結論與展望 - 使用重組蛋白製造對 HPV、IHHNV 和TSV殼蛋白有專一性之單株抗體

目前可利用所設計之引子，放大出所選之目標基因，並可利用基因重組方式，大量表現這些蛋白質並純化之。也可以使用這些純化的蛋白質，

在 BALB/c 老鼠體內，誘導出對重組蛋白具有免疫反應的多株抗體。所發展的單株抗體中，選擇 3-24 這株單株抗體，與未含 HPV 的蝦子檢體 (由 PCR 方式證實未含 HPV) ，進行西方墨點實驗，結果指出，3-24 這株單株抗體，並不會與蝦子檢體產生非特異性反應，僅會針對 HPV 重組蛋白產生反應，3-7 進行相同實驗，也有相似的結果。在 IHHNV 方面，目前已篩到 3-62 細胞株，可單一辨認出 IHHNV 重組蛋白，並且可在 IHHNV 病蝦中，辨認出此一殼蛋白。但也非特異性地辨認到蝦體粗抽液中其它蛋白質，因此該細胞株可能無法單一用於發展檢測試劑，仍需配合其它細胞株以增加專一性。另外，使用所得到之三株不同的抗體，針對不同之重組蛋白進行 ELISA 及西方點墨實驗也初步證實，這些抗體對不同的重組蛋白並不會有非特異性的辨認產生。

目前的結果中，僅使用未含 HPV 的蝦體，之後仍需使用含有 HPV 的蝦體，看能否使用單株抗體，認到 HPV 上的這個抗原。另外，TSV 尚有其它兩個殼蛋白、HPV 仍有一個殼蛋白，後續實驗中，可先利用同樣的方式，發展成單株抗體。再整合這些對蝦病毒殼蛋白具有專一性的抗體，作下列四種應用: (1) 發展出檢測工具； (2) 純化病毒，用以探討致病機制；

(3) 進行中和反應 (neutralization) ，以探討殼蛋白在病毒入侵宿主時，是否具有細胞吸附 (cell attachment) 的功能； (4) 發展口服疫苗。以下將就各應用作一簡略之介紹。

(1) 發展檢測工具，取得對病毒表面殼蛋白具有專一性之單株抗體後，

將應用於 immuno-strips 及 immuno-PCR 等檢測工具的開發。Immuno-strips (附錄七) 利用單株抗體，吸附在膜上當作捕捉抗體 (capture antibody) ，辨

認到病毒表面殼蛋白時，再利用結合金粒子之單株抗體當作二級抗體 (secondary antibody)，因為金粒子聚集時，會呈現紫紅色 (附錄八) ，以此作為判斷之用。Immuno-PCR (附錄九)，則是利用 PCR 的方式放大訊號，

藉此增加靈敏度。原理為，對殼蛋白 (抗原) 具有專一性的抗體作為捕抓抗體，再以 biotinylated anti-IgG antibody 當作二級抗體，透過 strepavidin 這個蛋白質，連結 biotinylated signaling DNA，如此，將抗原上連結上一段 DNA，之後可利用 PCR 將訊號放大。放大的 DNA 可用電泳分析，或是作比色分析。因為透過 PCR 放大訊號的關係，一般只要幾百個分子就可被偵測到。

(2) 純化病毒，病毒顆粒可用以探討致病機制。一般傳統的純化方法，

是利用不同的蔗糖梯度 (sucrose gradient) 或是氯化鍶梯度 (CsCl gradient) ，在超高速離心下，將病毒純化出來。我們利用抗體與抗原高度的專一性，

將抗體與 sepharose 這類載體結合，發展成親和性管柱。(附錄十)

(3) 中和反應實驗，探討病毒殼蛋白在病毒入侵宿主時，是否具有細胞吸附的功能。病毒在入侵宿主時，會先利用表面蛋白與宿主的細胞表面作結合，之後再入侵到細胞內，因此將利用抗體與殼蛋白具有專一性的特性作結合，若影響了病毒的感染能力，可推論該殼蛋白可能扮演了細胞吸附的功能。

(4) 發展口服疫苗。得到重組蛋白的過程中，建構了許多可以表現病毒殼蛋白的大腸桿菌。將這些大腸桿菌在大量表現後，將其吸附在飼料上。

因為這些飼料上含有細菌菌體及病毒殼蛋白，期待這些因子可以在蝦體引發類似 adaptive 的免疫反應，可對細菌或是病毒產生防禦能力。

第五章參考文獻

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附錄三、 IHHNV 選取之基因片段序列，及其蛋白質序列

