| Tuberculosis(TB)is the tenth leading cause of death worldwide.Globally in 2017,the number of TB deaths was about 1.57 million,of which was about37,000 in China.China is one of the 30 high TB burden countries in the world.In 2017,China had about 890,000 incident cases of TB which ranked second in the world.The current status of TB with high incidence and mortality has seriously increased the overall burden of TB disease control.The situation of TB prevention and control is severe,but the current common methods of TB laboratory testing methods can not meet the needs of clinical diagnosis of TB.Since 1994,when Australian scientists proposed proteome,proteomics research had gone through 25 years.In the 25 years,more than 51,000 proteome research papers had publicated in international journals,not only recorded the continuous development of proteomics technology,but also described the wide application prospects of proteomics technology in the post-genome era.Among the reserch papers,more than 8,000 studies on markers used proteomics technology to detect,analyze and identify the disease marker proteins and target proteins,to elucidate the relationship and regularity between the changes of disease protein expression level and the different stages of disease development,which showed that proteomic technology was currently a advantageous way to address disease markers.Our previous study found the potential markers in serum of TB patients by proteomics techniques,namely sex hormone-binding globulin(SHBG)and serum amyloid A4(SAA4),might provide auxiliary detection methods for clinical diagnosis of TB.However,the imported SHBG and SAA4 test kits are expensive,which limite their promotion and application in clinical detection of TB,especially in primary medical institutions.Therefore,the main purpose of this study was to construct a cost-effective kit for detecting the expression level of SHBG in serum of TB,to explore the value of combined detection of SHBG and SAA4 protein in the diagnosis of TB,and to prepare polyclonal antibodies against SHBG and SAA4.The first part of the research mainly used three kinds of raw material reagents to construct a cost-effective ELISA kit for detecting the expression level of SHBG protein in the serum of TB,and explored the efficiency of combined detection of SHBG and SAA4 protein to diagnose smear-negative pulmonary tuberculosis(SNP-PTB).The second part were to independently express and purify SHBG and SAA4 recombinant proteins by E.coli system,to prepare anti-SHBG and anti-SAA4 polyclonal antibodies to lay a foundation for independent research and development of economical SHBG and SAA4 detection kits,and to strive to provide new auxiliary diagnostic methods for the prevention and treatment.Part 1 Construction and Application of SHBG Detection KitObjective:To detect the levels of SHBG protein in 214 serum samples by two kinds of ELISA kits,and further explore the feasibility of serum SHBG in the diagnosis of pulmonary tuberculosis,and explore the efficiency of combination of SHBG and SAA4 for the diagnosis of smear-negative pulmonary tuberculosis.Methods:1.The serum levels of SHBG and SAA4 protein in 84 cases of smear-negative pulmonary tuberculosis(SNP-PTB),36 cases of smear-positive pulmonary tuberculosis(SPP-PTB),30 cases of multidrug-resistant pulmonary tuberculosis(MDR-PTB)and 64 cases of normal control(NC)groups in a total of 214 clinical serum samples of the four groups were detected by SHBG(A)kit and SAA4(B)kit.2.The optimal reaction conditions of the combined SHBG(C)kit were explored and performances of the kit were evaluated.The SHBG protein levels of the same 214 clinical serum samples were tested by combined SHBG(C)kit.The correlation and difference between the the combined SHBG(C)kit and SHBG(A)kit were analyzed.3.The diagnostic value of SHBG which detected by two kinds of SHBG(A and C)kits in 150 cases of PTB,16 cases of PTB-DM and 84 cases of SNP-PTB were assessed by mapping ROC curves.At the same time,the diagnostic value of SHBG combined with SAA4 in the diagnosis of SNP-PTB was analyzed.4.According to the operation requirements of the ELISA test kit,each serum sample was repeatedly tested twice,so the 96-well ELISA kit could detect44 serum samples and 8 standard samples.The price of each kit was converted to the cost of each serum sample tested,and the detection cost of testing each serum sample was compared.Results:1.Serum SHBG concentrations were detected by SHBG(A)kit:serum levels of SHBG in PTB patients were significantly higher than NC group(P<0.000),which were 46.87(28.24~65.61)nmol/L and 25.06(13.93~33.93)nmol/L,respectively.Compared with the SHBG concentration in NC group[25.06(13.93~33.93)nmol/L],levels of SHBG were significantly elevated in SNP-PTB,SPP-PTB and MDR-PTB groups,which were 51.59(25.02~77.67)nmol/L(P<0.000),47.25(34.22~58.47)nmol/L(P<0.000)and 39.26(26.37~53.37)nmol/L(P=0.002),respectively,but there was no significant difference in the SHBG expression levels in the three groups of PTB(P>0.05).Serum SAA4 concentrations were detected by SAA4(B)kit:serum levels of SAA4 in PTB patients were significantly higher than NC group(P<0.000),which were 47.30(36.96~70.52)ng/mL