發(fā)布時間：2018-03-17 02:33

  本文選題：家蠅　切入點：圍食膜蛋白　出處：《貴州醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:研究家蠅幼蟲圍食膜蛋白MdPM-17分子特點及生物學特性。方法:用RT-PCR方法從家蠅cDNA中獲取MdPM-17基因,運用生物信息學方法對該基因及其編碼蛋白序列進行預測和分析;構建原核表達質粒pET-32a(+)-MdPM-17,在大腸桿菌Transetta(DE3)中誘導表達并進行純化,純化產物經免疫新西蘭大白兔制備多克隆抗體;利用幾丁質結合實驗檢測重組蛋白的幾丁質結合特性。對家蠅幼蟲MdPM-17基因進行RNA干擾,探索最佳干擾時間及效果,通過實時熒光PCR方法檢測防御素defensin、天蠶素cecropins、雙翅肽diptericin等抗菌肽基因的表達變化情況,研究MdPM-17基因干擾后抗微生物感染反應。結果:成功克隆MdPM-17基因,MdPM-17基因全長635 bp,其ORF長477 bp,編碼158個氨基酸,理論分子量為17 kDa,等電點為4.55,蛋白質的N端含有一段信號肽,結構預測分析顯示其包含幾丁質Ⅱ型結構域。RT-PCR檢測顯示MdPM-17在家蠅幼蟲脂肪體、前腸、中腸、氣管以及馬氏管中均有表達,其中在中腸的表達量最高。雙酶切及SDS-PAGE電泳結果表明成功構建了pET-32a(+)-MdPM-17重組質粒并表達目的重組蛋白。幾丁質結合實驗表明MdPM-17蛋白具有幾丁質結合活性,并只能夠被強變性劑6M尿素洗脫,屬于PM第三類蛋白。RNA干擾家蠅MdPM-17基因,結果顯示在注射MdPM-17dsRNA后24 h達到有效沉默,MdPM-17基因沉默后家蠅幼蟲的存活率及化蛹率降低。RNA干擾MdPM-17基因后,抗菌肽基因(defensin、cecropins、diptericin)的表達均較對照組升高;微生物感染后抗菌肽基因的表達各有差異,大腸桿菌感染時,cecropins、diptericin的表達較對照組高;金黃色葡萄球菌感染時,defensin、cecropins、diptericin的表達較對照組高;白假絲酵母菌感染時,diptericin的表達較對照組高。結論:成功構建pET-32a(+)-MdPM-17重組質粒并獲得純化重組蛋白。MdPM-17具有幾丁質結合活性,屬于第三類圍食膜蛋白。MdPM-17基因在家蠅幼蟲的中腸組織表達量最高;RNA干擾后MdPM-17基因在24 h時達到有效沉默,并且使抗菌肽基因的表達升高。微生物感染后MdPM-17 RNAi組的抗菌肽基因的表達各有不同程度的代償性上調,表明MdPM-17在家蠅幼蟲腸道免疫調控中發(fā)揮重要作用。
[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.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R384.2

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