家蝇幼虫围食膜蛋白（MdPM-17）的基因克隆鉴定及其特性研究

发布时间：2018-03-17 02:33

本文选题：家蝇　切入点：围食膜蛋白　出处：《贵州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究家蝇幼虫围食膜蛋白MdPM-17分子特点及生物学特性。方法:用RT-PCR方法从家蝇cDNA中获取MdPM-17基因,运用生物信息学方法对该基因及其编码蛋白序列进行预测和分析;构建原核表达质粒pET-32a(+)-MdPM-17,在大肠杆菌Transetta(DE3)中诱导表达并进行纯化,纯化产物经免疫新西兰大白兔制备多克隆抗体;利用几丁质结合实验检测重组蛋白的几丁质结合特性。对家蝇幼虫MdPM-17基因进行RNA干扰,探索最佳干扰时间及效果,通过实时荧光PCR方法检测防御素defensin、天蚕素cecropins、双翅肽diptericin等抗菌肽基因的表达变化情况,研究MdPM-17基因干扰后抗微生物感染反应。结果:成功克隆MdPM-17基因,MdPM-17基因全长635 bp,其ORF长477 bp,编码158个氨基酸,理论分子量为17 kDa,等电点为4.55,蛋白质的N端含有一段信号肽,结构预测分析显示其包含几丁质Ⅱ型结构域。RT-PCR检测显示MdPM-17在家蝇幼虫脂肪体、前肠、中肠、气管以及马氏管中均有表达,其中在中肠的表达量最高。双酶切及SDS-PAGE电泳结果表明成功构建了pET-32a(+)-MdPM-17重组质粒并表达目的重组蛋白。几丁质结合实验表明MdPM-17蛋白具有几丁质结合活性,并只能够被强变性剂6M尿素洗脱,属于PM第三类蛋白。RNA干扰家蝇MdPM-17基因,结果显示在注射MdPM-17dsRNA后24 h达到有效沉默,MdPM-17基因沉默后家蝇幼虫的存活率及化蛹率降低。RNA干扰MdPM-17基因后,抗菌肽基因(defensin、cecropins、diptericin)的表达均较对照组升高;微生物感染后抗菌肽基因的表达各有差异,大肠杆菌感染时,cecropins、diptericin的表达较对照组高;金黄色葡萄球菌感染时,defensin、cecropins、diptericin的表达较对照组高;白假丝酵母菌感染时,diptericin的表达较对照组高。结论:成功构建pET-32a(+)-MdPM-17重组质粒并获得纯化重组蛋白。MdPM-17具有几丁质结合活性,属于第三类围食膜蛋白。MdPM-17基因在家蝇幼虫的中肠组织表达量最高;RNA干扰后MdPM-17基因在24 h时达到有效沉默,并且使抗菌肽基因的表达升高。微生物感染后MdPM-17 RNAi组的抗菌肽基因的表达各有不同程度的代偿性上调,表明MdPM-17在家蝇幼虫肠道免疫调控中发挥重要作用。
[Abstract]:Objective: to study the molecular characteristics and biological characteristics of the membrane protein (MdPM-17) of housefly larvae. Methods: the MdPM-17 gene was obtained from housefly cDNA by RT-PCR method, and the gene and its coding protein sequence were predicted and analyzed by bioinformatics. The prokaryotic expression plasmid pET-32a (MdPM-17) was induced and purified in E. coli TransettaDe3. The purified product was immunized with New Zealand white rabbits to prepare polyclonal antibodies. Chitin binding assay was used to detect the chitin binding characteristics of recombinant proteins. RNA interference was carried out on the MdPM-17 gene of housefly larvae to explore the best interference time and effect. The expression of defensin, cecropins, diptericin and other antimicrobial peptide genes were detected by real-time fluorescence PCR. Results: the total length of MdPM-17 gene of MdPM-17 gene was 635 BP, its ORF length was 477 BP, encoding 158 amino acids, the theoretical molecular weight was 17 kDa, the isoelectric point was 4.55, and the N-terminal of the protein contained a signal peptide. Structural prediction analysis showed that MdPM-17 was expressed in the fat body, foregut, midgut, trachea and Markov tube of Musca domestica larvae by reverse transcription-polymerase chain reaction (RT-PCR). The results of double enzyme digestion and SDS-PAGE electrophoresis showed that the recombinant plasmid pET-32a (MdPM-17) was successfully constructed and expressed the target recombinant protein. Chitin binding assay showed that MdPM-17 protein had chitin binding activity. It can only be eluted by 6M urea, which belongs to the third group of PM protein. RNAs interfere with the MdPM-17 gene of housefly. The results showed that the survival rate and pupation rate of Musca domestica larvae were reduced after 24 hours after the silencing of MdPM-17 gene. The results showed that the survival rate and pupation rate of Musca domestica larvae decreased after the silencing of MdPM-17 gene. The expression of antimicrobial peptide gene defensinincecropinsdiptericinwas higher than that of control group, the expression of antimicrobial peptide gene was different after microorganism infection, the expression of cecropinsdiptericin was higher than that of control group, and the expression of defensin cecropinsdiptericin was higher than that of control group in the case of Staphylococcus aureus infection. Conclusion: the recombinant plasmid pET-32a (MdPM-17) was constructed successfully and purified recombinant protein. MdPM-17 had chitin-binding activity. The expression of MdPM-17 gene in the midgut of housefly larvae was the highest, and the MdPM-17 gene was effectively silenced at 24 h after interference. The expression of antimicrobial peptide gene was up-regulated in MdPM-17 RNAi group, which indicated that MdPM-17 played an important role in the intestinal immune regulation of housefly larvae.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R384.2

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