TB vaccine .........................pptx

Vaccine.

1. Presented to : Mam Dr. Aqeela Ashraf Presented by : Hina Gul (002) Muhammad Wasif Khan (005) Muhammad Waseem Sajjad (009) Amina Saleem (014) Muhammad Asif (015) Assignment of Recombinant DNA Technology

2. Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study Article Title

3. ABSTRACT KEYWORDS ABBREVATIONS INTRODUCTION METHODOLOGY RESULTS DISCUSSION CONCLUSION CITATIONS REFERENCES CONTENTS

4. KEYWORDS Tuberculosis Cloning GST-MPT83

5. TB Tuberculosis MDR-TB Multi-drug resistant tuberculosis XDR-TB M. tuberculosis extensively drug resistant BCG Bacillus Calmette-Guerin IPTG Isopropyl β -D-1 thiogalactopyranoside X-GAL 5-Bromo-4-Chloro-3-Indolyltrophoresis G-A Gluthation-agarose SDS-PAGE Sodium dodecyl sulphate polyacryl amide gel electrophoresis ABBREVATIONS

6. Abstract Appearance of Mycobacterium tuberculosis strain leading to drugs resistance created new problem for TB treatment in various parts of the world. WHO declared TB as global emergency. With increase of TB drug resistance, its convinced that more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigen, one of which is MPT83, examined as TB vaccine candidate.

7. MPT83 antigen, which is very immunogenic in lipoprotein micro-bacteria , identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG(Bacillus Calmette-Guerin) vaccine MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. Purpose of this study is amplification of MPT83 antigen cloning, and expression of its antigen in E. coli bacteria . It is expected from the results of the research that raw material to produce TB vaccine as well as high quality antigen can be obtained.

8. Cont … The band of DNA in PCR the product is 660 BP, while the one in pGEMT-Easy-MPT83 recombinant plasmid is 3678 BP. This is expressed in E.coli BL21 strain and produce 48 KDA protein as well as GST-MPT83 fusion protein .

9. Introduction One of the big 3 of main contagious diseases in the world. It causes 2 million deaths per year with 9 new cases per year. In Indonesia, 70% TB occur at productive age, causing the economic and social problems. Mycobacterium tuberculosis multi-drug resistance(MDR) worsen the situation.

10. It is necessary to find an effective and affordable vaccine as well as its quick and appropriate diagnosis method of development. The treatment of TB is getting difficult following the appearance of M. tuberculosis that is resistant to TB drugs. The strain is resistant to two main antibiotics commonly used i.e. isoniazid and rifampicin. Its also found out that TB drug resistant to M. tuberculosis extensively drug resistant, XDR-TB. Increase of TB drug resistance , convinced that more effective vaccine development will stop the epidemic of TB : by decreasing the pain. stop TB reactivation

11. Bacillus Calmette-Guerin(BCG) currently only vaccine available against TB prophylaxis. Its quite safe, contain efficacy that prevent severe TB in children . However in some clinical experiments, BCG efficacy to prevent active TB in adults. lowest protection in highest TB cases countries like India, china and Indonesia. When given to someone with immunological system defiance, lack of BCG can lead infection.

12. More effective vaccine development is needed that provide protection toward active TB in adults. Antigen MPT64 can be isolated from nontoxic MBT, H37Ra. Large quantity of MPT64 was obtained from the expression in E.coli BL21. Purified using NI affinity Column. Resulting pure MPT64 with molecular weight was 23,497 Da. Recent studies showed RV1096 gene from the M. tuberculosis H37Rv can be expressed in both E.coli and M. smegmatis strain.

13. A recent studies shows the HspX / EsxS and MPT83 gene from M. tuberculosis H37Rv strain can be cloned and expressed in E.coli BL21. Research on MPT83 antigen conducted using recombinant DNA technology(rDNA ). Production of antigen carry out by cloning the gene encoding MPT83 recombinant antigen. Gene expression is conducted in vivo in bacteria . Purification has to be done in the last phase top ensure that MPT83 antigen can be used as vaccine candidate. Production of antigen reduce the production cost of TB vaccine.

14. Cont … Reduce rate of morbidity and mortality caused by TB. Present study, antigen MPT83 from M. tuberculosis local strain in Makassar was cloned and expressed in E.coli BL21 might be a vaccine candidate in future clinical studies, especially developing countries.

15. Materials and methods Reagents and chemicals Blood sample PGEX-2TK vector E.coli BL21 components PGEM-T-Easy vector ampicillin, IPTG X-Gal , Phenylbenzosulfonly fluoride, dithiothreitol Sarkosy and lysozyme

16. Materials Source Blood samples as source of local strain of M. tuberculosis were collected from pulmonary tuberculosis patient and Signed written informed consent was obtained, from the Wahidin Sudirohusodo Hospital, Makassar, Indonesia. pGEX-2Tk vector and E.coli BL21 were purchased from the biotech Pharmacia. PGEM-T Esay vector and E.coli JM109 purchased from Promega USA. Glutinous agarose beads were purchased from GE healthcare Hong Kong. Ampicillin, IPTG, X-gel, phenyl- benzosulfonly fluoride, dithiothreitol , sarkosyl and lysozyme were purchased from sigma.

