Cloning and Sequencing of the Infectious Bovine Rhinotracheitis virus (Yunlin strain) Envelope glycoprotein gIII gene

本實驗利用基因重組技術，選殖出牛傳染性鼻氣管炎病毒(台灣雲林株)封 套醣蛋白gIII基因並加以定序分析。首先，將雲林株病毒核酸使用內限制 酵素HindIII切割，再以適當載體進行選殖，以建造基因庫並行封套醣蛋 白gIII基因之選殖。gIII基因之選殖策略乃應用雲林株與美國Cooper株基 因體以HindIII、EcoRI及BamHI切割之電泳圖譜呈現共線性關係之模式， 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 的HindIII-HindIII片段上，且位於該片段之BamHI及EcoRI切割位間。 因 此，使用HindIII及BamHI切割含有不同病毒核酸片段之重組質體，將具有 BamHI切割位者以適當載體加以選殖，並進行限制酵素圖譜分析，証實含 有EcoRI切割位；且與Cooper株限制酵素圖譜比對，甚為相符。因此，再 以BamHI及EcoRI切割加以選殖，經核酸定序與Cooper株比對，証實 gIII 基因選殖成功。此重組質體經詳細限制酵素圖譜定位及次選殖後，進行核 酸定序，共定序出2431個鹼基對，含有72.25% 的G+C核甘酸，其中在 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 開讀框共有1563個鹼基對，可轉譯出521個氨基酸，含有四個潛在的N- linked之醣基位，與Cooper株核酸序列比對，結果完全相同。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