This paper should be cited as: Hashemi, N. Yazdani, Y.
Designing, Optimization and Construction of Myelin Basic Protein Coding Sequence Binding to the Immunogenic Subunit of Cholera Toxin
Hashemi, N. (BSc)1, Yazdani, Y. (PhD)2
1. MSc Student of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Science, Gorgan, Iran
2. Assistant Professor of Immunology, Infectious Diseases Research
Center and
Laboratory Science Research Center, Golestan University
of Medical Sciences, Gorgan, Iran
Abstract
Background and Objectives:
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease. Mucosal
feeding of myelin basic protein binding to the cholera toxin B subunit can
reduce the intensity of the immune response in MS patients. Expression system,
the domain composition of the fusion protein, accessibility of two domains,
codon adaptation index (CAI) and GC contents are very important for the large
scale production of fusion protein.
Material and Methods: we used DNA2, PSIPRED and ProtParam softwares for designing
the best form to produce fusion protein. Moreover, the correct open reading
frame of myelin basic protein was also considered.First
the coding sequence was verified and then synthesized.For
confirmation of the recombinant vector, PCR test was carried out using T7
primers. Finally it was inserted into the cloning site of pET28 expression
vector.
Results: After coding optimization, the CAI rate was increased
from 64 % to 80% and GC content from 41 % to 49%. The presence of a band near
700bp resulted from PCR amplification test demonstrates the correct cloning of
recombinant vectors in the cloning site of pET28 expression vector.
Conclusion:According to
software and experimental analysis, the designed sequence probably in the best
form could be used for production of recombinant protein.
Keywords: Multiple Sclerosis, Cholera Toxin, Myelin Basic
Protein
Corresponding
Author: Yazdani,
Y.
Email: yaghobyazdani59@yahoo.com
Received: 21 May 2014
Revised: 27 Jun 2014
Accepted: 1 Jul 2014