17. 2. Genomic DNA isolation and PCR: Genomic DNA isolated from M. tuberculosis using phenol, chloroform, and iso -amyl alcohol. Amplify the specific gene of our interest antigen MPT83, PCR use following three conditions: 1. Complete denaturation 94 °C for 3 min. 2. Annealing 55 ° C for 30 s. 3. Extension 72 ° C for 1 min followed 25 cycles of amplification .

18. Finally, elongation 72 C for 7 min using forward primer and reverse primer. PCR products were separated and analyzed on 1 % agarose gel Electrophoresis.

19. 3. Cloning of MPT83 gene PCR product antigen MPT83 were eluted from gel . Cloned in PGEM-T- Esay vector( Promega , USA ). Ligated mix transformed into competent E.coli JM109 cell by calcl2 transfection method. Transformant were plated on Luria Broth (LB ). Agar supplemented ampicillin, isopropyl B-D-1-thiogalactopyranoside, IPTG and X-gal. Cell were incubated 37 C for overnight. Blue-white screening colony selection method were performed.

20. BLUE- WHITE Screening

21. BLUE-WHITE Screening

22. Plasmid were isolated from positive clone by alkali-lysis method. 2 ML of over night culture were centrifuged. Cell pellet were resuspended in 200uL ice cold lysis solution 1( 15% glucose, 25m M tris, 10mM EDTA) followed by vortexing . 400 Ul freshly prepared solution of 2 (0.2 NAOH, 1% SDS) and 50ul solution 3 (3M sodium acetate) added in centrifuge for 10,000 rpm for 10 min. 4. Plasmid isolation

23. Equal amount iso -propanol added in supernatant and incubate at room temperature fo 10 min. After centrifugation the pellets were washed with 70% ethanol. Dried and dissolved in 50uL TE buffer for further use.

24. Recombined white colonies were isolated from LB- ampicillin agar plates. Then inoculated in LB ampicillin contain broth and incubate at 37 c overnight. Release of gene product check on 1 % agarose gel. 5. Confirmation of recombinant clone by plasmid isolation and restriction Digestion

25. MPT83 gene of pGEM-T-Easy-Mpt83 recombinant plasmid then sub cloned into pGEX-2TK expression vector. E. coli BL-21 cell were transformed with pGEX-2TK-MPT83. GST-MPT83 Cdna grown in LB medium supplemented with 200 ug /Ml ampicillin. Induction with 50uM IPTG overnight at 20 c, cell collected by centrifugation. 6. Expression and purification GST-MPT83 fusion protein:

26. Suspended in lysis buffer 10 Ml containing 0.1% phenylbenzosulfonly fluoride and lysozyme. 15 min incubation on ice , dithiothreithol and sarkosyl added to 5mM AND 1.5 % final volume . Sample was sonicated for 2 min on ice and water bath sonicator . Sample centrifuged and triton-X 100 was added to supernatant. GST fusion protein (GST-MPT83) were purified with glutathione-agarose beads.

27. 7. Data analysis : Data obtain from two reading of two method were use for comparison study. Data analysis from SDS-PAGE and DNA sequencing were later analyzed using bio-edit application.

28. Results Genomic DNA isolation and quantification: Blood sample incubated in Lowenstein-Jensen (LJ) MEDIUM FOR 2-3 weeks. Small colonies developed in plate. From colony 6 bacteria were transferred into LJ broth and used for DNA genome isolation. M. tuberculosis was cultured in the LJ broth media and genomic DNA was isolated by modified CTAB method .

29. Isolated DNA was electrophorized in 1% agarose gel. Quantity and quality of DNA was analyzed by UV-visible spectrophotometer. Isolated DNA with A260/280 shows highest pure DNA. Optimization of PCR condition and cloning of Mpt83 gene Optimizing PCR product at optimum temperature to get high density DNA amplification result. Primary concentration of 40 μ mol and 58.1°C on annealing phase.

30. After purification , DNA band was ligated into pGEM -T- Esay plasmid vector to produce pGEM -T Easy-mpt83 recombinant vector. Ligation between plasmid and PCR carefully done in uncontaminated condition . Incubated overnight at 4 C. Then transformed into E.coli JM109 competent cell. Finally, growth pattern of recombinant bacteria colony was obtained.

31. Seven white colonies of the recombinant plasmid isolated from the bacteria growing in LB media plate. Picked up and cultured in LB media. After using mini prep purification, plasmid isolation encoding MPT83 antigen from M. Tuberculosis successfully done 6 colonies. Meanwhile 3 rd colony did not grow. Result of recombinant DNA of six colonies tested using gel electrophoresis.

32. PGEM-T Easy-MPT83 recombinant plasmid was successfully constructed. Existence of 3678bp band which result of insertion of MPT83 gene into pgem -T Easy gene. Pgem -T EASY-MPT83 was sequenced. MPT83 gene of PGEX-2TK recombinant plasmid is sub clone into PGEX-2TK expression vector resulting PGEX-2TK-MP83 plasmid. Expression vector was expressed into BL-21 bacteria as GST-Mpt83 fusion protein.

