| Objective The effect of Shigella casein hydrolytic protease B(Clp B)on Rotavirus(RV)replication was clarified,which laid the foundation for further research on the molecular mechanism of Shigella Clp B protein affecting RV replication,and provided a reference for revealing the interaction between intestinal bacteria and enterovirus.Methods 1.Identification of Shigella superalbumin components: The frozen Shigella solution was taken out and evenly coated in the Salmonella Shigella medium(SS medium),and cultured at 37℃ for 12 h.Single colonies on SS medium were selected and added into the culture medium of Shigella augmenting broth-neomycin.Shigella amplification was cultured in a shaking bed at 37℃ at 200 rpm for 12 h.The supernatant of Shigella was obtained by centrifugation at 8,000 rpm at 4℃ for 30 min.The supernatant was filtered by ultrafiltration tube,and the protein components of the supernatant were identified by mass spectrometry.2.Expression and purification of ClpB protein: DNA of Shiga Bacillus was extracted and used as a template to amplify Clp B prokaryotic gene fragments,which were connected to the prokaryotic expression vector p ET-24 a.The constructed recombinant plasmid was transformed into BL21(DE3)receptor cells to induce expression.The recombinant prokaryotic expression plasmid Clp B-p ET-24 a was used to explore the optimal expression conditions of Clp B protein from three aspects of induction time,temperature and IPTG concentration.Then,a large number of Clp B proteins were induced,and high purity and concentration of Clp B protein were obtained by His label affinity chromatography.3.Construction and verification of Clp B eukaryotic expression vector: DNA of Shiga Bacillus was used as a template to amplify eukaryotic gene fragments and connect them to the eukaryotic expression vector Pc DNA3.1-myc.The constructed recombinant plasmid was transformed into DH5α receptive cells.After amplification,the recombinant eukaryotic expression plasmid Cl PbPc DNa3.1-My C was extracted and transfected into 293 T cells.The expression of Clp B protein was detected by Western Blot.4.Preparation and verification of Clp B polyclonal antibody: C57BL/6 mice were immunized by fully mixing purified Clp B protein with adjuvant in equal proportions,and the mice were immunized with normal saline as the control under the same conditions.The immunization was strengthened every two weeks,and the antisera of mice were collected at the 7th week for verification of titer,sensitivity and specificity.(1)Titer verification: enzyme-linked immunosorbent assay(ELISA)was used to detect the coating of 5 μg/m L,0.5μg/m L,0.05μg/m L,and 0.005μg/m L Clp B proteins with different dilution gradients,It can identify the highest dilution multiple of Clp B protein in Clp B polyclonal antibody serum with different dilution gradients of 1:1,000,1:2,000,1:4,000,1:8,000,1:16,000,1:32,000,1:64,000 1:128,000.(2)Sensitivity verification: Western Blot assay can detect the lowest concentrations of Clp B at 1:3,000 dilution of 8μg/m L,4μg/m L,2μg/m L,1μg/m L,0.5μg/m L,0.25μg/m L,and when the concentration of Clp B is 1μg/m L.The highest dilution ratio of Clp B polyclonal antibody was 1:1,000,1:2,000,1:3,000,1:4,000,1:5,000,1:6,000.(3)Specificity verification: Western Blot was used to detect the specificity of Clp B polyclonal antibody at 1:3,000 dilution,and Shigella superalbumin Dank and Gro EL were used as controls.5.Effects of Clp B protein on RV replication in vitro: Cell count Kit 8(CCK8)was used to detect the cytotoxicity of Clp B protein at concentrations of 100μg/m L,75μg/m L,50μg/m L,25μg/m L,10μg/m L,1μg/m L and 0.1μg/m L.Then the concentration of Clp B protein added to the cells was determined.ClpB protein with the RV function according to the following four different processing methods(1)Caco-2 cells were incubated with different concentrations of Clp B protein(100μg/m L,10μg/m L,1μg/m L,0.1μg/m L)for 2h at 37℃.RV with MOI=0.2 was used to infect the cells and maintained for 12 h.Clp B protein was not added as control group.(2)Different concentrations of ClpB protein(100μg/m L,10μg/m L,1μg/m L,0.1μg/m L)were co-incubated with RV with MOI=0.2 at 37℃ for 2h in vitro.Caco-2 cells infected with the protein treated virus were maintained for 12 h,with no Clp B protein added as the control group.(3)Caco-2 cells were infected with RV with MOI=0.2,and Clp B protein of different concentrations(100μg/m L,10μg/m L,1μg/m L,0.1μg/m L)was added for 2h.After washing,the cells were maintained for 12 h.The control group was treated without Clp B protein.(4)The recombinant eukaryotic expression plasmid ClpB-PcDNA3.1-myc optimization transfection Caco-2 cells after 48 h,adopt the MOI=0.2 the RV infected cells,maintain training 12 h cells.The empty eukaryotic expression plasmid Pc DNA3.1-myc was used as control group.The above four groups of cells with fluorescent quantitative real-time reverse transcription polymerase chain reaction(RT-q PCR)detection of RV gene copy number variations,immunofluorescence(IFA)detection of RV oven drop degree change,Western Blot detection of RV protein expression relative to the change.Results1.Identification of superalbumin components of Shiga Bacillus: A total of 103 kinds of proteins in the superalbumin of Shiga Bacillus were identified by mass spectrometry analysis,and the top 10 proteins were obtained according to the protein identification scores,and