本文選題：干擾素 + 干擾素刺激基因12�。� 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：動物的抗病毒作用主要通過機體的免疫系統(tǒng)產(chǎn)生的特異性免疫和非特異性免疫來實現(xiàn)。干擾素(Interferon,IFN)系統(tǒng)作為非特異性免疫系統(tǒng)的重要組成部分,是動物抗病毒感染的第一道防線,在先天性免疫和獲得性免疫中起關(guān)鍵作用。IFN本身對病毒無滅活作用,它主要通JAK-STAT信號途徑誘導(dǎo)IFN刺激基因(Instimulatedgene,ISG)的表達建立動物細胞的抗病毒狀態(tài),抑制病毒的復(fù)制和增殖。作為一種ISG,ISG12能受IFN-α誘導(dǎo)上調(diào)表達,它是一種新發(fā)現(xiàn)的先天免疫調(diào)節(jié)器,它能夠調(diào)節(jié)抗炎細胞核受體,并在細胞中起著特定的作用,一些研究發(fā)現(xiàn),ISG12基因可能參與了JAK/STAT信號途徑的信號傳遞。ISG12基因在病毒感染過程中已經(jīng)被監(jiān)測到,但是針對其抗病毒活性的研究卻很少。為了研究ISG12的抗病毒活性,根據(jù)已發(fā)表的ISG12蛋白的mRNA基因序列,設(shè)計一對特異性引物,采用反轉(zhuǎn)錄-聚合酶鏈式反應(yīng)(RT-PCR)的方法從雞胚成纖維細胞(CEF)中擴增一條分子量為352bp的特異性基因片段,經(jīng)測序分析,該基因序列與Genbank中登錄的禽屬ISG12基因序列相似性為100%,證明已經(jīng)成功擴增到正確的ISG12基因。將該基因亞克隆到原核表達載體pET-32a構(gòu)建出重組表達質(zhì)粒p32a-C12,工程菌p32a-C12/BL21經(jīng)IPTG誘導(dǎo)表達,SDS-PAGE分析獲得了分子量約30 ku的目的蛋白,重組蛋白經(jīng)Ni2+瓊脂糖凝膠柱層析純化得到的單一的目的蛋白。將純化的ISG12蛋白梯度稀釋(50μg/mL、25μg/mL、12.5μg/mL)后處理CEF細胞12h,結(jié)果50μg/m L和25μg/mL的ISG12蛋白都會引起細胞凋亡,而12.5μg/m L的ISG12蛋白在眼觀上對細胞沒有影響。因此將ISG12蛋白稀釋到高(12.5μg/mL)低(2.5μg/mL)兩個濃度處理CEF細胞和雞外周血淋巴細胞(Peripheral blood lymphocyte,PBL)12h,利用MTS法檢測ISG12蛋白對細胞活性的影響。結(jié)果發(fā)現(xiàn)高濃度(12.5μg/mL)的ISG12蛋白能夠顯著降低PBL細胞的活性,而低濃度(2.5μg/mL)的ISG12蛋白能夠顯著提高兩種細胞的活性。因此,我們得出結(jié)論,ISG12蛋白對細胞活力的影響具有劑量依賴性。以禽流感病毒(Avian influenza virus,AIV)1215株作為病毒模型進行抗病毒活性試驗。試驗一:將病毒感染ISG12蛋白預(yù)處理12h后的CEF細胞和PBL細胞;試驗二:將ISG12蛋白與病毒混合后一起感作CEF細胞和PBL細胞。以管家基因為參照,利用熒光定量PCR檢測病毒的載量。結(jié)果顯示AIV 1215株在CEF細胞上的復(fù)制水平幾乎沒有差異,而AIV 1215株在PBL細胞上的復(fù)制水平顯著降低。綜上所述,我們得出結(jié)論,合適濃度的重組ISG12蛋白在體外能夠抑制AIV 1215株在PBL細胞上的增殖但是在CEF細胞上卻沒有明顯作用,這或許與ISG12蛋白抗病毒的機理有關(guān),具體原因還有待進一步研究。
[Abstract]:The antiviral effect of animals is mainly realized by the specific immunity and non-specific immunity produced by the immune system. As an important part of the nonspecific immune system, IFNs are the first line of defense against virus infection in animals. IFNs play a key role in innate and acquired immunity. It mainly induces the expression of IFN- stimulated gene (ISG) through JAK-STAT signaling pathway to establish the antiviral state of animal cells and to inhibit the replication and proliferation of viruses. ISG- ISG12, a newly discovered innate immune regulator, can regulate anti-inflammatory nuclear receptors and play a specific role in cells, which can be up-regulated by IFN- 偽. Some studies have found that the ISG12 gene may be involved in the signal transduction of JAK / stat signaling pathway. ISG12 gene has been detected in the process of virus infection, but little research has been done on its antiviral activity. In order to study the antiviral activity of ISG12, a pair of specific primers were designed according to the published gene sequence of ISG12 protein. A specific gene fragment with molecular weight of 352bp was amplified from chicken embryo fibroblasts by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. The similarity between the sequence of ISG12 gene and that of Genbank is 100, which proves that the correct ISG12 gene has been amplified successfully. The gene was subcloned into prokaryotic expression vector pET-32a to construct the recombinant expression plasmid p32a-C12. The target protein with molecular weight of about 30 ku was obtained by IPTG induced expression of p32a-C12 / BL21. The recombinant protein was purified by Ni 2 agarose gel column chromatography. CEF cells were treated with 50 渭 g / mL of ISG12 protein and 25 渭 g / mL of ISG12 protein for 12 h. The results showed that 50 渭 g / mL of ISG12 protein and 25 渭 g / mL of ISG12 protein could induce apoptosis, but 12.5 渭 g / mL ISG12 protein had no effect on the cells. Therefore, the ISG12 protein was diluted to 12.5 渭 g 路mL ~ (-1) and 2.5 渭 g 路mL ~ (-1) for 12 h, and the effect of ISG12 protein on cell activity was detected by MTS method. The results showed that the ISG12 protein at high concentration (12.5 渭 g / mL) significantly decreased the activity of PBL cells, while the ISG12 protein at low concentration of 2.5 渭 g / mL significantly increased the activity of the two kinds of cells. Therefore, we conclude that the effect of ISG 12 protein on cell viability is dose-dependent. The antiviral activity of avian influenza virus strain Avian influenza virus1215 was tested. In experiment 1, CEF cells and PBL cells were pretreated with ISG12 protein for 12 h, and CEF cells and PBL cells were sensitized by mixing ISG12 protein with virus. The viral load was detected by fluorescent quantitative PCR using butler base as reference. The results showed that there was almost no difference in the replication level of AIV 1215 strain on CEF cells, while the replication level of AIV 1215 strain on PBL cells was significantly decreased. In conclusion, we conclude that the recombinant ISG12 protein at the appropriate concentration can inhibit the proliferation of AIV 1215 strain in vitro, but not in CEF cells, which may be related to the antiviral mechanism of ISG12 protein. The specific reasons need to be further studied.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.4

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本文編號：2022672