بررسی کلونینگ و بیان ژن های ‭F ‬و Hویروس سرخک با استفاده از سیستم بیانی باکولوویروس و انجام آزمایش نوترالیزاسیون در کشت سلول به کمک آنتی بادی تهیه شده بر علیه پروتیین های نوترکیب ویروس سرخک

Measles remainss a significant cause of global mortality, killing over 700000 children per year. Despite the availability of safe and effective attenuated vaccines against this infection it couldn t be use in infants under 9 months especially in the presence of maternal antibobies. Measles virus is a member of the family Paramyxoviridae, genus Morbillivirus with a non segmented negative strand RNA. The 16 Kb linear RNA encodes six structural (N,P,L,H,F,M) and two non structural (C,V) proteins. Two glycoproteins of hemagglutinin (H) and fusion (F) are present as envelope spikes. The H is responsible for receptor binding and has a determinant role in tropism and also induction of protective immunity against Measles virus. The F is responsible for the membranes fusion, and have a key role in infection process in the body. It can induce immune system to produce neutralizing antibody against virus. In this regards these two glycoproteins were considered as a target for new generation of protein-based measles vaccine. In this study, the baculovirus insect cell expression system has been used as a means of expressing Measles virus H and F genes. In such a system, the strong, efficient promoter from polyhedrin gene directs transcription of the genes First of all, Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. Then, H and F genes was amplified by specific primers (containing attB sites) during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculoviruses harboring H and F genes were consequently constructed by LR reaction. In the next step, insect cells (Sf9) were infected with recombinant baculoviruses. In order to increase viral titer, recombinant baculoviruses were passaged 4 times in Sf9 cells. Immunofluorescence experiments using rabbit anti goat antibody conjugated with FITC were performed to investigate the recombinant proteins localization. SDS-PAGE and western-blot using goat polyclonal antibody against Measles Virus, confirmed the presence of recombinant H and F proteins.Other assays showed that the expressed recombinant proteins were also biologically active and could neutralized the Measles viruses in cell culture. The titer of neutralization antibodies against recombinant H and F proteins respectively were 1:128 and 1:16 in comparison with <1:2 for pre immunization-control serum and 1:256 for whole particles virus immunizing serum These clones will be further used in development of recombinant vaccine against Measles.

Keywords:

پارامیکسوویریده , آگلوتینین ها , پروتیین ها , باکولوویریده , بیان ژن