桦褐孔菌酸性蛋白酶基因克隆与原核表达以及多克隆抗体制备

发布时间：2018-06-22 21:11

  本文选题：桦褐孔菌 + IO-AP基因　； 参考：《延边大学》2017年硕士论文

【摘要】：桦褐孔菌(Inonotus obliquus)是一种十分珍稀而名贵的野生药用真菌,其药用部分为菌核。目前虽然很多学者已成功人工驯化栽培桦褐孔菌,并能够形成菌核,但其生物学效率过低,严重地限制了桦褐孔菌的规模化生产。为了从分子水平研究影响菌核产量的因素,本研究采用RACE技术首次克隆得到桦褐孔菌酸性蛋白酶基因(IO-AP),对该基因进行生物信息学分析,并对该基因进行原核表达与多克隆抗体制备,结果如下:1.使用简并PCR和RACE技术从桦褐孔菌JL01菌株中克隆获得了IO-AP的cDNA全长序列。IO-AP基因cDNA全长序列大小为1178 bp,编码329个氨基酸。2.对IO-AP基因编码的氨基酸序列进行一致性搜索,结果显示IO-AP与其他真菌的AP 一致性较高,IO-AP与鲍姆桑黄菌的AP蛋白一致性最高为88%,与其它真菌AP蛋白一致性次之,进一步表明克隆得到的基因序列为目的基因序列。3.通过构建系统发育树发现,IO-AP蛋白与同为锈革孔菌科的鲍姆桑黄菌的AP蛋白亲缘关系最近,说明蛋白的进化关系和物种的进化关系存在一定的相关性。4.生物信息学分析表明,IO-AP蛋白无信号肽,属于稳定蛋白,在细胞中以自由态存在,该蛋白归属于胃蛋白酶超级家族的天冬氨酸蛋白酶A1家族,属于酸性蛋白酶。其生物学功能是参与蛋白质水解,具有天冬氨酸类型蛋白内切酶活性。5.本研究成功构建桦褐孔菌酸性蛋白酶原核表达载体pGEX-4T1-IO-AP。在大肠杆菌BL21(DE)中获得稳定表达,且以包涵体形式存在,重组蛋白分子质量约为60 KDa。6.本研究采用原核重组表达IO-AP的方法,并获得较高纯度的IO-AP作为免疫原免疫4只B5lb/c小鼠,免疫过程采用传统3-2-2-2免疫方式,以重组IO-AP作为抗原,通过间接ELISA法测定抗血清效价,4免之后,4只小鼠抗血清效价均大于121500。7.本研究用制备的IO-AP抗血清初步建立检测重组蛋白IO-AP的ELISA方法,通过棋盘滴定试验确定抗原最佳包被浓度为1μpg/mL,抗血清最佳稀释比例为 1:4000。8.用IO-AP抗血清检测重组蛋白,可以检测到ng级别的重组蛋白(5ng),说明制备的抗血清效价高,成功制备IO-AP多克隆抗体。
[Abstract]:Inonotus obliquus is a rare and valuable wild medicinal fungus, the medicinal part of which is sclerotia. Although many scholars have successfully domesticated and cultivated Betula obliquus and form sclerotia, their biological efficiency is too low, which seriously limits the scale production of Betula obliquus. In order to study the factors affecting sclerotia yield at molecular level, the acid protease gene (IO-AP) of Betula obliquus was cloned by race technique for the first time, and the gene was analyzed by bioinformatics. Prokaryotic expression of the gene and preparation of polyclonal antibody were carried out. The results were as follows: 1. 1. The full-length cDNA sequence of IO-AP and the full-length cDNA sequence of IO-AP gene were obtained by degenerate PCR and race from strain JL01 of Betula obliquus. The full-length cDNA sequence of IO-AP gene was 1178 BP, encoding 329 amino acids. The sequence of amino acid encoded by IO-AP gene was searched. The results showed that the consistency of AP between IO-AP and other fungi was higher than that between IO-AP and Pommulia baumsanguilli. The highest consistency of AP protein between IO-AP and other fungi was 88%, followed by AP protein consistency with other fungi. It is further indicated that the cloned gene sequence is the target gene sequence. 3. By constructing phylogenetic tree, we found that the phylogenetic relationship between IO-AP protein and AP protein of P. baumsanguinae is close, which indicates that there is a certain correlation between protein evolution relationship and species evolution relationship. 4. Bioinformatics analysis showed that the IO-AP protein belongs to the stable protein, which belongs to the aspartate protease A1 family of pepsin superfamily and belongs to the acidic protease family. Its biological function is to participate in protein hydrolysis, with aspartic acid type protein endonuclease activity. In this study, the prokaryotic expression vector pGEX-4T1-IO-APwas successfully constructed. The recombinant protein was stably expressed in E. coli BL21 (DE) and existed in the form of inclusion body. The molecular weight of the recombinant protein was about 60KDa.6. In this study, a prokaryotic recombination method was used to express IO-AP, and 4 B5lb / c mice were immunized with high purity IO-AP. The immune process was carried out by traditional 3-2-2-2 immunization, and the recombinant IO-AP was used as antigen. The antiserum titers of 4 mice were all higher than 121500.7 by indirect Elisa. In this study, an Elisa method for the detection of the recombinant protein IO-AP was established by using the prepared IO-AP antiserum. The best coating concentration of antigen was 1 渭 g / mL and the best dilution ratio of antiserum was 1: 4000.8 by chessboard titration. The ng level recombinant protein (5ng) can be detected by using IO-AP antiserum, which indicates that the prepared antiserum has high titer and successfully prepared IO-AP polyclonal antibody.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.3
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本文编号：2054242