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Construction of an Full-Lengh Genomic cDNA Clone of Hev SAAS-JdY5

Author: WangZuo
Tutor: YangZuo;LiZhen
School: Nanjing Agricultural College
Course: Zoology
Keywords: Hepatitis E virus full-lengh genomic cDNA overlap pcr In-fusion
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 21
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Abstract

Hepatitis E (HE) is a zoonotic disease causing severe health problem. The pathogen of the disease is Hepatitis E virus(HEV) which is a single positive-sense RNA virus and classified into a new genus Hepevirus of the Hepeviridae. The HEV genome is 7.2 kb long and consists of a short 5’nontranslated region, three open reading frames (ORFs), and a short 3’nontranslated region followed by a poly(A) tract.ORF1 encodes a nonstructural protein. ORF2 encodes the capsid protein. The ORF3 gene overlaps with ORF2 and encodes an immunogenic protein with an unknown function.At present there is no effective therapy method for HE, so preventive measures including use of vaccine gets lots of attention. No commercial vaccine was available so far and the candidate vaccine based on the DNA recombinant technology was under study. Construction of HEV infectious cDNA clone and establishment of HEV cell infection model were very helpful tools for the study on the virus as well as vaccine development.In this study we constructed the full-lengh genomic cDNA clone of HEV strain SAAS-JDY5 with different technology of molecular biology. This research was composed of three parts:overlap pcr to make 4 longer fragments from 9 fragments which covered the entire HEV genome, in-fusion of the 4 fragments to make HEV full-length cDNA, sequence correction with In-fusion technique.The research was initiated with overlap PCR of the 9 overlapping cDNA fragments. At last the 9 fragments were ligated into 4 fragments which were about 2kb (1-2474,2094-4664,4340-6645,5186-7232). In the process concentration and ratio of templates as well as anneal temperation were important and needed to adjust to proper values.At beginning of the second part of the research, the two segments in the middle were ligated together to make a larger segment (2094-6645). Then the two fragements were ligated together to make the largest fragments of 1-5318 with in-fusion tenique which is based on the principle of gene homologous recombination. The last step in this stage was to ligate the fragment 5186-7232 to the largest fragment 1-5318 by restriction enzyme Fse I at site 5245 to make the full length genomic cDNA. Sequencing of the full length genomic cDNA showed 94 bp at 5’end were senseless sequence and there were 5 extra nucleotides at the sequence position of 2467. So the last part of the work was to make corrections of the full-lengh genomic cDNA clone. For correction of the full-length cDNA clone, swine HEV RNA was extracted from an infectious stock of swine HEV by use of the Trizol reagent. Promoter of T7 RNA polymerase was introduced when the new 5’fragment was amplified with RT-PCR and Nest-PCR. At last the fragment containing the 94bp nonsense sequence and 5 extra nucleotides was replaced with the new amplified fragment by In-fusion technique. Sequencing of this part confirmed the correctness of this fragment of the genomic cDNA.In the research of viral reverse genetics DNA recombination technique based on restriction enzyme and ligase was most frequently used to construct full-lengh genomic cDNA of the virus. In this research teniques of overlap pcr, ligase and In-fusion was integrated creatively to construct HEV full-lengh genomic cDNA. Advantages and disadvantages of these methods were also compared in the research.

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