鸡IFN-α-IL-18酵母表达及其与中药单体联合抗IBDV作用的研究

发布时间：2018-05-10 10:16

本文选题：α干扰素 + 白介素18　；参考：《河南科技大学》2015年硕士论文

【摘要】：1鸡传染性法氏囊病(IBD)是由传染性法氏囊病病毒(IBDV)引起的一种急性、高接触性传染病。近年来,由于超强毒株和变异株的出现,传统疫苗的免疫效果已不尽理想,病发后的治疗难度也随之增强,因此,新型抗IBDV制剂的研究已成为当前养禽业的关注热点。本研究将鸡的IFN-α和IL-18基因融合后于毕赤酵母中表达,通过对表达产物的生物学活性检测和抗IBDV中药单体的筛选,研究重组鸡IFN-α-IL-18融合蛋白、中药单体以及二者联合抗IBDV效果和抗病毒机制,为研究抗IBDV新型制剂提供新的思路。1.重组鸡IFN-α-IL-18酵母表达载体的构建与表达为探讨鸡IFN-α和IL-18及其融合蛋白的结构和功能,本研究将鸡IFN-α、IL-18及其融合基因分别置于酵母表达载体p PICZ-αA的AOX1启动子下游,构建重组质粒p PICZ-IFN-α、p PICZ-IL-18和p PICZ-IFN-α-IL-18。将重组质粒分别用Pme I酶切线性化,电转入X-33酵母感受态细胞中,以1%甲醇诱导表达,SDSPAGE和Western blot检测表达蛋白。结果表明,在酵母培养上清液中存在分子量分别为22 KD、23 KD和43 KD的目的蛋白,且该蛋白具有良好的免疫原性。2.重组鸡IFN-α-IL-18融合蛋白的生物学活性研究为探讨鸡IFN-α-IL-18融合蛋白对鸡外周血淋巴细胞转化及核因子κB(NF-κB)的影响。本研究应用淋巴细胞转化试验初步检测重组鸡IFN-α-IL-18融合蛋白的生物学活性,ELISA法测定淋巴细胞中总NF-κB和细胞核中NF-κB的含量。结果表明,表达的IFN-α-IL-18和IL-18蛋白均能明显促进鸡外周血淋巴细胞的转化(P0.05),IFN-α对淋巴细胞转化有促进作用,但总体差异不显著(P0.05)。与对照组相比,经IFN-α-IL-18和IL-18作用的鸡淋巴细胞中总NF-κB含量明显增加(P0.05),细胞核中NF-κB含量差异极显著(P0.01),IFN-α作用的淋巴细胞中总NF-κB含量与对照组相比差异不显著(P0.05),细胞核中NF-κB含量显著增加(P0.05)。IFN-α-IL-18及IL-18能够明显促进淋巴细胞转化,IFN-α促进淋巴细胞转化活性较弱,这种促淋转机制与NF-κB的表达、活化及核质转运有关。3.重组鸡IFN-α-IL-18融合蛋白的抗IBDV效果研究及抗IBDV中药单体的筛选为进一步探讨重组鸡IFN-α-IL-18融合蛋白的抗IBDV活性并筛选出抗IBDV的中药单体,本研究通过鸡胚接种试验对IFN-α-IL-18及连翘苷元、黄芪苷、黄芩素、绿原酸、木犀草酸、马钱酸6种中药单体的抗IBDV效果进行评价。结果表明,IFN-α-IL-18融合蛋白能够显著抑制IBDV在鸡胚中的增殖(P0.01),IFN-α和IL-18酵母表达产物对IBDV也有明显的抑制作用(P0.05)。六种中药单体都能一定程度地发挥抗IBDV活性,其中,黄芩素和绿原酸作用极显著(P0.01)。4.重组鸡IFN-α-IL-18融合蛋白与中药单体的联合抗IBDV效果研究为探索重组鸡IFN-α-IL-18融合蛋白与中药单体的联合抗IBDV效果,本研究将筛选出的具有明显抗IBDV的中药单体黄芩素和绿原酸,分别与不同浓度的重组鸡IFN-α-IL-18蛋白进行组合,接种9日龄鸡胚。IBDV抗原检测结果表明,联合使用黄芩素和重组蛋白IFN-α-IL-18对IBD的预防效果极显著(P0.01),且优于中药单体组及重组蛋白组。攻毒保护试验中,黄芩素和重组鸡IFN-α-IL-18蛋白组发病率最低,且无病鸡死亡,起到了较好的保护作用。联合使用时,采用10μM黄芩及300μg IFN-α-IL-18的组方能够发挥最佳的抗病毒活性。这为进一步寻找防治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.

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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