33. Purified using glutation agarose matrix. Result analysis of SDS-PAGE was GST protein band of 26KDa. GST-MPT83 fusion protein molecule was 48 kDa . Size of MPT83 alone 22 kDa . Protein marker . Total protein whole extract from E.coli containing pGEX-2TK MPT83 Cdna with IPTG induction. Total protein rough extract from E.coli containing pGEX-2TK MPT83 Cdna without iptg induction.

34. Discussion The OP of PCR product carried out to get high density DNA amplification. By doing PCR gradient on product result positive control (H37Rv) and consider product concentration. Result gained on 40 umol . Temperature of 58.1 c at annealing phase shows a thick band. Amplify the DNA on positive sample (H37Rv) and clinical sample by using PCR. Then analyze them by using electrophoresis .

35. After electrophoresis, positive control show higher intensity clinical sample (S1 and S2) with 663 bp. This shows higher the cloning phase possibility. During PP the positive control (H37Rv) showed higher intensity than the clinical sample (S1 and S2 ). Final step of ligation B/w pGEM-T Esay plasmid /vector and purified DNA product. Incubated overnight at 4 c. Ready to be transformed into E. coli JM109 competent cells. Transformation on the competent cells done by creating lysis on the cell wall of E. coli JM109. Result of recombinant plasmid ligation could be inserted into the bacteria cell without killing it.

36. Next, recombinant bacteria bred on the selective breeding media, i.e. solid LB medium in order to: 1. Multiply recombinant bacteria. 2. To learn weather the recombinant bacteria had been inserted by pGEM-T-Easy-MPT83 recombinant plasmid or not . IPTG and X -Gal are added to the agar LB media to obtain blue and white colonies. Bacteria colony inserted by recombinant plasmid would show white color. While the one without recombinant plasmid would show blue color. Recombinant plasmid isolated from white colony.

37. Mini-prep kit purification is used to obtain pure recombinant plasmid. A pure DNA needed. By adding IPTG and X-Gal, pGEM-T-Easy-MPT83 recombinant plasmid is found in five white colonies, that were chosen from breeding of recombinant bacteria. Five colonies bred in a liquid LB media. After isolation using Miniprep kit purification. A product of plasmid isolation encoding MPT83 antigen from M. Tuberculosis obtained . Further tested using agarose gel on electrophoresis.

38. Results gain in PCR cloning and subsequent PCR show the length of the band is 3678 bp. With this length, cloning of pGEM-T-Easy-MPT83 recombinant plasmid genes considered successfully carried out. Having 3015 bp pGEM-T plasmid size and 663 MPT83 antigen size. Concluded that pGEM-T-Easy plasmid and MPT83 antigen were already fused. Product of recombinant plasmid was ready for sequencing . Sequencing process of pGEM-T-Easy-MPT83 plasmid was carry out using applied ABI PRISM 310 Bio-system. Data analysis done using bio edit v.7.0.9.

39. Nucleotide order data gained were compiled and translated into ordered amino acid i.e. 219 amino acid starting. 660 bp including ATG start codon and TAA stop codon. 220 amino acid for M. Tuberculosis H37Rv wild type. After pGEM-T-Easy-MPT83 plasmid had been cloned, protein expressed to form fusion protein. i.e. GST-MPT83 fused protein as vaccine candidate of tuberculosis.

40. Conclusion: Objective of this study was to clone a TB vaccine by using PCR . Name of the vaccine is MPT83 ANTIGEN(NOVEL ANTIGEN). Its size is 660 bp. Vector used for cloning is PGEM-T Easy vector. Its size with inserted gene to be cloned is 3678bp.

41. Resulting Recombinant plasmid is called as pGEM-T Easy-Mpt83. Recombinant plasmid is cloned in Expression system of E.COLI–BL 21. It produces the fusion protein GST-MPT83 of 48kDA (48000 grams per mole ) size . GST-MPT83 is the Tb vaccine candidate that will protect people against Tb at young age.

42. Flow sheet of conclusion STEP 1 STEP 2 STEP 3 STEP 4 STEP 5 CLONE TB VACCINE (MPT 83/660 bp )BY PCR CLONE DONE BY VECTOR (PGEM-t) 3678 bp RECOMBINANT PLASMID ( PGEMt - MPT 83 ) CLONED IN EXPRESSION SYSTEM OF E.COLI–BL 21 . PRODUCTION OF (GST-MPT 83) PROTEIN

43. PRODUCT OF ARTICLE CONCLUSION STEP 6 GST-MPT83 is the Tb vaccine candidate that will protect people against Tb at young age

44. CITATIONS Cloning and expression of recombinant protein CFP21 from  Mycobacterium tuberculosis  as a tuberculosis vaccine candidate Cloning and characterization of Rv1980c gene encoding MPT64 Protein from  Mycobacterium tuberculosis  as a new candidate vaccine of tuberculosis

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