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Cloning Sequencing and Functional Identification of Long Terminal Repeat U3 Region from Donkey Leukocyte Attenuated Equine Infectious Anemia Virus Vaccine and Its Parental Virus Strain

Author: YangWei
Tutor: LiuBaoQuan
School: Northeast Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Equine Infectious Anemia Virus Long Terminal Repeat Nucleotide Sequence Transcription Regulation
CLC: S852.65
Type: Master's thesis
Year: 2000
Downloads: 63
Quote: 1
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Abstract


Equine infectious anemia virus (Equine Infectious Anemia Virus, EIAV) is a lentivirus reverse transcriptase member of the genus . The virus is a serious threat to human and animal health, an important pathogen . Especially a human AIDS (AIDS), the disease that is the people scared . This type of virus genome structure , the molecular mechanism of replication , antigenic drift , and the interaction between virus and host , etc. are extremely similar. Of EIAV donkey leukocyte attenuated vaccine successfully developed and applied in the world and created a slow virus disease immunization of precedent. Thus, at the molecular level reveals Chinese EIAV strong and weak regulation of gene expression between strains , antigenic variation , mRNA molecules in vivo shear mode , and other basic rules , no doubt others , including HIV disease and animal lentiviruses immunization with treatment has important theoretical significance and reference value. In this experiment, the transcriptional regulation of ELAV focus , select EIAV strong and weak strain transcriptional regulatory region of the LTR U3 part of the cloning , sequencing and identification of some functions . In EIAV donkey leukocyte attenuated (DLA-EIAV) and its parents donkey virulent (D-A EIAV) as experimental material, the use of RT-PCR method , amplified long terminal repeat (LTR) U3 portion of cDNA, and cloned into the plasmid vector pUC19 in ABI377DNA sequencing automatic sequencing instrument . 3-5 times respectively independent cloning and sequencing , to finalize the nucleotide sequence of U3 . Thereafter, DNase Ⅰ Footprinting and EMSA methods identified EIAV U3 enhancer region of some of the cis- regulatory elements and trans-acting factors . DLA EIAV genome with DA EIAV U3 part by 197 nucleotides, the difference between the two rates of 7.1% , with the U.S. clone 1369 U3 zone differences rates as high as 22 % -26 % . DNase Ⅰ Footprinting and EMSA results show that , DLA EIAV and D-A EIAV U3 portion of cis- regulatory elements are arranged distributed accord EIAV general rule . In the enhancer region at both ends of MDBP and PU. 1, AP-1 is conserved motifs present in the middle area and large variation , the frequency of transcription initiation control CAAT motifs disappeared and there has been a consistent GATA transcription factor binding sequences TGATAA. Experiments confirmed , GATA transcription factor on horse blood mononuclear cells to express , and with the U3 region of the corresponding cis-elements combined. Furthermore attenuated vaccine RNA in -115nt of A → G mutation adds another cis- regulatory element motifs CAACTG, meet consensus transcription factor binding sequences bHLH family . DLA EIAV and D-A EIAV in the negative regulatory region (NRE) nucleotide sequence differences are significant , via DNase Ⅰ Footprinting analysis of the NRE area DLA EIAV protein factor binds protected , while D-AEIAV not. In summary , EIAV attenuated vaccine and donkeys virulent U3 enhancer region varied greatly, and the emergence of some new transcription factor binding sites and CAAT element is missing. These changes imply that the strength as well as with foreign EIAV strains in cell tropism between strains , transcription initiation frequency and virulence gene expression levels may exist a huge difference . This experiment reveals our EIAV Donkey Leukocyte Attenuated Vaccine their parents donkey virulent LTR U3 region nucleotide sequence and carried out functional identification and found the new Equine viral cis- regulatory elements and the unique lentivirus genome structure , rich lentivirus basic theory and applied research .

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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
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