本文選題：α干擾素 + 白介素18��； 參考：《河南科技大學(xué)》2015年碩士論文

【摘要】：1雞傳染性法氏囊病(IBD)是由傳染性法氏囊病病毒(IBDV)引起的一種急性、高接觸性傳染病。近年來,由于超強毒株和變異株的出現(xiàn),傳統(tǒng)疫苗的免疫效果已不盡理想,病發(fā)后的治療難度也隨之增強,因此,新型抗IBDV制劑的研究已成為當前養(yǎng)禽業(yè)的關(guān)注熱點。本研究將雞的IFN-α和IL-18基因融合后于畢赤酵母中表達,通過對表達產(chǎn)物的生物學(xué)活性檢測和抗IBDV中藥單體的篩選,研究重組雞IFN-α-IL-18融合蛋白、中藥單體以及二者聯(lián)合抗IBDV效果和抗病毒機制,為研究抗IBDV新型制劑提供新的思路。1.重組雞IFN-α-IL-18酵母表達載體的構(gòu)建與表達為探討雞IFN-α和IL-18及其融合蛋白的結(jié)構(gòu)和功能,本研究將雞IFN-α、IL-18及其融合基因分別置于酵母表達載體p PICZ-αA的AOX1啟動子下游,構(gòu)建重組質(zhì)粒p PICZ-IFN-α、p PICZ-IL-18和p PICZ-IFN-α-IL-18。將重組質(zhì)粒分別用Pme I酶切線性化,電轉(zhuǎn)入X-33酵母感受態(tài)細胞中,以1%甲醇誘導(dǎo)表達,SDSPAGE和Western blot檢測表達蛋白。結(jié)果表明,在酵母培養(yǎng)上清液中存在分子量分別為22 KD、23 KD和43 KD的目的蛋白,且該蛋白具有良好的免疫原性。2.重組雞IFN-α-IL-18融合蛋白的生物學(xué)活性研究為探討雞IFN-α-IL-18融合蛋白對雞外周血淋巴細胞轉(zhuǎn)化及核因子κB(NF-κB)的影響。本研究應(yīng)用淋巴細胞轉(zhuǎn)化試驗初步檢測重組雞IFN-α-IL-18融合蛋白的生物學(xué)活性,ELISA法測定淋巴細胞中總NF-κB和細胞核中NF-κB的含量。結(jié)果表明,表達的IFN-α-IL-18和IL-18蛋白均能明顯促進雞外周血淋巴細胞的轉(zhuǎn)化(P0.05),IFN-α對淋巴細胞轉(zhuǎn)化有促進作用,但總體差異不顯著(P0.05)。與對照組相比,經(jīng)IFN-α-IL-18和IL-18作用的雞淋巴細胞中總NF-κB含量明顯增加(P0.05),細胞核中NF-κB含量差異極顯著(P0.01),IFN-α作用的淋巴細胞中總NF-κB含量與對照組相比差異不顯著(P0.05),細胞核中NF-κB含量顯著增加(P0.05)。IFN-α-IL-18及IL-18能夠明顯促進淋巴細胞轉(zhuǎn)化,IFN-α促進淋巴細胞轉(zhuǎn)化活性較弱,這種促淋轉(zhuǎn)機制與NF-κB的表達、活化及核質(zhì)轉(zhuǎn)運有關(guān)。3.重組雞IFN-α-IL-18融合蛋白的抗IBDV效果研究及抗IBDV中藥單體的篩選為進一步探討重組雞IFN-α-IL-18融合蛋白的抗IBDV活性并篩選出抗IBDV的中藥單體,本研究通過雞胚接種試驗對IFN-α-IL-18及連翹苷元、黃芪苷、黃芩素、綠原酸、木犀草酸、馬錢酸6種中藥單體的抗IBDV效果進行評價。結(jié)果表明,IFN-α-IL-18融合蛋白能夠顯著抑制IBDV在雞胚中的增殖(P0.01),IFN-α和IL-18酵母表達產(chǎn)物對IBDV也有明顯的抑制作用(P0.05)。六種中藥單體都能一定程度地發(fā)揮抗IBDV活性,其中,黃芩素和綠原酸作用極顯著(P0.01)。4.重組雞IFN-α-IL-18融合蛋白與中藥單體的聯(lián)合抗IBDV效果研究為探索重組雞IFN-α-IL-18融合蛋白與中藥單體的聯(lián)合抗IBDV效果,本研究將篩選出的具有明顯抗IBDV的中藥單體黃芩素和綠原酸,分別與不同濃度的重組雞IFN-α-IL-18蛋白進行組合,接種9日齡雞胚。IBDV抗原檢測結(jié)果表明,聯(lián)合使用黃芩素和重組蛋白IFN-α-IL-18對IBD的預(yù)防效果極顯著(P0.01),且優(yōu)于中藥單體組及重組蛋白組。攻毒保護試驗中,黃芩素和重組雞IFN-α-IL-18蛋白組發(fā)病率最低,且無病雞死亡,起到了較好的保護作用。聯(lián)合使用時,采用10μM黃芩及300μg IFN-α-IL-18的組方能夠發(fā)揮最佳的抗病毒活性。這為進一步尋找防治IBD的最佳方法做了有益的探索。
[Abstract]:1 infectious bursal disease (IBD) is an acute, high contact infectious disease caused by the infectious bursal disease virus (IBDV). In recent years, the immune effect of the traditional vaccine has been not ideal because of the emergence of the super virulent and variant strains, and the difficulty of the treatment after the disease is also enhanced. Therefore, the research of the new anti IBDV preparation has become the current breeding. In this study, the IFN- alpha and IL-18 genes of chicken were fused in Pichia pastoris, and the recombinant chicken IFN- alpha -IL-18 fusion protein was studied by the biological activity detection of the expression products and the screening of the anti IBDV Chinese medicine monomer. The anti IBDV effect and the anti-virus mechanism were combined to study the anti IBDV new system for the study of the anti IBDV system. The construction and expression of recombinant chicken IFN- alpha -IL-18 yeast expression vector,.1., was constructed and expressed to explore the structure and function of chicken IFN- alpha and IL-18 and its fusion protein. This study put chicken IFN- alpha, IL-18 and its fusion gene in the downstream of AOX1 promoter of yeast expression vector p PICZ- alpha A respectively. P PICZ-IFN- alpha -IL-18. used Pme I enzyme to tangent, respectively, into X-33 yeast receptive cells, induced by 1% methanol, SDSPAGE and Western blot to detect the expression protein. The results showed that the molecular weight of the yeast culture supernatant was 22 KD, 23 KD and 43 KD, and the protein had good immunity. The biological activity of the recombinant.2. recombinant chicken IFN- alpha -IL-18 fusion protein was studied to investigate