ARCHIT SOOD Dr.

ANEETA MALHOTRA
M.Sc. Bioinformatics ( PROJECT GUIDE)

SUBMITTED TO : PUNJABI UNIVERSITY

ABSTRACT

PROJECT TITLE : Immunocharacterisation and In-silico analysis of Culture Filtrate

Proteins of Mycobacterium tuberculosis with specific reference to Antigen 85 Complex

Presently, our objective was to Immuno-characterise the secretory proteins of

Mycobacterium tuberculosis by SDS-PAGE , Western Blotting followed by

Immunoblotting for 30-32 KDa proteins (Antigen 85 complex) and to perform in-silico

analysis of Antigen 85 complex of culture filtrate and then to co-ordinate the immuno-

reactive characteristics between them.

The isolated secretory proteins of Mycobacterium tuberculosis were concentrated

through milipore filter assembly (0.22um). Proteins were estimated by Lowery’s method

and were fractionated and separated by SDS-PAGE as described by Lamelli (1970)

using 4% stacking gel and 16% resolving gel at 15mA for 30 minutes and then at 30 mA

for 6 hours along with marker. ( Genei PMW-M). Six corresponding bands were

appeared in the region of 14.3 KDa – 97.6 KDa. Then Western Blotting was performed

(400mA) in which separated proteins from SDS-PAGE were transferred to nitrocellulose

membrane sheets ( BIORAD). Then Immunoblotting assay was performed in which

components of Antigen 85 complex were identified using relevant specific antibodies.

By the In-silico analysis ,the amino acid sequences of proteins of Antigen 85 complex

were analysed for their secretory nature by using a computational tool i.e. SRTPred.

Further,the amino acid sequences of relevant proteins were analysed for subcellular

location byusing subcellular localization prediction tools such as TBPred. Proteins were

further analysed for predicting Signal Peptides and T-cell Epitopes by using various

prediction tools such as Sig-Pred, SignalP, CTLPred. Amino acid sequences of

different components of Antigen 85 complex were aligned using sequence alignment

programmes for finding the similarity searches. ( using BLAST, CLUSTALW).

In-silico analysis of the proteins of Antigen 85 complex showed its immunoreactive and

secretory nature as in the immunoblot. Such type of studies in secretory proteins of

Mycobacterium tuberculosis may aid in the better understanding of the organism at the

genome/ proteome level and may also support in the basic understanding for developing

new anti TB drugs/Subunit vaccines.