| Objective:At present,the methods used for tuberculosis screening are mainly based on the skin test of tuberculin purified protein derivative(PPD),but its specificity and sensitivity need to be improved.The Region of Difference(RD)is a genome-wide comparison of the different strains found in the Mycobacterium tuberculosis complex missing fragments in the genome.Due to the universality of BCG vaccination,using the protein antigen encoded by the RD region that BCG does not have can significantly improve the specificity of the skin test.The study expressed 12 kinds of MTB genomic proteins which can stimulate T cell immune response,including ESAT6-CFP10,Rv0222,Rv1985c,MPT64,Rv3117,Rv3872,Rv1738,Rv3425,Rv2626c,Rv3619c,Rv3120 and Rv2346c,mainly in RD region.Compared with recombinant tuberculosis fusion protein(EC)and traditional PPD used in clinical practice,those proteins with strong T cell response were further screened for better delayed-type hypersensitivity(DTH)at the cell and animal level.At the same time,the DTH cell types caused by EC and PPD were identified.This study hopes to use the above-mentioned research to guide how to improve the diagnosis of latent tuberculosis infection(LTBI).Methods:1.Twelve proteins including EC,Rv0222,Rv1985c,MPT64,Rv3117,Rv3872,Rv1738,Rv3425,Rv2626c,Rv3619c,Rv3120 and Rv2346c were expressed in E.coli,laying a certain foundation for subsequent cell and animal experiments.2.To determine the optimal does of skin reagent for the antigen used in the guinea pig skin test,the MTB standard strain(H37Rv,ATCC27294)was used to sensitize the guinea pigs.After successful sensitization,the guinea pigs were injected with phytohemagglutinin(PHA-L),TB-PPD,EC and 11 kinds of MTB antigens(including recombinant antigens Rv3872,MPT64,Rv1985c,Rv0222,Rv3117,Rv3120,Rv2346c,Rv3619c,Rv3425,Rv1738 and Rv2626c),each 0.1 ml.The skin reaction was recorded at 24 h and 48 h after the injection,and the average diameter of the induration[(transverse diameter+longitudinal diameter)/2]was calculated.The average diameter of the induration≥ 5 mm was defined positive.The difference in skin responses of 11 MTB antigens compared with TB-PPD and EC.3.To determine the effect of the dominant antigen in stimulating the proliferation of lymphocytes,the MTB standard strain(H37Rv,ATCC27294)was used to sensitize mice,and a single spleen lymphocyte was obtained.Different antigens were added to stimulate the proliferation of a certain number of spleen lymphocytes.The absorbance of OD450 was measured after 72 h.Add 10μl of CCK-8 reagent to each well 4 h before the measurement;at the same time,the cell proliferation effect was observed under the microscope after 72 h.4.In order to determine the indurated cell groups in the skin test of TB-PPD and EC,the sensitized guinea pigs by MTB H37Rv were injected with 0.1ml each of TBPPD and EC on the back and cut them out at 24 hours.Two methods,flow cytometry and single-cell sequencing,were used to determine the cell group.Results:1.The prokaryotic expression plasmids of the above antigens were constructed,and IPTG was further used to induce large amounts of protein expression.It was found that Rv3117,Rv3 872,Rv 173 8 and Rv2626c were all expressed in the supernatant,and purified proteins were isolated and purified at a concentration of 2mg/ml,1.1mg/ml,1.5mg/ml and 2mg/ml.EC,MPT64,Rv1985c,Rv0222,Rv3425,Rv3619c,Rv3120 and Rv2346c were all in the precipitation,so the protein was purified after washing,and the inclusion body was dialyzed and renatured to obtain a higher purified protein with a concentration of 1.3mg/ml,0.8mg/ml,1.4mg/ml,1mg/ml,1.8mg/ml,1.1mg/ml and 1.1mg/ml.2.After 24 hours of antigen injection,the positive rates of Rv3120 and Rv3619c were 9/9 and 8/9,respectively at the dose of 5μg;the skin reaction of EC and MPT64 were all positive at a dose of 0.5μg.In the skin reactions,the average diameters of induration(M(Q1,Q3))of TB-PPD(5 μ)and EC(at the dose of 0.5μg),MPT64(at the dose of 0.5μg),Rv3120(at the dose of 5μg)and Rv3619c(at the dose of 5μg)24 h after injection were all significantly bigger than those 48 h after injection(the average diameter of induration 9.00(7.00,11.00)mm vs.6.50(5.50,8.25)mm,13.00(12.75,14.25)mm vs.9.00(8.00,9.75)mm,9.50(8.50,12.50)mm