蝦類白點症病毒結構性蛋白質之鑑別及研究

Abstract

白點症病毒 (white spot syndrome virus, WSSV) 純化自人工感染的螯蝦(Procambarus clarkii) 血淋巴 (hemolymph)，以8-18 % 梯度聚丙醯硫胺膠體電泳 (gradient SDS-PAGE) 分離組成病毒顆粒 (virion) 之結構性蛋白質，得到電泳分析圖譜。膠體經Sypro Ruby染色，將可見之蛋白質帶切出，並進行蛋白質還原及烴基化反應，再以胰蛋白酶水解，得到各蛋白質帶之水解胜肽片段，經液相層析串聯式質譜儀 (LC-nanoESI-MS/MS) 進行胺基酸定序。將得到的序列與NCBInr資料庫比對，共辨識出33個WSSV結構性蛋白質基因，其中20個為著者首次發現並發表之。加上6個之前已知，本研究無法辨識的結構性蛋白質，故WSSV至少有39個結構性蛋白質基因，著者據此建立白點症病毒之結構性蛋白質基因組成資料庫。 至於病毒結構及各主要結構性蛋白質所在之部位如今仍有所爭議。本研究接著將純化之病毒先以1% Triton X-100處理，溶解病毒之外套膜 (envelope)，再經氯化銫 (CsCl) 梯度離心，得到純化之核蛋白質鞘 (nucleocapsid)。經過梯度膠體電泳分離及串聯式質譜儀做蛋白質辨識，進而解出5個核蛋白質鞘的結構性蛋白質，包括:VP664, VP160A, VP160B, VP60B, VP51C。其中VP160A及VP160B則是初次被發現之結構性蛋白質，並證實VP664不只是目前已知分子量最大的病毒蛋白質，也是構成核蛋白質鞘的最主要單元。 為了辨識其它結構性蛋白質位於病毒顆粒上的組成部位，將純化之病毒以1% Triton X-100處理後，再經低至高不同濃度的鹽類 (NaCl) 分別處理，發現病毒除了先前已知的部位，即外套膜及核蛋白質鞘外，還有首次被證實的夾層 (tegument) 存在，介於前述二者之間。輔以西方氏點墨法 (Western blot analysis) 可將結構性蛋白質歸屬三類不同之蛋白質，即夾層蛋白質包括目前已知VP26, VP36A, VP39A及VP95，VP19, VP28, VP31, VP36B, VP38A, VP51B及VP53A之外套膜蛋白質，最後為VP664, VP51C, VP60B及VP15之核鞘蛋白質。此外本研究也發現當VP15在高濃度鹽類下，會從核蛋白質鞘被解離而溶解，此時病毒DNA仍未完全與核蛋白質鞘分離。輔以穿透式電子顯微鏡 (TEM) 觀察，胰蛋白酶水解分析 (trypsin sensitivity assay) 及免疫金標定 (immuno-electron microscopy, IEM) 等實驗下，進一步證實WSSV夾層部位之存在及其與核蛋白質鞘的關係。

White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 ＋ 6) WSSV structural protein genes. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. After isolating WSSV nucleocapsids by treatment with Triton X-100 and CsCl isopycnic equilibrium centrifugation, mass spectrometry identified only VP664 and four other minor structural proteins, VP160A, VP160B, VP60B, VP51C. Surprisingly, VP15 was not detected in this fraction. To locate the other structural proteins, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained: envelope and tegument proteins; tegument and nucleocapsid proteins; or nucleocapsid proteins only. From the protein profiles and Western blotting, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins as distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations even though DNA was still present. These results were confirmed by the above identified nucleocapsid component proteins using proteomic methods, by a trypsin sensitivity assay, and by an immunogold assay.