and 36.22(29.16~41.69)ng/mL,respectively.Compared with NC group[36.22(29.16~41.69)ng/mL],SAA4protein expression level were significantly increased in SNP-PTB[53.61(41.17~74.00)ng/mL](P<0.000),while there were no significant difference in the SAA4 levels between both SPP-PTB and MDR-PTB compared with NC group(P>0.05).2.Optimized conditions of the combined SHBG(C)kit included overnight coated at 4°C,anti-SHBG capture antibody at 6?g/mL,anti-SHBG detection antibody at 1?g/mL,HRP-streptavidin diluted in a ratio of 1:150 followed by incubation for 20 min and incubated the substrate for 15 to 20 minutes.The combined SHBG(C)kit had a detection range of 2.56 nmol/L to 0.04 nmol/L,R~2?0.999,and a sensitivity of 0.0042 nmol/L that was higher than SHBG(A)kit(0.006 nmol/L).The intra-assay CV%was 2.365~5.215%,and the inter-assay CV%was 4.913~10.013%.The combined SHBG(C)kit was used to detect the same 150 cases of PTB and 64 cases of NC group.The results showed that the serum SHBG concentration of PTB patients[60.87(32.40~90.61)nmol/L]was higher than that of NC group[24.14(14.61~47.32)nmol/L](P<0.000).Compared with the SHBG protein concentration in NC group[24.14(14.61~47.32)nmol/L],the levels of SHBG were significantly elevated in SNP-PTB,SPP-PTB and MDR-PTB groups,which were 56.90(31.63~91.42)nmol/L(P<0.000),70.98(46.22~92.47)nmol/L(P<0.000)and 50.21(30.07~67.42)nmol/L(P=0.006),respectively.It could be seen that the trend of detecting SHBG expression levels in PTB and NC groups by using the combined SHBG(C)kit was consistent with the result of SHBG(A)kit,and the correlation coefficient was 0.869(P<0.000).3.Results of clinical application of the combined SHBG(C)kit:(1)The AUC,sensitivity,and specificity of the kit in the diagnosis of 150 cases of PTB were 0.770,0.687 and 0.719,respectively(P<0.000).(2)Compared with the SHBG protein concentration in 64 cases of NC group[24.14(14.61~47.32)nmol/L],the expression levels of serum SHBG protein in 16 cases of PTB-DM group and 134 cases of simple PTB group were 57.17(19.72~106.55)nmol/L(P=0.017)and 60.87(34.44~90.32)nmol/L(P<0.000),respectively.The AUC of the kit in the diagnosis of PTB-DM was 0.698(P=0.015).(3)The AUC,sensitivity,and specificity of combination of SHBG and SAA4 for the diagnosis of SNP-PTB were 0.889,0.829 and 0.812,respectively(P<0.000)and the combination of SHBG and SAA4 had higher diagnostic efficiency for the diagnosis of 84 cases of SNP-PTB than them alone(P<0.01).4.The cost of testing each serum sample using the combined SHBG(C)kit required only 21 RMB,which was only 17%(21/123 RMB)of the cost of the SHBG(A)kit.Conclusion:1.A cost-effective combined SHBG(C)kit was developed,which could be initially applied to the detection of serum SHBG in PTB patients,and had obvious advantages compared with SHBG(A)kit.2.SHBG and SAA4 exhibited a good performance in diagnosis of SNP-PTB,and combined detection was contributed to obviously improving the detection rate of SNP-PTB.Part II Prokaryotic Expression of SHBG and SAA4 and Preparation of Their Polyclonal AntibodiesObjective:To express SHBG and SAA4 recombinant proteins through E.coli expression system,and to prepare high titer anti-SHBG and anti-SAA4polyclonal antibodies.Methods:1.Using HL-7702 human hepatocytes as template,SHBG and SAA4 gene fragments were amplified by RT-PCR to construct pET30a-SHBG and pET30a-SAA4 recombinant expression vectors.After optimized the conditions of IPTG concentrations(0.2 mM,0.4 mM,0.8 mM and 1.0 mM IPTG)and temperatures(18?,28?,37?and 40?),recombinant proteins were induced by a large number of bacteria.2.After determined the optimum imidazole elution concentration of the Ni sepharose affinity chromatography by imidazole gradient elution method,recombinant proteins were purified by Ni sepharose affinity chromatography and SDS-PAGE gel-recovery method using 250 mM KCl negatively stained.3.BALB/c mice were immunized subcutaneously with purified recombinant protein as immunogen,and the obtained anti-SHBG and anti-SAA4polyclonal antibodies were identified by indirect ELISA and Western blot.Results:1.The 25KD recombinant protein of SHBG was efficiently induced under the conditions of 37?and 1.0 mM IPTG.The 19KD recombinant protein of SAA4 was efficiently induced at 40?and 0.2 mM IPTG.2.The optimal imidazole elution concentration of both SHBG and SAA4recombinant proteins was 150 mM by Ni sepharose affinity chromatography.The purified recombinant proteins of SHBG and SAA4 obtained by inducing300 mL bacterial fluid of E.coli were about 2.5 mg and 1.5 mg,respectively,and the purity of SHBG and SAA4 recombinant protein were above 95%.3.The serum titers of anti-SHBG and anti-SAA4 polyclonal antibodies detected by indirect ELISA were 1:243000,and both antibodies showed antigen specificity by Western blot.Conclusion:1.TherecombinantexpressionvectorsofpET30a-SHBGand pET30a-SAA4 were successfully constructed,and the high purity recombinant proteins of SHBG and SAA4 were obtained.2.High titer anti-SHBG and anti-SAA4 polyclonal antibodies were successfully prepared,which laid a good foundation for further independent development of SHBG and SAA4 detection kits. |
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