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Molecular Cloning and Sequencing of Heavy and Light Chain cDNAs from Papaya ringspot and Cymbidium mosaic viruses-Specific Monoclonal Antibodies

木瓜輪點與喜姆蘭嵌紋病毒單元抗體基因選殖與核苷酸序列譯讀

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摘要


木瓜輪點病毒(Papaya ringspot virus, PRSV)和喜姆比蘭崁紋病毒(Cymbidium mosaic virus, CymMV)鞍蛋白單元抗體,分別為9G2D3具IgG1重鍵和kappa輕鍵之單元抗體。由產生單元抗體之融合瘤細胞萃取獲得抗體基因之訊息核酸(mRNA),並建立兩病毒抗體基因之互補去氧核醣核酸(cDNAs)。分別選殖對抗體基因重鍵和kappa輕鍵固定區(constnat region)核苷酸序列具專一性之寡核探針,成功選殖到木瓜輪點病毒全長度之鞘蛋白單元抗體重鍵(PRSV-H 10-9, 1571 bp; accession number: AY571285)和輕鍵(PRSV-L 3-8, 966 bp; accession: AY571286),喜姆比蘭崁紋病毒全長度之鞘蛋白單元抗體重鍵(CymMV-H 10-1, 1545 bp; accession number: AY571287)和輕鍵(CymMV-L 23, 948 bp; accession number: AY571284)。PRSV-H 10-9全長度核苷酸序列包含一個34 bp(1-34)之5’端非轉譯區,一個1380 bp(35-1414)之開放轉譯架構(open reading frame)轉譯成459個胺質酸序列,其中包含57 bp(35-91)轉譯成19個前導胺基酸序列(leader peptide sequence),348 bp(92-439)變異區(variable region)序列和975 bp(4-720)之開放轉譯架構轉譯成238個胺基酸序列,其中包含60 bp(4-63)轉譯成20個前導胺基酸序列,333 bp(64-396)變異區序列和324 bp(397-720)固定區序列,一個208 bp(721-928)3’端非轉譯區和一個poly A尾區序列(929-966)。CymMV-H 10-1全長度核苷酸序列包含一個37 bp(1-37)之5’端非轉譯區,一個1386 bp(38-1423)之開放轉譯架構轉譯成461個胺基酸序列,其中包含57 bp(38-94)轉譯成19個前導胺基酸序列,339 bp(95-433)變異區序列和990 bp(434-1423)固定區序列,一個102 bp(1424-1525)3’端非轉譯區和一個poly A尾區序列(1526-1545)。CymMV-L 23全長度核苷酸序列包含一個2 bp(1-2)之5’端非轉譯區,一個720 bp(3-722)之開放轉譯成239個胺基酸序列,其中包含60 bp(3-62)轉譯成20個前導胺基酸序列,336 bp(63-398)變異區前導序列和324 bp(399-722)固定區序列,一個208 bp(723-930)3’端非轉譯區和一個poly A尾區序列(931-948)。本試驗結果可提供進一步合成重鍵和輕鍵變異區單鍵抗體分子,進行轉殖植物之育成,達到防治木瓜輪點病毒和喜姆比蘭嵌紋病毒之目的。

並列摘要


Two cDNA libraries were constructed from mRNAs isolated hybridoma cell lines, 9G2D3 and 11A12F5, producing monoclonal antibodies specific to the coat proteins of Papaya ringspot virus (PRSV) and Cymbidium mosaic virus (CymMV), respectively. 9G2D3 hybridoma cell was determined to be a secretor of the IgG1 heavy chain and kappa light chain class. A full-length heavy and a light chain cDNAs (1572 and 966 bp), PRSV-H 10-9 and PRSV-L 3-8, were selected by hybridization with specific oligonucleotide probes, respectively. The sequence of PRSV-H 10-9 cDNA clone (accession number: AY571285) contained a 5’ untranslated region (34 bp, 1-34), an open reading frame (1380 bp, 35-1414) including a 19 amino acid leader peptide sequence (57 bp, 35-91), a variable region (348 bp, 92-439) and a constant region (957 bp, 440-1414). The sequence of the 3’ end of this clone contained the untranslated region (100 bp, 1415-1514) with a prototype sequence of AATAAA (1482-1487) at position 27 nucleotides before the poly A region, and a poly A region (1515-1571). The sequence of PRSV-L 3-8 cDNA clone (accession number: AY571286) contained a 5’ untranslated region (3 bp, 1-3), and open reading frame (717 bp, 4-720) including a 20 amino acid leader peptide sequence (60 bp, 4-63), a variable region (333 bp, 64-396) and a constant region (324 bp, 397-720). The sequence of the 3’ end of this clone contained the untranslated region (208 bp, 721-928) with a prototype sequence of AATAAA (906-911) at position 17 nucleotides before the poly A region, and a Poly A region (929-966). 11A12F5 hybridoma cell was determined to be a secretor of the IgG3 heavy chain and kappa light chain class. A full-length heavy and a light chain cDNAs (1545 and 948 bp), CymMV-H 10-1 and CymMV-L23, were selected by hybridization with specific oligonucleotide probes, respectively. The sequence of CymMV-H 10-1 cDNA clone (accession number: AY571287) contained a 5’ untranslated region (37 bp, 1-37), an open reading frame (1386 bp, 38-1423) including a 19 amino acid leader peptide sequence (57 bp, 38-94), a variable region (339 bp, 95-433) and a constant region (990 bp, 434-1423). The sequence of the 3’ end of this clone contained the untranslated region (2bp, 1-2), an open reading frame (720 bp, 3-722) including a 20 amio acid leader peptide sequence (60 bp, 3-62), a variable region (336 bp, 63-398) and a constant region (324 bp, 399-722). The sequence of the 3’ end of this clone contained the untranslated region (208 bp, 723-930) with a prototype sequence of AATAAA (908-913) at position 17 nucleotides before the poly A region, and a poly A region (931-948). The results in this study bring us closer to the use of transgenic plants expressing a functional single-chain variable fragement antibody specific to the viral coat proteins for controlling PRSV and CymMV.

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