the effect of chicken IFN- alpha -IL-18 fusion protein on lymphocyte transformation and nuclear factor kappa B (NF- kappa B) in chicken peripheral blood. The biological activity of the recombinant chicken IFN- alpha -IL-18 fusion protein was preliminarily detected by lymphocyte transformation test, and the ELISA method was used for the determination of lymph finer. The total NF- kappa B in the cell and the content of NF- kappa B in the nucleus showed that the expression of IFN- alpha -IL-18 and IL-18 protein could obviously promote the transformation of peripheral blood lymphocytes of chickens (P0.05). IFN- alpha could promote the transformation of lymphocytes, but the overall difference was not significant (P0.05). The content of total NF- kappa B increased significantly (P0.05), and the difference in the content of NF- kappa B in the nucleus was very significant (P0.01). The total NF- kappa B content in the lymphocyte of IFN- alpha was not significantly different from that of the control group (P0.05). The activity is weak, the mechanism of the spray transfer and the expression of NF- kappa B, activation and nuclear transport related to the anti IBDV effect of the.3. recombinant chicken IFN- alpha -IL-18 fusion protein and the screening of the anti IBDV Chinese medicine monomer to further explore the anti IBDV activity of the recombinant chicken IFN- a -IL-18 fusion protein and screen out the IBDV traditional Chinese medicine monomer. This study was inoculated by chicken embryo inoculation. The effects of IFN- alpha -IL-18 and forsythia glycoside, astragaloside, baicalein, chlorogenic acid, oxalic acid, and pennic acid on the resistance to IBDV were evaluated. The results showed that IFN- alpha -IL-18 fusion protein could significantly inhibit the proliferation of IBDV in chicken embryos (P0.01), IFN- A and IL-18 yeast expression products had obvious inhibitory effect on IBDV (P0.05). Six All kinds of Chinese medicine monomers can exert anti IBDV activity to a certain extent, among which, the effect of baicalein and chlorogenic acid on the combined anti IBDV effect of recombinant chicken IFN- alpha -IL-18 fusion protein and traditional Chinese medicine monomer is studied to explore the combined anti IBDV effect of recombinant chicken IFN- alpha -IL-18 fusion protein and traditional Chinese medicine monomer, which will be screened by Ju Youming. The traditional Chinese medicine scutellarin and chlorogenic acid, which showed anti IBDV, were combined with different concentrations of recombinant chicken IFN- alpha -IL-18 protein respectively. The results of.IBDV antigen detection at 9 day old chicken embryos showed that the combined use of baicalein and recombinant protein IFN- alpha -IL-18 was very effective in the prevention of IBD (P0.01), which was superior to the traditional Chinese medicine group and the recombinant protein group. In the nursing test, the incidence of baicalein and the recombinant chicken IFN- alpha -IL-18 protein group was the lowest, and the disease free chicken died. The combination of 10 mu M Scutellaria and 300 g IFN- alpha -IL-18 can play the best antiviral activity. This is a useful exploration for the best way to find the best way to prevent and cure IBD.

【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3

【參考文獻】

相關(guān)期刊論文 前2條

1 張春杰,程相朝,李銀聚,吳庭才,趙德明;雞IL-18真核表達載體的構(gòu)建及其對IBD滅活疫苗免疫增強作用的研究[J];中國免疫學(xué)雜志;2004年09期

2 程俊貞;閆若潛;吳志明;趙明軍;閆志玲;劉梅芬;趙雪麗;孔剛銳;陳慧娟;楊曉璐;張書陽;劉毅;;重組雞干擾素/白細胞介素-2融合蛋白體內(nèi)抗傳染性法氏囊病病毒作用研究[J];中國預(yù)防獸醫(yī)學(xué)報;2014年02期

相關(guān)碩士學(xué)位論文 前1條

1 張棟;復(fù)方中藥“毒菌殺”對雞傳染性法氏囊炎的防治試驗[D];山東農(nóng)業(yè)大學(xué);2005年

，

本文編號：1868908