Guideline for creating Immunotation of Mtb

From DrugPedia: A Wikipedia for Drug discovery

Revision as of 08:41, 5 January 2010

This page provide the instruction to the users participating in Immunotation of M.tb projects. Please read the instruction carefully given in this page.

Purpose

The functional annotation of protein is most important task for future prediction of drug/vaccine candidate. Thus CRDD of OSDD lunched a project named as Immunotation of M.tb means functional annotation of Mycobacterium tuberculosis protein from immunological point of view like prediction of B-cell, T-cell epitopes etc.

Protein Sequence

You may download Mtb sequences from here in FASTA format Bold text==DrugPedia Account Instruction== 1. User must follow the link to create a account on Drugpedia Create account.
2. Login on Drugedia using the user name and password that have been provided to you by click on the login/create account on top right side on ht edrugpedia page or by the foolowing link Login.
3. Now you have succesfully login on Drugpedia. In the search box on left side of page you will search the page that you want to create. If this page is already existing you click on the title and can add more information by clicking edit buttion on the top of page. But if this page is not exist then search result will give no link and your search title will appear in red color, where you can click on create page link to create a new page by the title that you have searched.
. 4. Now you can add information by click on edit buttion about this page title.

Example

Below is an example of added information about a particular record entitled "Immunotation of Rv2753C(dihydrodipicolinate synthase)".

1. Create the page using Immunotation of Rv2753c (dihydrodipicolinate synthase).
2. click on edit button to add information.
3. Add general information about the Rv2753c
4. User have to search the biohealth database to find the information about the Rv2753c like Gene Symbol,Name,Locus Name,Entrez Gene ID and save this information in immunebox.
5. Click on detail link and get information about the PDB id , subcellular localization and essential gene information.
6. Go to Genbank by the link provided in biohealthbase and get Swissprot id from this page, also include it in immunebox of your record.

Blast Search

7. Go to Human Homologue and perform blastp against SwissProt database and select organism as 'Homo sapiens', get the following information for top five hits}
8. Do literature search for its allergencity and then go to Algpred (Allergen prediction server. Here paste the protein sequence in the box and select the sequence format respectively. Then chose 'hybrid approach' for prediction.

Database Search

9. Go to AntigenDB database, paste the Locus ID eg. 'Rv2753c' or gene name eg 'dapA' or SwissProtID eg. P63945 in the keyword Search box and select 'Mycobacterium tuberculosis' in the 'Antigen Source' option and get information like B cell T cell Epitopes and also got the respective literature (From the NCBI related articles find out latest information regarding epitope and immunogenecity if not compiled in these databases). If epitope found, search in the Antibacterial peptide database (AntiBP2) for more antibacterial activity of the peptide.
10. Go to IEDB database , paste Locus name in the 'keyword search' box at the top right of the page, if get some positive results browse the database and get all the B and MHC Class-I and MHC Class-II T cell epitopes and respective literature.

Predict B-cell Epitope by following steps

a) Go to site [1] and paste sequence in raw format, select all methods/properties using 'Ctrl' key then submit. At the result page go to 'Overlap Display' and select peptides/amino acids which are blue/predicted in atleast 2 properties.
b)Similarly go to ABCpred and paste sequence in raw format, the submit and get the top 10 predicted epitopes.
c) Go to site IEDB and paste sequence in raw format, select 'Bepipred Linear Epitope Prediction' (6th) then submit. At the result page copy the predicted peptides from the table.

Prediction of MHC Class-I binder

a)Go to the site nhlapred paste sequence in 'raw format ie only amino acid code and no header', select 'ALL' in Allele selection and put the 2 proteosomes filters 'ON' then submit after which you will get the result page at the bottom of the page click on the 'Sorted Binding Regions' links and now copy the whole page starting from ALLELE Table, paste in word pad or notepad the run the following script and get the epitopes and its frequency, now select all epitopes with atleast 2 allele frequency.
b) Go to the site IEDB paste the sequence in raw format, select All alleles in 'MHC allele' field, select Total score in 'Sort peptides by' field, then submit, copy ~ top 100 hits starting from HLA and paste to the word pad or notepad, then follow the script and follow the same procedure as in nHLApred.

Prediction of MHC Class-II binder

a) Go to the site NetMHCII paste sequence in 'FASTA format', select all alleles using 'Ctrl' button, check sort by affinity, Turn of P1 amino acid preference then submit, select top 10 for each allele, paste in word pad or notepad and run the frequency script.
b) Go to the site Propred paste the sequence in raw format, select All alleles in 'allele' field, then submit after which you will get the result page at the bottom of the page click on the 'Sorted Binding Regions' link and now copy the whole page starting from 'ALLELE' Table, paste in word pad or notepad then run the following script and get the epitopes and its frequency, now select all epitopes with atleast 2 allele frequency