蝗虫微孢子虫孢壁蛋白的鉴定及功能分析

【作者】 冷赫；

【导师】 王贵强； 张龙；

【作者基本信息】 黑龙江大学 ， 农业昆虫与害虫防治， 2012， 硕士

【摘要】 孢壁蛋白（spore wall protein）是一类存在于微孢子虫最外层孢子壁上的蛋白。最近的研究表明这类蛋白在微孢子虫侵染寄主的过程中发挥着重要的作用。本研究提取蝗虫微孢子虫的孢壁蛋白，并结合双向电泳技术对其进行分离，采用质谱技术鉴定出多种可能的孢壁蛋白，通过生物信息学的分析手段，最后对其中的一种孢壁蛋白（ALSWP1），进行了深入的研究：包括序列分析、基因克隆、原核表达、免疫荧光及免疫电镜的定位分析，之后又对其表达图谱进行了研究，并通过GST-pull down及三维结构模拟初步推测了其潜在功能。主要研究结果如下：运用SDS-boiling法和Brosson三步法提取了蝗虫微孢子虫的总蛋白，并通过双向电泳技术共分离出了111个蛋白点。绝大部分（70%）蛋白均为碱性蛋白，分子量在30-50kDa，pH值在5-8。将SDS-boiling法提取的蛋白送去做质谱鉴定，共鉴定出了5种蛋白，对这些蛋白进行生物信息学分析后，选定了其中分子量为22.4kDa的假定孢壁蛋白进行深入的研究。实验对假定孢壁蛋白经行了基因的克隆，原核表达及免疫印迹的研究。在蝗虫微孢子虫体内成功的克隆了此蛋白的编码基因。之后我们构建原核表达载体pGEX4T-1/ALSWP1，然后在大肠杆菌中诱导表达，并利用GST-Tag进行蛋白的纯化。同时，将纯化的蛋白免疫兔子，制备抗血清，并进行Western blotting分析，该结果表明所制备的抗血清可以用于后续对其进行体内表达定位的研究。通过间接免疫荧光和免疫电镜试验对假定的孢壁蛋白ALSWP1在蝗虫微孢子虫生活史不同时期的表达特异性和亚细胞定位特点进行了研究，两项研究结果均表明该蛋白是一种孢壁蛋白。对已完成精确定位的ALSWP1在东亚飞蝗中肠组织中的表达谱进行了分析。RT-PCR结果表明：β-tubulin基因的转录本在感染后11d才能检测到其转录本，且随着感染时间的延长扩增的目的条带亮度也随着感染时间的增长；在感染后15d才能检测到ALSWP1目的条带，且都有较高的转录水平。以该重组融合蛋白ALSWP1为诱饵蛋白，采用GST沉淀试验分离互作的靶蛋白，然后利用质谱技术进行鉴定，获得了3个假定的互作的靶蛋白。使用生物信息学的方法，以3M4F_A（14%）的晶体结构为模板，模拟了ALSWP1的三维结构。更多还原

【Abstract】 Spore wall proteins are a kind of proteins which exist on the most outer layer ofmicrosporidia. Recent studies suggest that their surface proteins play important roles inhost tissue infection and invasion process. In this study, a combination of certainextract methods and two-dimensional electrophoresis techniques were employed by,using mass spectrometry to identify multiple possible spore wall proteins. Andfortunately, the first spore wall protein from Antonospora Locustae was found throughbioinformatics analysis means, named ALSWP1. Then ALSWP1was studied further:including sequence analysis, gene cloning, prokaryotic expression,immunofluorescence(IFA)and immunoelectron microscopy (IEM) analysis wereemployed on protein localization, then the level of RNA expression profiles wereanalyzed. Finally, function analysis of the spore wall protein ALSWP1was conductedthrough GST-pull down and its three-dimensional model. In this dissertation, the mainresults are as follows:Protein extraction was performed on two methods, SDS-boiling and Brossonmethod, respectively. The two extract method was analyzed by a2-DE proteomicmethod, and obtained111protein spots. Wide pH gradient gels (pH3–10) revealedabout100different spots for more than70%of these being composing of the30-50kDa/pI5-8range. However, only SDS-boiling extract were used on the MALDItarget, as well as to data acquisition and interpretation. For some spots, theidentification was confirmed or obtained by LC-MS/MS analysis.As a result,5proteins were identified, and the22.4kDa putative spore wall protein were selected tostudy after analyzing these proteins.Researches were carried on gene cloning, prokaryotic expression andimmuneblotting study. The spore wall protein was successfully cloned of the gene encoding, and constructed of prokaryotic expression vector of pGEX4T-1/ALSWP1,and expressed in E. coli. Fusion proteins were purified by affinity chromatographyusing a GST-Tag Purification Kit. At the same time, the ALSWP1-specific polyclonalantibody was generated by immunizing rabbit with the purified fusion proteins andused in western blot analysis.The results clearly demonstrated that the preparedantiserum could be used to localization study.In order to further determine expression specificity and subcellular localizationcharacteristics of the protein during the different development stages of AntonosporaLocustae, intraeellular Parasites were examined by the indirect immunofluorescence(IFA) and immune electron microscopy (IEM). Both results of the two studies showedthat the putative ALSWP1is a spore wall protein.The expression pattern of the spore wall protein ALSWP1was presented in theinfected midgut of Locusta migratoria manilensis.The results indicate that the transcripts of β-tubulin could be detected at11d, and asthe infection time of growth, correspondingly the brightness of the band increasedsignificantly. Meanwhile, ALSWP1only can be detected at15d, and the transcripts stayat an obvious expression all the time.Using the recombinant fusion protein ALSWP1as the bait protein, using GSTprecipitation test to separate the host target proteins which have the interaction withALSWP1, and three hypothetical target interacting protein were identified with massspectrometry, preliminary results obtained with3putative interaction of the targetprotein.A3-dimensional model of ALSWP1was built by homology modeling with3M4F_A（14%）.更多还原