NC002190, Gene ID: 1457864

2701 acttcctagg actcttccaa gaatacgaaa aggaaatcga cgacatcata gacaaccatg 2761 tgcgccgatt caacaagagc aagcccaagg aaaagatcca ggagggatgc acataatgaa 2821 gacgaagaac acgccgaagg atcaagtgga ccagacccac acagatgtct acaattcaat 2881 actggagact caatacatat tactttccaa acaagaagat acttcgaatt cgacgctgcc 2941 aatgatggaa acttcgacgg aaaaaattta tactgcctcc cactacattg gatgaactta 3001 tatctctatg gtctaaagag cagcgacagt tcagcaacag aaacacaacg atataagatg 3061 gtaaaatcaa tgatgaagac ctacggatgg aaagtacata aagcaggcgt agtgatgcac 3121 tcgatggtac cccttatgaa agacttaaaa gtatcaggag gcacatcatt tgagactctc 3181 acatttacag acaccccata tttagaaata tttaaggata ctactggact acataatcaa 3241 ctatcaacta aggaagccga cgtaacattg gcaaaatgga tacaaaatcc ccaacttgtg 3301 accgtacaat caacagcagc aaactatgaa gacccaatcc aacaatttgg attcatggaa 3361 caaatgcgaa ccggtgacag aaaagcctat acaatccatg gtgacactag aaattggtat 3421 ggcggagaaa taccaacaac cggacccacc ttcatcccaa aatggggtgg tcaattaaaa 3481 tgggacaaac catcccttgg aaacctagtc tacccagcag accaccatac aaacgactgg 3541 caacagatct tcatgagaat gtcaccaatc aaaggaccaa atggagacga acttaaactt 3601 ggctgcagag tacaagccga cttcttccta cacctagaag tacgactccc accacaagga 3661 tgtgtcgcaa gtttggggat gttacaatat cttcacgcac catgtactgg acaacttaac 3721 aaatgttata ttatgcatac taactaaata tattcgatgt gcaatatata cccgattata

Product: 37 kDa coat protein, protein id: NP 039239.1

MCADSTRASPRKRSRRDAHNEDEEHAEGSSGPDPHRCLQFNTGDSIHITF

附錄四、 HPV 選取之基因片段序列，及其蛋白質序列

NC007218, GENE ID: 3562190

181 tctccgattg gtctagcagt gaagttctca gcacgatggc gagcaaaggt gactattatt 241 attttactcc tagagatcta gtgaactttg taaatacctt gctctgtcat aagattaatg 301 ctatagctgt gaaaatgcta accatgaagg aggaggattt cttgagccaa gtctggttgc 361 catacataca aggctttcag acagctcatg acttgggtag agacagtgat aagtgcggtg 421 cggcggggga tgaatttcaa gaggtttttc tggatgaacg taaattcata gatttcatga 481 catctttccg ccgcagtgta tttatcagct atggtgacga gtggagacat agatcatctc 541 actttatggc agatgtattt tggagtgata atctaaggca attttggatg atgatatttg 601 gagagtgttt tcacgtaaat atgtcaccat gtaagagatt gtatgtagac atgttaccat 661 actattattt agcaaagatg actacggaaa acagacatct tatagaatgg gaatacatga 721 atccatgtcc tgccacacat gtgcggcgga ataagatgac tggtatgaac ttttgtagtc 781 agggagtagt tatagacaac gaatatccag acaatcagat gggttgttac aacatagacg 841 aacatccatt accaggaggt attagatgga gtggtaacac agaatatcgt acaggttatg 901 tacacgttaa taaggtaaaa tggttgggag ttacagataa agtcagtgac atggaggaga 961 cttccagtga tgaggaggtg cctagcagtc aggagaagta tatgaagagt aaggagaaga 1021 aggaacagcc aaagacatcg gagaagaaag acgatgagcc agcaaacaag aagaggaagt 1081 tctgcctcac aagcgcagct ctggagaagc agaagttaga actgggaaaa tttttccgga 1141 tggaagagga gccgatcaac atcaaattat acgacttaga ggaagggaag gaacaccacg 1201 tacatgaagc tatacgtatc gatggtacta actctaagtt tgctaagaag aaagacgaac 1261 atggtaacgt tatcgatgat ttcaaggtaa ttgtttgtga tggagagaac aacttgtatg 1321 gtttttttgc taacacacag ttgaacaaat tgtttaacaa atggcacagt acaaagaaat 1381 atagtatgaa gccagaacac aacataagct taaaggtttc acagattcag gaggttagga 1441 atggaaagat gtgtattgta aagatggcca ttaacgatga tgtaaaatgt ttcgccaggt 1501 aatgaagctc aagagagaag ggatactgga tcataaccct cttgttacat tttactctgg

