bacterium BL21/p ET-28a(+)-HP16118 P.The strains were added to Luria-Bertani(LB)solid plate(containing kanamycin 100μg/m L)at 37℃ and cultured overnight at 220 rpm.The third passage was induced with IPTG for expression overnight,and the supernatant was collected after the fragmentation of the cells.The protein was purified by three Ni column affinity chromatography,and the protein quality was detected by polyacrylamide gel electrophoresis.2.In vitro experiments to verify the immune characteristics of HP16118P(1)The number of IFN-γ+T lymphocytes induced by HP16118 P was analyzed by ELISPOT assay Twenty-three Healthy controls(HC),24 LTBI patients,and 19 ATB patients were included in the study.Peripheral blood mononuclear cells(PBMC)were isolated from 5ml EDTA anticoagulated blood.PBMC were stimulated with phosphate buffer solution(PBS),fusion protein CE,and HP16118 P,and the number of IFN-γ+T lymphocytes induced by HP16118 P was detected by ELISPOT kit.(2)The levels of cytokines induced by HP16118 P were analyzed by high-throughput liquid phase protein analysis Seven HC,eight LTBI,and seven ATB patients were included in the experiment.Peripheral blood mononuclear cells(PBMC)were extracted from 10 ml EDTA anticoagulated blood and stimulated with PBS,CE,and HP16118 P.After 48 hours,the cell culture supernatant was collected,and the levels of 35 inflammatory cytokines induced by HP16118 P were analyzed by high-throughput liquid phase protein analysis.Results 1.15 antigens were screened: The 15 antigens with the most potential for differential diagnosis of LTBI-RD were screened out,including Rv1736 c,Rv1737c,Rv2626 c,Rv2656c,Rv2659 c,Rv1511,Rv1980 c,Rv1981c,Rv3873,Rv3878,Rv3879 c and Rv3 425,Rv1978,Rv2031 c,and Rv3429.2.LTBI-RD multi-epitope molecule HP16118 P nomenclature: 16 dominant HTL cell epitopes,11 CTL cell epitopes,and 8 B cell epitopes were predicted and screened.LTBI-RD was obtained by linking all the epitopes with the epitope linkers GPGPG,AAY,and KK,adjuvant PSMα4,adjuvant linkers EAAAK,and peptide PADRE and HBD-3.It was designated HP16118 P.3.The results of bioinformatics and immunoinformatic analysis: HP16118 P model was generally feasible: it predicted the physical and chemical properties,immune properties,and spatial structure of epitope molecules and simulated immune response.The results showed that HP16118 P was a hydrophilic and relatively stable essential protein with a molecular weight of 90265.44 Da.HP16118P has good antigenicity and immunogenicity,no toxicity and no sensitization,and has the potential to induce immune responses.HP16118 P contains 41% α-helix,7% β-fold,and 50% random coil.Immune response simulations showed that HP16118 P successfully stimulated the immune system,which induced an increase in activated B lymphocytes with high levels of Ig G and Ig M antibodies,an increase in the number of activated cytotoxic T cells that peaked on day 50 after stimulation and also induced high levels of IFN-γ and IL-2.4.Purified expression epitope molecule HP16118P: the target gene of HP16118 P was inserted into the p ET28a(+)plasmid vector for cloning,the recombinant protein was expressed and purified,and the quality of purified protein was detected by polyacrylamide gel electrophoresis.5.In vitro experiments were performed to verify the immune characteristics of HP16118P(1)ELISPOT assay showed HP16118 P could induce IFN-γ+T lymphocytes to rise,indicating that HP16118 P had immunogenicity(2)The high throughput liquid phase protein analysis showed that HP16118 P induced PBMC cells to secrete different cytokines.Among them,the concentration of cytokines more than 10000pg/ml was TIMP-1.GM-CSF,IL-6,IL-8,MCP-1,MIP-1β,and TNF-α were higher than 1000pg/ml.The levels of IL-1α,IL-10,IL-23,TIM-3,and VEGF-A were higher than 100pg/ml.IFN-α,IL-12p70,IL-17 F,and IL-31 were found to be the most common cytokines with a concentration of less than 10pg/ml.These data again confirmed the strong immunogenicity of HP16118 P.Further analysis of the differences in HP16118P-induced cytokines among the three groups showed that Cytokines induced by HP16118 P included IL-1α(P=0.0020),IL-1β(P=0.0106),IL-17F(P=0.0076),IL-2(P=0.0004),IL-5(P=0.0009),MIG(P=0.0151)and HGF(P=0.0065)The levels of TNF-α in LTBI group were significantly lower than those in HC group(P=0.0117).The levels of IL-17F(P=0.0171)and TIM-3(P=0.0224)induced by HP16118 P in the ATB group were significantly lower than those in the HC group.The level of IL-5 induced by HP16118P(P=0.0372)in the LTBI group was significantly lower than in the ATB group.Conclusions Bioinformatics and immunoinformatics analysis results showed that the multi-epitope molecule HP16118 P constructed by the dominant epitopes of LTBI-RD related antigen was a relatively stable basic protein molecule with strong antigenicity and immunogenicity,which could induce the proliferation of B and T lymphocytes and produce high levels of antibodies and cytokines.In vitro ELISPOT and high-throughput protein analysis experiments showed that HP16118 P was immunogenic and could induce IFN-γ+T lymphocytes and various cytokines.This study laid a theoretical foundation for screening potential candidate target molecules for the diagnosis and differential diagnosis of LTBI.