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Cloning and Sequencing of the Infectious Bovine Rhinotracheitis virus (Yunlin strain) Envelope glycoprotein gIII gene
|關鍵字:||Infectious Bovine Rhinotracheitis virus:glycoprotein gIII|
推論雲林株gIII基因位於HindIII所切割的病毒核酸基因體圖譜 11.7 kb
gIII基因之上游區，含有潛在的CAAT box及TATA box，而在開讀框(open
readiing frame)之下游區則具有潛在的poly A訊息序列(AATAAA)。另外
Using the recombinant DNA technique, I have cloned the gene encoding infectious bovine rhinotracheitis virus (Yunlin strain, Taiwan) envelope glycoprotein gIII and sequence it in the ex- periment. First, I used the restriction enzyme HindIII to di- gest viral DNA of Yunlin strain and to clone it with appropriate vector. The goal was going to construct IBRV genomic library and clone envelope glycoprotein gIII gene. The cloning strategy of gIII gene was according to the colinear model of HindIII, EcoRI and BamHI digested genomic electrophoretic map of Yunlin and American Cooper strain. I suppose the gIII gene of Yunlin strain locates between BamHI and EcoRI restriction enzyme site in 11.7 kb HindIII-HindIII fragment that was genomic map of viral DNA by HindIII digestion. Thus, I used HindIII/BamHI to digest recombinant plasmids that contained different viral DNA fragments and inserted the one that contain BamHI site fragment to suitable vector. I also continued analysis of restriction mapping that it contained EcoRI site and similar to gIII gene restriction mapping og Cooper strain. Moreover, I used BamHI/ EcoRI digestion, cloning and sequencing to compare nucleotide sequence with Cooper strain. It was find the cloning strategy worked. I continued completely nucleotide sequencing after de- tailed restriction mapping and subcloning. The result was 2431 bps that contained 72.25% G+C nucleotide. The upstream of gIII gene contained potential CAAT and TATA box. The downstream of open reading frame contained potential poly A signal sequence (AATAAA). The open reading frame has 1563 bps that could trans- late into 521 amino acid, which had 4 potential N-linked glyco- sylation sites. It shared completely homology in the DNA level by comparing DNA sequence of Yunlin and Cooper strain.
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