應用第一型與第二型貓冠狀病毒型別特異性棘蛋白於感染之區分

Abstract

貓冠狀病毒 (Feline coronavirus, FCoV) 是致命性之貓傳染性腹膜炎之病原。FCoV可區分為第一型 (Type I) 及第二型 (Type II)。世界各地FCoV病毒分型調查結果皆以Type I感染為主，然Type II感染卻顯示與疾病較為相關。 冠狀病毒藉由病毒上的棘蛋白 (Spike) 辨識細胞上的特異性receptor進入標的細胞中，此蛋白除了造成病毒與細胞間融合，亦是誘導中和抗體產生的重要醣蛋白。由於棘蛋白在第一型與第二型FCoV中具相當大的差異，因此為目前用以區分此兩型病毒之主要標的。由於台灣不曾有過全省大規模FCoV血清學之調查，第一型與第二型FCoV之盛行率亦仍不明。本研究以桿狀病毒表現系統針對兩型FCoV棘蛋白上差異區間，進行型別特異性重組蛋白之表現，分析其特異性，並將之運用於兩型別病毒感染貓隻血清之檢測。首奶尷R兩型FCoV棘蛋白基因發現其receptor結合位之基因序列區間相似性極低 (19.2％)，將此段約500 bp之基因序列以PCR進行增幅與純化，選殖入桿狀病毒表現載體，製作具表現兩型FCoV部分棘蛋白之重組桿狀病毒，並順利獲得力價達2 × 106 PFU/ ml之高效價重組病毒。以此重組病毒生產兩型FCoV之部分棘蛋白利用相關抗體，辨認出符合預期大小約為23-25 kDa之兩型重組蛋白質。進一步發現兩型重組病毒以MOI 0.1，分別感染Sf9細胞72小時以及96小時後可獲得最高蛋白質的表現量。利用免疫沈澱法確立第一型重組蛋白之特異性。運用兩重組抗原免疫螢光染色分析確定感染型別之貓隻臨床血清，發現各型別之抗血清可辨認其相對應重組蛋白，而隨著血清稀釋可見螢光訊號之遞減，表示所生產之兩型別重組蛋白具有特異性，同時具定量抗體力價之功能。未來可將此兩重組蛋白純化後進一步應用於ELISA檢測的發展，進行台灣兩型貓冠狀病毒盛行率之調查。

Feline infectious peritonitis (FIP), is a fatal disease caused by feline coronavirus (FCoV). According to the serum neutralizing capacity and the antigenicity relation to canine coronavirus, FCoVs can be divided into two serotypes, namely, Type I and Type II. Type I FCoV is predominant in the field. Despite the higher prevalence of Type I FCoV infection, infection of Type II FCoV appears to be highly significantly correlated to FIP. Coronavirus infection is determined by the interaction between the spike protein and its corresponding receptors on the target cells. S gene has been used in the differentiation of different type(s) of FCoV infection due to the low similarity between the two viruses. The seroprevalence of different types of FCoV infection in Taiwan is unknown yet. This study aim to investigate the seroprevalence of different types of FCoV infection in Taiwan through the expression of type specific spike proteins from Type I and II FCoV. Spike gene of two types of FCoV were aligned, and a low similarity region corresponding to the receptor binding domain of spike protein from Type II virus and the related region of Type I virus were selected, amplified and cloned into a baculovirus expression system. Two recombinant viruses with the titer up to 2 × 106 PFU/ml have been obtained. Cells were infected with these recombinant viruses and total protein was harvested, the type-specific recombinant proteins could be recognized by anti-serum with the expected size of 23-25kDa. While Sf9 cells were infected with these two recombinant viruses at a multiplicities of infection 0.1, the highest protein yield can be obtained at 72 hour and 96 hour post infection for type I and II RBD recombinant viruses, respectively. The specificity of type I recombinant protein could be evaluated by co-immunoprecipitation. Sera from cats infected with different types of FCoV could specifically identify corresponding recombinant proteins by immunofluorescence assay. The intensity of fluorescence was declined through serial dilution of anti-serum, which indicates the proteins is type-specific and could be used for the titration of the specific antibody. These type-specific recombinant proteins will be purified and used for the establishment of ELISA for further serological surveillance.