Screening Of Diagnostic Antigen Markers In RD Region Of Mycobacterium Tuberculosis And Construction Of Mutant Library Of BCG

Tuberculosis?TB?is a chronic and consumptive zoonotic disease that is caused by Mycobacterium tuberculosis complex?MTBC?.In 2016,the World Health Organization?WHO?estimated 10.4 million cases and 1.3 million deaths resulting from TB.TB is the leading cause from a single infectious agent.Recently,WHO made “The END TB Strategy” and targeted to end TB till 2035.It is well known that most deaths from TB could be prevented with early diagnosis and appropriate treatment.Therefore,there is an urgent need to develop an efficient diagnosis method for tuberculosis.Indirect ELISA based on serodiagnosis technology is simple and rapid,and it has become a research hotspot in the diagnosis of tuberculosis with the sputum-free samples advocated by WHO.However,there is no successfully commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity,especially for the diagnosis of smear-negative pulmonary tuberculosis?PTB-SN?and extra-pulmonary tuberculosis?EPTB?.One of the potential strategies in developing a new diagnostic method involves the identification of novel antigenic candidates.This study was aimed to clone and express all the 129 ORFs within 16 RDs among the M.bovis,BCG and M.tb.The RD proteins were screened by using clinical samples from PTB and EPTB patients to comprehensively evaluate the ability of RD proteins to react to sera from different groups.We shall hope to screen and identify new diagnostic markers of M.tb in order to provide a new method for the diagnosis of tuberculosis especially for PTB-SN and EPTB.At the same time,a library of BCG mutants was constructed,and biofilm-enhanced mutants were screened from the mutation library.The main results are as follows:?1?The cloning,expression and purification of RD genes129 differential genes among the M.bovis,BCG and M.tb were obtained from relevant literature.The p ET-32 a vector was selected as the fusion vector.After digestion,ligation and sequencing,78 recombinant plasmids were successfully constructed,which involved genes from 15 other RD regions except RD12.After induced by IPTG,68 proteins were highly expressed,and the size of the obtained protein was consistent with the expected by SDS-PAGE.These proteins were divided into eight categories according to their functions.The top five were hypothetical proteins,phage proteins,membrane/transmembrane proteins,secreted proteins and transposase.Finally,the proteins were purified and only seven proteins were soluble,including Rv0222,Rv0311,Rv1766,Rv1767,Rv1976 c,Rv2660c and Rv3120,respectively,the other proteins were insoluble.?2?The preliminary screening of RD proteinsStandard positive sera from PTB and EPTB patients determination: The clinical sera from PTB and EPTB patients with obvious clinical symptoms were collected from Wuhan Medical Treatment Center.IGRA and TB-DOT commercial kit were used to further test the clinical samples.Six positive sera which were determined by these two methods were selected to mix together as a standard positive sera?every three equal amounts of mixing?.Standard negative sera from volunteers determination: PPD test,IGRA and TB-DOT commercial kit were used to further test the clinical samples.Six negative sera which were determined by these three methods were selected to mix together as a standard negative sera?every three equal amounts of mixing?.Sixty-eight proteins were screened by the IELISA with OD630 positive sera / OD630 negative serum?P / N??2 as the preliminary screening standard,and 29 proteins had higher recognition reactions with the standard positive sera.Among them,19 antigens were screened in PTB group,including Rv3872,Rv3874,Rv3875 and Rv1981 c,which located in RD1-RD4,RD6,RD8,RD10-RD11,RD13 and RD15-RD16;14 antigens were screened from EPTB group,including Rv1985 c,Rv1577c,Rv1508 c and Rv1514 c,which located in RD1-RD4?RD6?RD8?RD10?RD13-RD14?RD16.Four proteins out of the selected 29 proteins were shared by PTB group and EPTB group,namely,Rv3872?RD1?,Rv0222?RD4?,Rv0309?RD8?and Rv3403c?RD16?.?3?The confirmation of potential proteinsReceiver Operating Characteristic?ROC?analysis showed that Rv0222?Area Under the Curve?AUC?: 0.8247;95%CI,0.7496-0.8997?and Rv3403c?AUC,0.8223;95%CI,0.7431-0.9015?performed better than ESAT6/CFP10?AUC,0.7468;95%CI,0.6576-0.8360?.Rv0222 and Rv3403 c demonstrated the highest diagnostic ability in PTB-SP group?83.3%/80%,79.1%/80%?,while Rv3403 c demonstrated the highest diagnostic ability in PTB-SN group?sensitivity: 70%,specificity: 80%?.In EPTB diagnosis,Rv0222 exhibited the highest diagnostic value?AUC,0.7706;sensitivity: 70%,specificity: 82.5%?.And the sensitivity and specificity of ESAT6/CFP10 were 60% and 70% with a diagnostic value?AUC,0.7394;95%CI,0.6092-0.8695?.The combination of Rv0222 and Rv3403 c could improve the detection rate in PTN-SP group,and the corresponding sensitivity and specificity were 86.6% and 72.5%.?4?Immunological evaluation of Rv0222 and Rv3403cMice were injected subcutaneously with each purified proteins Rv0222 and Rv3403 c respectively.The total Ig G titer of anti-Rv0222 in the sera of mice after the third immunization was 2¹¹ × 100,and the titers of Ig G1 and Ig G2 a were 2¹² × 100 and 2¹⁰ × 100 respectively.The total Ig G titer of anti-Rv3403 c was 2¹² × 100,and the titers of Ig G1 and Ig G2 a subtypes were 2¹² × 100 and 2¹⁰ × 100 respectively.Antibody titers were not detected in the PBS immunized group.Stimulation index of protein immune group and PBS immune group are different?p<0.05?.The secretion of IFN-? and TNF-? by mouse spleen cells stimulated by endotoxin-free proteins were detected by sandwich ELISA.IFN-? and TNF-? production in response to stimulation of Rv0222 and Rv3403 c in the immunized mice was significantly higher than that in negative control mice?p<0.01 or p<0.001?.?5?Construction of Biofilm-associated Mutant Library of BCGThe library containing more than 3170 BCG random mutants were obtained using the Myco Mar T7 transposon system.1000 of these mutants were identified by PCR amplification,and 960 mutants were correct.The transduction efficiency was estimated to be 96%.The 960 mutants successfully identified by PCR were transferred to Sauton liquid medium and cultured to screen biofilm-forming ability,of which 148 strains of biofilm formation ability were enhanced compared to BCG.In summary,this study confirmed that Rv0222 from RD4 and Rv3403 c from RD16 region have potential ability to serve as serological diagnostic markers,especially for EPTB and PTB-SN diagnosis.Moreover,these two antigens can induce increased Th1/Th2 type immune response in mice,characterized by an elevated concentrations of IFN-? and TNF-? and high total Ig G,Ig G1 and Ig G2 a antibody titers.The related results of the unknown functional protein Rv3403 c were reported for the first time.In addition,a library of BCG-associated mutants was constructed to provide the basis for the study of the correlation between mycobacterial biofilms and vaccines.