Wajihul Hasan Khan1, VL N S. Raghuram2, Shama Parveen1, Shobha Broor2
1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.
2Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
ABSTRACT
Respiratory syncytial virus (RSV) is important viral pathogen of acute respiratory tract infection (ARI) with high occurrence in infants and childhood in both developed and developing countries. RSV has been classified into two antigenic groups (group A and group B) based on antigenic and genetic analysis. RSV G protein is a type II integral membrane protein and shows highest degree of divergence both between and within the two groups. The G protein is glycosylated and is the target for neutralizing and protective immune response and is the vaccine candidate along with F protein. Membrane proteins are often difficult to express in large quantities, which is a significant obstacle for further structural and biophysical investigation. One way to improve the throughput of membrane protein is to optimize the codon sequence so that it reliably facilitates the expression.Two different approaches (full length G protein gene sequence and codon optimized full length G protein gene sequence) were used to clone full length G protein gene of prototype RSV strain (18537) in eukaryotic expression system. The full G protein gene was optimized bycommercially (GeneArt, GmbH, Regensberg, Germany) to increase mRNA stability, alter suboptimal codon usage for mammalian t-RNA bias, remove AT- rich region and to improve secondary mRNA structure.The sequence optimizations were performed with the GeneOptimizer software suite, which was developed in-house by the GeneArt Corporation. Full length G protein gene of RSV was cloned into pcDNA3.1 eukaryotic expression vector with C terminal his-tag. The codon optimized sequence of G protein gene of prototype RSV was synthesized and was inserted in to the PUC57 vector by GeneArt. The optimized sequence was digested and sub-cloned in to the pcDNA 3.1. Both the pcDNA 3.1 vectors with wild type RSV G protein gene sequence as well as the vector with codon optimized G protein gene sequence were transfected in HEK-293T cells and expression was assayed by indirect immunofluorescence assay using anti- RSV G protein antibody and anti-His antibody. The codon optimized G protein gene was expressed at a significantly higher level as compared to the wild type sequence. This is the first report of cloning and expression of full length G protein gene of group B RSV in mammalian system. Further the expressed protein will be purified and characterized by biophysical methods. Cloning, expression and characterization of G protein gene of RSV will have implications for vaccine development and implementation. Read more…
