| RNA virus is difficult to operate than DNA virus.Its study must use reverse genetics technology to build its infectious clone. With the various types of research methods and experimental techniques of continuously updated, reverse genetics technology are advancing with the times, some new model of rescure systems have been established.The limitation of traditional prokaryotic promoter system is needing to generate RNA polymerase by other tedious steps,this increase the difficulty of the experimental operation, and some means to generate polymerase, such as the use of auxiliary virus may be interference experiment and even lead to cytopathic effect on cells (CPE) and this will cause the defeat of virus rescure at last.However,Eukaryotic cells can synthesis its own RNA polymerase, these RNA polymerase is a eukaryotic cell's endogenous protein, so the staring of eukaryotic expression promoter is very convenient with no needs of auxiliary means (such as the above helper virus, helper plasmid,Construction of cell lines, etc.)and it has fundamentally put an end to the generation of CPE.Addition, some eukaryotic RNA polymerase located in the nucleus, some others located in the cytoplasm,so the rescure of various types of virus are possible.At present, the virus rescure using eukaryotic RNA polymerase system has become increasingly obvious advantages.Coxsackievirus B3 (CVB3) belongs to the genus of human enteroviruses in the Picornaviridae family. Due to it is closely related to poliovirus (PV), it shares a high degree of structural homology. CVB3 has a single positive stranded (+)RNA genome of 7433 nucleotides (nt) which encodes a large single open reading frame (ORF) of 2207 aminoacids (aa) flanked by a 5'non-translated region (5'NTR) and a polyadenylated 3'NTR. The 5'-NTR contains a type I independent ribosomal entrysite (IRES) which enables 5'cap independent translation initiation of the viral polyprotein. The viral RNA genome can be translated immediately by cellular ribosomes to produce viral polypeptides necessary for the next steps of the rapid viral lifecycle.Our Objective is to construct a CMV promoter-based infectious clone of coxsackivirus B3. First, the plasmid pcDNA3.1(+)was used and the downstream sequence of its CMV promoter is replaced with multiple clone site sequence we designed, and then we inserted two element (the core sequence of HdvRz and polyA tail) into its multiple clone site, this lead to plasmid pc-H-A. We transfected pc-H-A into Hela cells to evaluate its availability; Second, Hela cells is infected with wild type CVB3 (nancy), and the viral RNA is extracted from supernatant. Then, the full length CVB3 cDNA was amplified by long RT-PCR and long PCR through condition optimization. At last we insert this full length cDNA into plasmid pc-H-A and the recombinant particles is verified by sequencing.The framework vector pc-H-A was constructed based on CMV promoter and its availability was initially evaluated. A full length pc-CVB3-H-A cDNA clone was obtained with its sequence similarity 98% compared with CVB3 sequence in gene bank,but the efficiency of this system still needs futher study. This work will be of great value for the further study of RNA polymeraseâ…¡driven system. |
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