本文選題：樺褐孔菌 + IO-AP基因��； 參考：《延邊大學》2017年碩士論文

【摘要】：樺褐孔菌(Inonotus obliquus)是一種十分珍稀而名貴的野生藥用真菌,其藥用部分為菌核。目前雖然很多學者已成功人工馴化栽培樺褐孔菌,并能夠形成菌核,但其生物學效率過低,嚴重地限制了樺褐孔菌的規(guī)�；a(chǎn)。為了從分子水平研究影響菌核產(chǎn)量的因素,本研究采用RACE技術首次克隆得到樺褐孔菌酸性蛋白酶基因(IO-AP),對該基因進行生物信息學分析,并對該基因進行原核表達與多克隆抗體制備,結(jié)果如下:1.使用簡并PCR和RACE技術從樺褐孔菌JL01菌株中克隆獲得了IO-AP的cDNA全長序列。IO-AP基因cDNA全長序列大小為1178 bp,編碼329個氨基酸。2.對IO-AP基因編碼的氨基酸序列進行一致性搜索,結(jié)果顯示IO-AP與其他真菌的AP 一致性較高,IO-AP與鮑姆桑黃菌的AP蛋白一致性最高為88%,與其它真菌AP蛋白一致性次之,進一步表明克隆得到的基因序列為目的基因序列。3.通過構(gòu)建系統(tǒng)發(fā)育樹發(fā)現(xiàn),IO-AP蛋白與同為銹革孔菌科的鮑姆桑黃菌的AP蛋白親緣關系最近,說明蛋白的進化關系和物種的進化關系存在一定的相關性。4.生物信息學分析表明,IO-AP蛋白無信號肽,屬于穩(wěn)定蛋白,在細胞中以自由態(tài)存在,該蛋白歸屬于胃蛋白酶超級家族的天冬氨酸蛋白酶A1家族,屬于酸性蛋白酶。其生物學功能是參與蛋白質(zhì)水解,具有天冬氨酸類型蛋白內(nèi)切酶活性。5.本研究成功構(gòu)建樺褐孔菌酸性蛋白酶原核表達載體pGEX-4T1-IO-AP。在大腸桿菌BL21(DE)中獲得穩(wěn)定表達,且以包涵體形式存在,重組蛋白分子質(zhì)量約為60 KDa。6.本研究采用原核重組表達IO-AP的方法,并獲得較高純度的IO-AP作為免疫原免疫4只B5lb/c小鼠,免疫過程采用傳統(tǒng)3-2-2-2免疫方式,以重組IO-AP作為抗原,通過間接ELISA法測定抗血清效價,4免之后,4只小鼠抗血清效價均大于121500。7.本研究用制備的IO-AP抗血清初步建立檢測重組蛋白IO-AP的ELISA方法,通過棋盤滴定試驗確定抗原最佳包被濃度為1μpg/mL,抗血清最佳稀釋比例為 1:4000。8.用IO-AP抗血清檢測重組蛋白,可以檢測到ng級別的重組蛋白(5ng),說明制備的抗血清效價高,成功制備IO-AP多克隆抗體。
[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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S567.3
，

本文編號：2054242