vs.8.50(5.50,9.50)mm,8.50(6.25,9.25)mm vs.5.00(0.00,5.50)mm,6.50(5.50,8.25)mm vs.3.50(1.50,4.75)mm);Z values were-2.4494,-2.677,-2.207,-2.63 and-2.670,respectively;P values were 0.013,0.007,0.027,0.008 and 0.008,respectively).At 24 h of skin reaction,MPT64(at the dose of 0.5μg),Rv3120(at the dose of 5μg)and Rv3619c(at the dose of 5μg)were all similar to that of TB-PPD(at the dose of 5 IU),with no statistical differences(H were-0.515,0.835 and 1.896,respectively and P were 1.000,1.000 and 0.869,respectively);and PHA-L(at the dose of 2μg)mean induration diameter was also not statistically different(H were-1.574,-0.297 and 0.765,respectively;P were 1.000,1.000 and 1.000,respectively).The size of skin reactions between MPT64(at the dose of 0.5μg)and EC(at the dose of 0.5μg)were similar,with no significantly difference(H=2.149,P=0.474).The average diameter of induration of Rv3120(at the dose of 5μg)and Rv3619c(at the dose of 5μg)were both significantly smaller than that of EC(at the dose of 0.5μg)24 h after injection(H were 3.683 and 4.744,respectively;P were 0.003 and 0.000,respectively).3.There were significant statistical differences between the stimulating effects of EC,Rv3120,MPT64,PHA-L and ConA antigens on cells(χ2=17.200,P=0.004).Each antigen could induce stimulating effect on lymphocytes.The cell proliferation effect was significantly greater than that of the negative control,with statistical difference(P<0.05).With ConA as the positive control,each antigen was significantly less than the stimulation of ConA on cells effect(P<0.05).There was no statistical difference between the PHA-L,EC,Rv3120 and MPT64 antigens(P>0.05).Observed under the microscope,there were no obvious proliferating cell clusters in the control group,all of which were single cells.In the positive control with ConA,there were obvious proliferating cell clusters,and the number was large.The number of cells stimulated by PHA-L,EC,Rv3120 and MPT64 was less than that of ConA cells.4.The proportion of leukocytes in CD45 labeled that TB-PPD and EC induration detected by flow cytometry was 53.5%and 44.9%,respectively.After single-cell sequencing,the cell viability of TB-PPD and EC skin induration tissues were 71%and 74%,respectively.After barcode correction in bioinformatics,the ratio of Reads with valid barcodes were both greater than the threshold 75%,and the Q30 of UMI bases were also relatively high,96.1%and 95.6%,respectively.The final number of single cells obtained from EC and TB-PPD sclerosis was 13221 and 12201 after the removal of low quality cells.The results of Singler’s final annotation of EC and TB-PPD in mice showed that TB-PPD was divided into 7 cell types,including fibroblasts(54.88%),dendritic cells(10.21%),T cells(15.67%),macrophages(2.89%),endothelial cells(13.83%),granulocytes(1.48%)and NK cells(1.05%).The final result of EC annotation was 6 cell types,including fibroblasts(63.85%),dendritic cells(2.01%),T cells(5.66%),macrophages(13.07%),endothelial cells(13.57%)and NK cells(1.66%).Among them,the number of T cells and dendritic cells in TB-PPD was significantly more than that in EC.The number of macrophages was significantly lower than that in the EC group,and granulocytes were only found in TB-PPD.Differential genes of EC and TB-PPD were enriched by KEGG,and there were differences in cell signaling pathways mediated by them in the same cell population,among which Il1b,Cd3e,Ccr7,LOC100736180,Cc15,Clqb,etc.were related to the immune regulation function.Conclusion:1.After 11 kinds of MTB antigens were reacted by guinea pig skin test,the skin reaction of MPT64(at the dose of 0.5μg)and Rv3120(at the dose of 5μg)were relatively better.2.EC(50μg/ml),Rv3120(50μg/ml)and MPT64(10μg/ml)can all stimulate lymphocyte proliferation.Proliferating cell clusters were observed under the microscope.3.Single-cell sequencing revealed TB-PPD and EC cell types include fibroblasts,T cells,dendritic cells,endothelial cells,macrophages and NK cells,as well as granulocytes in TB-PPD.There are also certain differences between the characteristic genes of TB-PPD and EC.It shows that there is a certain gap in the delayed type hypersensitivity induced by different skin test agents,which provides a direction for finding new skin test agents. |
PDF Full Text Request