product: non-structural protein 2，protein id="YP 271914.1

MASKGDYYYFTPRDLVNFVNTLLCHKINAIAVKMLTMKEEDFLSQVWL

附錄五、 TSV 選取之基因片段序列，及其蛋白質序列

AY826054

6901 ctttgggcaa agtagacagc cgcgcttgcg tggtgggact taattaatgc ctgctaaccc 6961 agttgaaatt gataattttg atacaacaac cagtggagga ctaattccag gaggtagtgt 7021 tacaaacagt gaaggttcta caatcttgat gaatgatatc ccaatcacta atcagaatgt 7081 agtgctgtct aagaatgtaa cagataacct gtttgaagtc caggaccaag ctctcattga 7141 atctctctct cgcgacgttt tacttcataa cgacagttgg acatctagtg atgatgaaat 7201 tggcacaact atgacgcagg aacagcttgc aacagaattc aatcagccac acttatatga 7261 aatttcccta cctgatgaca ttgtacgtaa atcgctgttt atgtctaata aattagcgaa 7321 tattgcatat atgcgatgtg attacgaggt tactgtacga gtacaagcca cgcccttttt 7381 acaaggagca ttgtggctgt ggaataagat gaatgctaag cagacatcaa ttattcgacg 7441 cactcttaca gaacacctac gctctattac atcatttcct ggcattgaga tgaacttgca 7501 gtctgaagct cgagctatta ctctttcgat tccttatact agtgagttgc aggtttttaa 7561 ccccagaaat gtgaataacc taaattctat tcggctttcc gttctgagtc aattgcaagg 7621 ccctgaagat gtagaatccg catcttattc tatttatggc aggttgaaaa acatcaagct 7681 atatgggcat gcgccatctg tgacatcttc agtatatccg tctactcagt ccggatatga 7741 tgatgattgt cccattgtgc atgcgggaac tgatgaggat tcttctaaac aggggattgt 7801 ctcaagggtt gcagacaccg ttggtgcggt ggcaaatgta gtagatgggg taggagtacc Product: Viral coat protein 2, protein id: AAX35818.1"

ANPVEIDNFDTTTSGGLIPGGSVTNSEGSTILMNDIPITNQNVVLSKNVTD NLFEVQDQALIESLSRDVLLHNDSWTSSDDEIGTTMTQEQLATEFNQPH LYEISLPDDIVRKSLFMSNKLANIAYMRCDYEVTVRVQATPFLQGALWL WNKMNAKQTSIIRRTLTEHLRSITSFPGIEMNLQSEARAITLSIPYTSELQV FNPRNVNNLNSIRLSVLSQLQGPEDVESASYSIYGRLKNIKLYGHAPSVT SSVYPSTQSGYDDDCPIVHAGTDEDSSKQGIV"

附錄六、桿狀病毒表現系統

(pBacPAK8-MTEGFP)

Homologous Recombination (SF9 cell)

Infected Hi5 insect cell

附錄七、immunostrips

金粒子單株抗體二級抗體

(Secondary antibody)

膜捕捉抗體

(Capture antibody)

附錄八、抗原與免疫金膠抗體結合示意圖

捕捉抗體抗原免疫金膠抗體

Y Y

抗原與免疫金膠抗體結合示意圖。捕捉抗體正免疫金膠抗體均為對抗原有專一性的抗體，但認的 epitope 不同，以增加對抗原的專一性(可由排列組合找到最佳化)，因為免疫金膠抗體上帶有金顆粒，當其聚集在一起時，即可呈現出紫紅色，可明顯觀察之。

附錄九、immuno-PCR

在文檔中使用重組蛋白製造對 HPV、IHHNV 和TSV殼蛋白有專一性之單株抗體 (頁 64-79)