Production of transgenic algal strain harboring novel design for human norovirus G.II.4 shell for vaccine and diagnosis improvement

Abstract

In this study, we produced novel design for a chimeric relatively conserved of Norovirus (G II.4) and fusion (F) domain of NDV (FLNS). The recombinant protein is supposed to be safe and economic. It is also proposed to confer broader immune response against HuNoV. We used the synthetic construct in C.reinhardttii transformation, producing transgenic algal strain. Besides other strains, we adapted algal strain; C.reinhardtti CC-3395, to our lab conditions, using home prepared growth media (TAP media), first report in Asia and Africa. Additionally, we devised a simple, rapid, economic, and sensitive method for algal DNA isolation This targets were achieved as following: a) To design the hybrid construct we 1- Retrieved the Egyptian sequences of both HuNoV (G.Ⅱ.4) and NDV 2- Selection of domains to be utilized from both viruses 3- In silico, validation of both domains functionality (Norovirus VP1 antigenic conservancy and NDV fusion antigenicity) 4- Sewing of both domains using flexible linker 5- In silico testing of the whole designed protein safety

b) To produce the algal transgenic strain we 1- Synthesized the designed construct after codon optimization to nuclear expression in C.reinhardttii. 2- Cloned the insert into pChlamy_4 (nuclear algal expression vector), after identity and freedom from other vectors contamination 3- Verification of the whole insert using sequencing 4- Transformation of different C.reinhardttii two strains (CC-3395 and CC-125) by electroporation (using different parameters) 5- Selection of algal strains and testing for transgenesis by PCR using novel devised method for extraction