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|標題:||Expression of the dengue 2 virus ribozyme using double-subgenomic Sindbis virus
|關鍵字:||dengue virus;登革病毒;Sindbis virus;ribozyme;Aedes aegypti;辛德比病毒;核糖核酸酶;埃及斑蚊||出版社:||昆蟲學系||摘要:||
本試驗成功構築以雙次基因辛德比表現載體(dsSIN virus expression system ; pTE/3’2J)表現兩端帶有辨識登革病毒原膜蛋白區序列之核糖核酸酶(Rz-DprM)，此為一段形成二級結構之酵素性RNA，具有辨識並可切割登革病毒核酸GUC序列之功能。
試驗首先構築含有Rz-DprM的pTE/rib載體，再經由體外轉錄作用(in vitro transcription)得到重組之pTE/rib dsSIN virus之mRNA，將此mRNA以電穿透作用(electroporation)感染BHK-21細胞，以獲得重組之pTE/rib dsSIN virus。
以此pTE/rib dsSIN virus感染C6/36細胞後，可藉由RT-PCR偵測到Rz-DprM的表現，以dot blot偵測pTE/rib dsSIN virus感染C6/36細胞12、24、48、72小時後Rz-DprM之表現量，結果顯示Rz-DprM會隨時間的增加而增多，以感染後72小時的表現量達最高。
令pTE/rib dsSIN virus或pTE/3’2J dsSIN virus分別與登革二型病毒同時感染C6/36細胞，結果顯示pTE/rib dsSIN virus及pTE/3’2J dsSIN virus兩處理皆抑制登革病毒對C6/36細胞之感染，且差異性不大。而依次感染登革二型病毒及pTE/rib dsSIN virus或pTE/3’2J dsSIN virus的試驗結果發現，登革病毒與辛德比病毒間可能存在感染競爭之現象。以埃及斑蚊(Aedes aegypti)雌蚊進行辛德比重組病毒抑制登革病毒複製之蚊體試驗，結果與C6/36細胞感染試驗相似，pTE/rib dsSIN virus及pTE/3’2J dsSIN virus皆會降低登革病毒對雌蚊之感染率。埃及斑蚊雌蚊若先感染pTE/rib dsSIN virus或pTE/3’2J dsSIN virus 7天後，再感染登革二型病毒，則登革病毒之感染率皆比同時感染登革病毒及辛德比病毒低。
綜合pTE/rib dsSIN virus於C6/36細胞中以及在埃及斑蚊體中抑制登革病毒複製之結果，雖然Rz-DprM可藉由辛德比病毒於C6/36細胞中表現，但其在活體中之功能，可能因辛德比病毒與登革病毒之間存在競爭現象，目前尚無法證實，有待進一步探討。
The double-subgenomic Sindbis virus (dsSIN) expression system was applied to express the dengue 2 virus specific-ribozyme (Rz-DprM) in vivo. The ribozyme is a catalytic RNA forming secondary structure, it is able to recognize and cleave dengue virus sequence at GUC site.We first constructed the plasmid containing the specific ribozyme for dengue virus prM region sequence, Rz-DprM. The mRNA of recombinant Sindbis virus was acquired by in vitro transcription, and then transfected into BHK-21 cells by electroporation. The recombinant Sindbis virus was collected, and was allowed to infect C6/36 cells. The expression of Rz-DprM in C6/36 cells was measured by RT-PCR and dot blot at 12, 24, 48, or 72 hr postinfection, the highest level of Rz-DprM occurred at 72 hr postinfection.
The C6/36 cells were coinfected with the recombinant Sindbis virus (pTE/rib dsSIN virus or pTE3'2J dsSIN virus) and dengue 2 virus. The dengue 2 virus infection rates were all decreased in C6/36 cells when infected with pTE/rib dsSIN virus and pTE/3'2J dsSIN virus, while the degree of decrease was not different significantly between these two types of cells. These results suggest that there is a competition of infection between dengue virus and Sindbis virus existing in our study model. Similar results were observed in Aedes aegypti females. In this study, the females were infected with pTE/rib dsSIN virus or pTE/3'2J dsSIN virus 7 days before dengue 2 virus inoculation. Meanwhile, a coinfection of dengue virus and Sindbis virus was also performed. The inhibition rates of dengue 2 virus replication were lower in females infected with the recombinant Sindbis virus earlier than in the coinfection ones. However, the inhibition rates of pTE/rib dsSIN virus and pTE/3'2J dsSIN virus were similar.
In conclusion, we were able to express Rz-DprM in C6/36 cells using pTE/rib dsSIN virus expression system. However, its inhibition function of dengue 2 virus replication in vivo was not clear. To design an effective method of inhibiting dengue virus infection, further study is required to clarify the infection competition between dengue virus and Sindbis virus in C6/36 cells.
|Appears in Collections:||昆蟲學系|
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