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Construction of Report Gene with Untranslation Region Sequences of CSFV and Screening of 3’ UTR Sequences Subclone

Author: DaiChen
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus 3'UTR 5'UTR Reporter gene microRNA Transfection
CLC: S852.651
Type: Master's thesis
Year: 2009
Downloads: 89
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Abstract


microRNA (miRNA) is a small non-coding regulatory molecule RNA, in a different regulatory pathway plays a key role. microRNA by complete or incomplete base pairing with the mRNA target molecules with non-coding region, thereby causing the level of transcription of the target mRNA or translation inhibition of shear. Recent studies have shown that many viruses can encode produce microRNA, while it is also a variety of viral infections play an important regulatory role of miRNA in order to speculate that the pathogenicity of the virus and the virus and host interactions may have some The role, then it is possible for classical swine fever virus (CSFV) infection plays a regulatory role. Through bioinformatics prediction, found in a variety of microRNA CSFV non-coding region may have an effect on the sites. Classical swine fever virus microRNA in order to study the possible role of infection in this study was constructed green fluorescent protein reporter system to take advantage of the green fluorescent protein to screen for microRNA in CSFV 3 'and 5' non-coding region of the action sites; also established expressing the 3 'non-coding region sequence was subcloned expression vectors to find the specific role of microRNA, to study its potential role CSFV infection basis. The results of this study are the following: (a) blood poisoning in CSFV Shimen strain infected cells extracted RNA; design primers by RT-PCR method was successfully amplified in line with the expected size of CSFV 3'UTR and 5'UTR fragment , PCR product was digested recovery, it even into the eukaryotic expression vector pEGFP-C1, by bacteria PCR, restriction enzyme digestion and sequencing correct that successfully constructed fusion of classical swine fever virus strain Shimen 3'UTR and 5'UTR Fragment of green fluorescent protein gene plasmid vector. (2) using lipofectamine method efficiently 3'UTR-pEGFP-C1 and 5'UTR-pEGFP-C1 vector into two recombinant porcine vascular endothelial cells and successfully expressed by the green fluorescence intensity Changes Initial found to CSFV its site of action of the 3 'non-coding region. (3) RNA22 and miRanda1.9 using software such as, respectively, with the known human and mouse microRNA the 3'UTR of classical swine fever virus genome scan was obtained of which may act on the non-coding region of CSFV 28 microRNA, select one of the 11 design and synthesis of its target sites, built into the eukaryotic expression vector pEGFP-C1, direct sequencing of the cloned and expressed the correctness of the carrier.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology > Classical swine fever virus
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