傳染性胰臟壞死病毒A段基因產物在昆蟲細胞內的合成與分析

Abstract

PT7是利用加州苜蓿尺蠖核多角體病毒的強勢啟動子－多角體蛋白基因?動子驅動外源基因的桿狀病毒表現載體。pAcMP2是一個與其類似的表現載體，但以鹼性蛋白基因?動子取代其多角體蛋白基因?動子，所以本載體在病毒感染過程中的晚期即驅動外來基因的表現；亦即較屬於最晚期基因的多角體蛋白基因?動子驅動的時間為早；已有證據顯示，以鹼性蛋白基因?動子驅動的外源基因轉譯後修飾較完全。本實驗以這二個桿狀病毒表現載體進行 IPNV A段基因在昆蟲細胞內的表現研究。 結果顯示，以這二個表現載體驅動的IPNV A段基因，均能夠在昆蟲細胞內被表現。經由西方墨點法和蛋白質放射性標定，發現經由鹼性蛋白?動子所驅動的IPNV A段基因在感染後十二個小時即可偵測到產物的生成，而由多角體蛋白?動子所驅動的則遲至二十四小時才可被發現。從西方墨點法得知二者表現出來的IPNV A段基因的產物無極大差異，包括有106K聚合蛋白、pVP2、VP2和VP3等；利用放射性標定分析，得知在標定的時間中，VP2與VP3的合成量與累積量均無法與同時標定的野生型病毒之多角體蛋白相比。在昆蟲細胞中表現的pVP2受到處理加工(process) 的情況不像在TO-2細胞中明顯。此外，就產量而言，VP3和VP2間的比例在昆蟲細胞中高於在魚細胞中的表現。此結果暗示IPNV A段基因表現機制在昆蟲細胞與魚細胞中不完全相同。經由tuicamycin的處理，顯示即使利用較早驅動外源基因表現的?動子，仍無法在昆蟲細胞內觀察到IPNV A段基因產物的醣化現象。

The pT7 baculovirus vector is used to express foreign gene under the strong AcNPV (Autografha califprnica nuclear polyhedrin virus) polyhedrin promoter in a baculovirus expression system. The pAcMP2 baculovirus vector is similar to the polyhedrin locused-based vectors, but contains the AcNPV basic protein promoter in place of the polyhedrin gene promoter. This vector permits foreign gene expression in the course of the late phase of virus infection, i.e. prior to the very late phase when polyhedrin gene and p10 gene are expressed, and there is evidence that post-translational modification are more readily accomplished in this period. These two vectors were used in the present study in order to investigate the synthesis of IPNV (Infectious pancreatic necrosis virus) A segment gene products in insect cells. The results show that the IPNV A segment genes have been expressed in insect cells by using both vectors. With western blot technique and radiolabelling of recombinant proteins in insect cells, it reveals that the basic protein promoter driven expression was detected at 12 hours postinfection while the polyhedrin promoter driven expression was not detected until 24 hours postinfection. Differences in the recombinant proteins produced by two vectors, including 106KDa polypeptide, pVP2, VP2, VP3, were not observed. The yield of recombinant proteins expressed by both vectors was similar, but was much lower than polyhedrin protein produced by wild type AcNPV. The processing of pVP2 to mature form in insect cells was not so efficient as in fish cells. In addition, the ratio of VP3 to VP2 (pVP2) in insect cell was higher than that in fish cells. This result implied that the gene expression mechanism of IPNV A segment in insect and fish cells is not the same. The results of tunicamycin treatment show that the glycosylation of IPNV A segment gene products was not occurred in insect cells even in basic protein promoter-driven expression..