Clp B protein was one of them.2.Expression and purification of Clp B protein: successfully constructed recombinant prokaryotic expression plasmid Clp B-p ET-24 a,Clp B protein expression of the optimal conditions for 12 h,28℃,the tendency for 0.6 mmol/L IPTG.The purity and concentration of Clp B protein were obtained by His label affinity chromatography,and the concentration of Clp B protein was 2.03mg/m L.3.Construction and validation of Clp B eukaryotic expression vector: successfully constructed recombinant eukaryotic expression vector Clp BPcDNA3.1-myc,Western Blot detection to the size of a 95 kda protein ClpB.4.Preparation and validation of ClpB polyclonal antibody: successful polyclonal antibody in serum of the mice.The titer of polyclonal antibody can reach 1:64,000 when the concentration of Clp B recombinant protein is 0.05μg/m L.The lowest Clp B recognition concentration of polyclonal antibody was 0.5μg/m L in 1:3,000 dilution.At the Clp B of 1μg/m L,at least 1:6,000 antibodies can be identified.The polyclonal antibody can specifically recognize the recombinant eukaryotic expression vector Clp B protein,and does not cross react with other Shigella superalbumin proteins Dank protein and Gro EL protein.5.Effect of Clp B protein on RV replication in vitro: CCK8 showed that Clp B protein had no obvious toxic effect on Caco-2 cells,and the working concentrations of Clp B protein in subsequent experiments were determined to be 100μg/m L,10μg/m L,1μg/m L and 0.1μg/m L,respectively.When 100μg/m L ClpB protein was incubated with Caco-2 cells for 2h at 37℃,RV infected cells and RV replication were significantly different from the control group.The results of viral copy number,viral titer and viral protein detection showed that the RV gene copy number was 4.36x109 copies/m L higher than that of the control group.RV titer higher than 1x103FFU/m L;The relative expression level of RV protein was 0.444 higher than that of the control group,with statistical significance(P≤0.05).ClpB when different concentration of protein with the RV 37℃ in vitro incubation optimization infection Caco-2 cells after 2h,the RV replication and the control group there were significant differences.(1)Viruses copy number test results shows that: Caco-2 cells were infected with Clp B protein of 100μg/m L,10μg/m L,1μg/m L and 0.1μg/m L after coincubation with RV at 37℃ in vitro for 2h.RV copy numbers in each group were 2.61x1010 copies/m L,2.24x1010 copies/m L,1.67x1010 copies/m L,1.41x1010 copies/m L,respectively.The RV copy numbers were 1.92 x1010 copies/m L,1.59 x1010 copies/m L,9.89x109 copies/m L and 7.29 x109copies/m L higher than those of the control group at 6.81x109 copies/m L.All of them were statistically significant compared with the control group(P≤0.05).Meanwhile,with the increase of Clp B protein concentration,RV gene copy number increased,and there was a positive correlation between them(P≤0.05).(2)Virus titer test results showed that: Caco-2 cells were infected with Clp B protein of 100μg/m L,10μg/m L,1μg/m L and 0.1μg/m L after co-incubation with RV at 37℃ in vitro for 2h.Virus titers in each group were 4.083x104 FFU/m L,2.250x104 FFU/m L,3.167x103 FFU/m L,2.583x103 FFU/m L,The RV titers of the control group were 3.925 x 104 FFU/m L,2.109 x 104 FFU/m L,1.584x103FFU/m L and 1.010x103FFU/m L higher than those of the control group.In addition,100μg/m L and 10μg/m L Clp B protein increased the virus titer significantly compared with the control group(P≤0.05).(3)Virus protein expression test results showed that: Caco-2 cells were infected with 100μg/m L,10μg/m L,1μg/m L,0.1μg/m L Clp B protein after incubation with RV at 37℃ in vitro for 2h.The relative expression levels of RVVP6 protein in each group(compared with β-actin)were 1.463,0.797,0.734,0.645.The relative expression of RV protein was 1.054,0.388,0.325 and 0.236 higher than that of control group 0.409,respectively.All of them were statistically significant compared with the control group(P≤0.05).With the increase of Clp B concentration,the relative expression of RV-VP6 protein increased,and there was a positive correlation between them(P≤0.05).When host cells overexpressed ClpB protein,RV infected Caco-2 cells,RV replication was significantly different from control group.The results of viral copy number,viral titer and viral protein detection showed that the RV gene copy number was 4.48x1010 FFU/m L higher than that of the control group.RV titer 2.083x103FFU/m L higher;The relative expression level of RV protein was 0.353 higher than that of control group,with statistical significance(P≤0.05).Conclusions1.ClpB prokaryotic recombinant plasmid and eukaryotic recombinant plasmid can be constructed to obtain Clp B protein with high purity and concentration.2.Clp B polyclonal antibody can be prepared,and the antibody has good titer,sensitivity and specificity.3.Confirmed that 100μg/m L Clp B protein was incubated with host cells for 2h at 37℃,RV infected cells and effectively promoted RV replication in vitro.4.Confirmed that Clp B protein can effectively promote RV replication in vitro after co-incubation with RV at 37℃ for 2h before infecting host cells,and the intensity of Clp B protein promoting RV replication is positively correlated with the concentration of Clp B protein.5.Confirmed that Clp B protein was highly expressed in host cells,which could effectively promote RV replication in vitro. |
