PURIFICATION AND CHARACTERIZATION OF AN ANTIVIRAL PROTEIN FROM THE SILKWORM ...

78 Pages·2012·1.51 MB·English

PURIFICATION AND CHARACTERIZATION OF
AN ANTIVIRAL PROTEIN FROM THE
SILKWORM Bombyx mori L.

ASHISH MARATHE
PAL 0214

DEPARTMENT OF PLANT BIOTECHNOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
GKVK, BANGALORE-560065
2012
PURIFICATION AND CHARACTERIZATION OF
AN ANTIVIRAL PROTEIN FROM THE
SILKWORM Bombyx mori L.

ASHISH MARATHE
PAL 0214

Thesis Submitted to the
UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE
in partial fulfillment of the requirements
for the award of the degree of
MASTER OF SCIENCE (Agriculture)
in
PLANT BIOCHEMISTRY
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DEPARTMENT OF PLANT BIOTECHNOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
G.K.V.K., BANGALORE-560065
CERTIFICATE
This is to certify that the thesis entitled “Purification and
Characterization of an antiviral protein from the silkworm Bombyx
mori L.” submitted in partial fulfillment of the requirements for the
degree of MASTER OF SCIENCE (Agri.) in PLANT BIOCHEMISTRY of
the University of Agricultural Sciences, GKVK, Bangalore, is a bonafide
record of research work done by Mr. ASHISH MARATHE, ID No. PAL
0214 during the period of his study in this University under my
guidance and supervision and the thesis has not previously formed the
basis for the award of any degree, diploma, associate ship, fellowship or
other similar titles.
Bangalore
July, 2012 Dr. ANITHA PETER
Associate Professor
Department of Plant Biotechnology
APPROVED BY,
CHAIRMAN : _____________________________
(ANITHA PETER)
MEMBERS : 1. _____________________________
(P. H. RAMANJINI GOWDA)
2. _____________________________
(C. K. SURESH)
3. _____________________________
(R. CHANDRU)
ACKNOWLEDGEMENT
I feel extremely honoured for the opportunity bestowed by Almighty upon me to
work under the versatile guidance of Dr. Anitha Peter, Associate Professor, Dept. of
Plant Biotechnology, University of Agricultural Sciences, GKVK, Bangalore-65,
Chairman of the Advisory Committee, for her excellent guidance and encouragement in
completion of this thesis. It is my privilege to record a deep sense of gratitude for the
invaluable guidance, constant inspiration and help, kind and constructive criticism,
unfailing interest, meticulous planning right from suggesting the problem till the
completion of this thesis.
I am very much grateful to my advisory committee members,
Dr. P. H. Ramanjini Gowda Professor, Dept. of Plant Biotechnology, University of
Agricultural Sciences, GKVK, Bangalore, Dr. C. K. Suresh, Professor, Dept. of Plant
Biotechnology, University of Agricultural Sciences, GKVK,, Bangalore-65, and
Mr. R. Chandru, Professor of Biochemistry, Dept. of Plant Biotechnology, University
of Agricultural Sciences, GKVK, Bangalore for their constant supervision, invaluable
guidance and all the facilities extended during the course of this investigation.
I express my sincere thanks to Dr. Ashok T. H. Professor & Head of the Dept.
of Plant Biotechnology for his invaluable administrative support during course of
investigation.
I am extremely grateful to Dr. Nataraj Karaba and Dr. M. S. Sheshshayee for
their willing and ceaseless help during the entire period of investigation. I express deep
sense of gratitude and a sincere feeling of indebtedness to Mrs. K. Shanti,
Mrs. M. Radha, Mrs. Hima Bindu and Mrs. Suchitra Naidu for their kind hospitality
and co-operation during my undergraduate studies.
It seems one uses the choicest words for kind help during the course of
investigation to measure the boundless love and fearless sacrifice of someone, I find no
such measure in adequate quantity to express my heartfelt gratitude to my father
Mr. Ramesh Marathe, my mother Mrs. Rekha Marathe and my beloved granny, Pushpa
Marathe, who always inspired me with love and affection, which enabled me to
withstand all types of stresses and strains to reach this milestone in my life.
This thesis would be incomplete if I do not reckon the sacrifice, love, affection
and support of my brother, Anup and my sister in law, Pooja. Without their efforts,
love and care, it wouldn’t be possible for me to complete my degree programme.
Man needs an offbeat to relieve himself of tension that it was here my friends
came in so handy, who are source of my strength and corners of spirit.I owe thanks from
depth of my heart to my best Friends, Sulochana, Sushil, Anila and Avinash Ambatkar
for their kind and genuine support throughout my academics till date and without
whose help I would not be what I am today.
I also thank Sagar, Sangram, Druvashree, Deepa, Dhananjay, Dalpat, Kheta,
Joy, Nazia, Pramod, and my lovely Juniors, Rini, Shweta, Bharath, Narendra, Ashwin
for their cooperation throughout the study. My special thanks to my extremely talented
seniors Hemlata, Subhada, Prashant, Sandeep, Chayanika, Pavan, Shyam and Eknath,
my friends- Bhagya, Geeta, my labmates- Supreeth, Shyamala, Manjula, Terminal and
Kumar for their valuable and unforgettable help.
I greatly acknowledge the ICAR-JRF, for providing essential fellowship to
carry out my research work.
Last but not the least I thank my rabbits Justin and Dexter without whom my
work would be incomplete
……. Any omission in this acknowledgment does not mean lack of gratitude.

Bangalore
July 2012 (Ashish Marathe)
“Purification and Characterization of an antiviral protein from the
silkworm Bombyx mori L.”
ABSTRACT
Sericulture is an important agrobased industry. India stands
second next to China among the silk producing countries of the world.
Silkworm is susceptible to fungal, bacterial, viral and protozoan diseases.
The Bombyx mori nucleopolyhedrosis virus (BmNPV) is the most harmful
virus in the sericulture industry. The Antiviral Red Fluorescent Protein
from the silkworm was purified from the digestive juice and the excreta of
the silkworm. The red fluorescent protein (RFP) was isolated and purified
by ammonium sulfate saturation, organic solvent precipitation and gel
filtration chromatography on sephadex G-100 column. The fractions that
fluoresce red in U.V were used for further analysis. The protein from the
digestive juice and excreta were subjected to Native and Denaturating
PAGE. The electrophoregram of the purified protein from the digestive in
native PAGE has shown two bands and the SDS – PAGE pattern for the
RFP from the gut fluid revealed the presence of four different bands. The
electrophoregram of the purified protein form the feacal matter in native
PAGE showed a single band which coincided with the first band in the
protein from the gut juice. The SDS-PAGE pattern revealed two bands of
which one was found to be around 28 kDa and the other band was
smaller than 14.4 kDa. Dot Blot was carried out to detect the specificity
between the antigen and the antibody. ELISA was also done to fix the
titre of the antigen and antibody. The titre obtained was 1:25 of antigen
and 1:2000 of primary and secondary antibody dilutions.

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CONTENTS
CHAPTER PARTICULARS PAGE No.
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-14
III MATERIAL AND METHODS 15-28
IV EXPERIMENTAL RESULTS 29-32
V DISCUSSION 33-37
VI SUMMARY 38-39
VII REFERENCES 40-48
LIST OF TABLES
Table Page
Contents
No. No.
Mean O.D values for Standardization of ELISA using the
1 purified RFP from the gut fluid with 1:1000 dilution of 32
secondary antibody
Mean O.D values for Standardization of ELISA using the
2 purified RFP from the gut fluid with 1:2000 dilution of 32
secondary antibody