本文選題：BmNPV + 復(fù)制��； 參考：《浙江理工大學(xué)》2017年碩士論文

【摘要】：眾所周知,家蠶桿狀病毒(BmNPV)多角體啟動(dòng)子具有超高效的啟動(dòng)功能,在昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)中常被用來(lái)啟動(dòng)外源基因的表達(dá)。通過(guò)酵母單雜交技術(shù)篩選出多種與多角體啟動(dòng)子存在相互作用的蛋白因子,其中ORF60蛋白被認(rèn)為是Bm NPV的DNA結(jié)合蛋白,且可能具有調(diào)控病毒基因轉(zhuǎn)錄的功能,但其調(diào)控機(jī)制還不是很清楚。Red同源重組技術(shù)是研究桿狀病毒基因功能常用的基因敲除的方法,運(yùn)用此技術(shù)我們成功的構(gòu)建了orf60缺失型病毒orf60-ko-Bacmid。Bac-to-Bac技術(shù)將敲除的片段再次補(bǔ)回到家蠶桿狀病毒(BmNPV)基因組中,通過(guò)此技術(shù),我們又成功的構(gòu)建了orf60補(bǔ)回型病毒orf60-re-Bacmid。然后將三種病毒(wtBacmid、orf60-ko-Bacmid和orf60-re-Bacmid)分別轉(zhuǎn)染家蠶Bm N細(xì)胞,病毒滴度和熒光定量PCR(qPCR)實(shí)驗(yàn)結(jié)果表明,orf60是BmNPV基因組復(fù)制的必需基因,也是包裝成成熟病毒的必需基因。反轉(zhuǎn)錄實(shí)時(shí)定量PCR(RT-qPCR)分析orf60基因的轉(zhuǎn)錄時(shí)相,結(jié)果表明orf60基因是BmNPV的一個(gè)晚期基因。進(jìn)一步通過(guò)qPCR技術(shù)分析orf60基因缺失對(duì)野生型病毒(wtBm NPV)3個(gè)不同時(shí)期(早期、晚期和極晚期)基因轉(zhuǎn)錄的調(diào)控作用,結(jié)果表明orf60基因的缺失導(dǎo)致病毒各個(gè)時(shí)期基因的轉(zhuǎn)錄水平均顯著降低。最后,構(gòu)建了一個(gè)由Bm NPV多角體啟動(dòng)子驅(qū)動(dòng)的螢火蟲(chóng)螢光素酶和家蠶胞質(zhì)肌動(dòng)蛋白A3啟動(dòng)子驅(qū)動(dòng)的海腎螢光素酶構(gòu)成的雙螢光素酶檢測(cè)系統(tǒng),實(shí)驗(yàn)結(jié)果表明orf60基因在BmNPV多角體啟動(dòng)子啟動(dòng)轉(zhuǎn)錄的過(guò)程中起正調(diào)控作用。
[Abstract]:It is well known that the polyhedrosis promoter of Bombyx mori baculovirus (BmNPV) has super efficient function and is often used to activate the expression of foreign genes in insect baculovirus expression system. A variety of protein factors interacting with polyhedrosis promoter were screened by yeast single hybrid technique. The ORF60 protein is considered to be the DNA-binding protein of BmNPV, and it may have the function of regulating the transcription of virus gene. However, its regulatory mechanism is not very clear. Red homologous recombination technology is commonly used to study the gene function of baculovirus gene knockout method, Using this technique, we successfully constructed the orf60 deletion virus orf60-ko-Bacmid.Bac-to-Bac technology to repair the knockout fragment back into the genome of Bombyx mori baculovirus (BmNPV). By using this technique, we successfully constructed the orf60 complementary virus orf60-re-Bacmid.Using this technique, we successfully constructed orf60-re-Bacmidvirus. Then three kinds of viruses (wtBacmidorf60-ko-Bacmid and orf60-re-Bacmid) were transfected into Bombyx mori BmN cells, respectively. The results of virus titer and fluorescence quantitative PCR (qPCR) showed that the virus titer and the fluorescence quantitative PCR (qPCR) showed that the three viruses (wtBacmida orf60-ko-Bacmid and orf60-re-Bacmid) were essential genes for the replication of BmNPV genome and for packaging them into mature viruses. The transcriptional phase of orf60 gene was analyzed by reverse transcription real-time quantitative PCR (RT-qPCR). The results showed that orf60 gene was a late gene of BmNPV. Furthermore, the effects of orf60 gene deletion on the transcription of wild type virus (wtBm NPV) at three different stages (early, late and very late) were analyzed by qPCR. The results showed that the deletion of orf60 gene resulted in a significant decrease in transcription level at all stages of the virus. Finally, a double luciferase detection system was constructed, which was driven by Bm NPV polyhedrosis promoter and silkworm cytoplasmic actin A3 promoter. The results show that orf60 gene plays a positive role in the initiation of transcription of BmNPV polyhedrosis promoter.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q78

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