九孔鮑疱疹病毒ORF44部分重組蛋白之表現與應用

Abstract

2003 年冬天，台灣東北角養殖九孔鮑（Holiotis diversicolor supertexta）因鮑魚疱疹病毒（Abalone herpesvirus, AbHV）的感染引起大量死亡。組織病理學顯示，AbHV 主要感染神經系統，造成神經元的壞死並引發神經節神經炎。本研究的目的是表現九孔鮑魚疱疹病毒膜蛋白ORF44部分重組蛋白及製備其多株抗體，並應用於免疫組織化學染色作為診斷工具。以發病之九孔鮑組織萃取DNA，進行聚合酶鍊反應(Polymerase chain reaction, PCR)選殖膜蛋白基因並將此DNA片段經限制酶修剪後，構築外膜蛋白原核表現質體 pET 24a(+) 載體，轉形入大腸桿菌（BL21（DE3））誘導表現具有110個胺基酸之重組外膜蛋白，以IPTG誘導表現的重組蛋白質可溶於水，可以重組蛋白質C端6X-His tag之特性與Ni2+-NTA金屬離子親和性管柱進行層析純化。所得純化之重組蛋白質在SDS-PAGE蛋白質電泳分析下為13 kDa。進一步將此純化的重組蛋白質混合佐劑後，接種於兔子製備多株抗體。此多株抗體可以西方墨點轉漬法上辨認ORF44蛋白質以及在免疫組織化學染色上辨認感染鮑魚疱疹病毒之九孔神經節病變。

Abalone herpesvirus (AbHV) infection of cultured abalone Haliotis diversicolor supertexta has induced high mortality rates in Taiwan in 2003. The histopathologic study has revealed AbHV is prone to infect neurotic tissues and has caused ganglioneuritis in moribund abalone. The purpose of this study is to express the outer membrane recombinant protein fragment ORF44 of AbHV and use this protein as an antigen to prepare the polyclone antibody, then apply in the immunohistochemistry study as diagnostic tool. We have designed a specific primers and amplified this ORF44 fragment by PCR. The amplifed DNA fragment was cleaved by restriction enzymes and inserted into pET24a (+) vector. The ORF44 expression plasmid was transformed into E. coli （BL21（DE3）） to construct the recombinant ORF44 protein with 110 amino acid residues. The IPTG-induced soluble recombinant protein was purified by a nickel-nitrilotriacetic acid (Ni2+_NTA) agarose metal ion column affinity chromatography via 6X-histidines on its C-terminal. By SDS-PAGE analysis, the molecular mass of the purified recombinant protein was about 13 kDa. The polyclonal anti-ORF44 antiserum was prepared from rabbit immunized with the adjuvant-emulsified recombinant ORF44 protein. By using the Western blotting hybridization and immunohistochemistry, the antiserum was proved to recognize the abalone hepesvirus ORF44 proteins and the neurologic lesion of AbHV-infected abalone.