【作者】 韩琦；

【导师】 何宁佳；

【作者基本信息】 西南大学 ， 生物化学， 2012， 硕士

【摘要】 无论是在脊椎或无脊椎动物中,胰岛素／胰岛素样生长因子信号系统调控着生物个体的代谢与生长发育。作为该系统中一种重要的效应因子,胰岛素样生长因子结合蛋白(IGFBPs)家族对维持体内血糖平衡,促进细胞的增殖分化等生命过程发挥着至关重要的作用。昆虫也具有胰岛素样肽,而目前对无脊椎动物IGFBPs的功能同系物少有报道。作为昆虫先天性免疫系统中一个重要的组成部分,酚氧化酶原级联反应在宿主抵御外源微生物侵染的过程中发挥着重要的作用。其通过产生黑色素,以毒性的醌类物质和活性氧、氮中间产物对微生物进行杀灭。但过度黑化会导致机体损伤,所以需要如丝氨酸蛋白酶抑制剂这样的负调控因子对黑化反应进行精确控制。家蚕具有开放式血液循环系统,血淋巴中的蛋白会参与新陈代谢,物质能量转运以及免疫防控。本文通过对家蚕血淋巴蛋白BmCP8的功能进行了初步分析,发现其可能是一种具有双重功能的家蚕血淋巴碱性小分子量蛋白,即参与了两种不同的生命进程：胰岛素样肽通路和黑化反应。主要的研究结果如下：1.家蚕BmCP8蛋白的生物信息学分析根据BmCP8的氨基酸序列,通过Uniprot的UniprotKB数据库进行BLASTP发现BmCP8与柞蚕真菌蛋白酶抑制剂AmFPI-1以及哺乳动物的IGFBPs的N端保守区域相似,其序列上都富含半胱氨酸。进行多重序列比对后发现,BmCP8具有AmFPI-1的活性区域,而BmCP8的“GCGCCXXCV"序列与IGFBPs N端结构域中保守的GCGCCXXCA"序列相似。2.家蚕BmCP8蛋白的纯化与富集为了大量富集BmCP8蛋白,本研究在原有纯化策略的基础上进行了改进,即将五龄三天的家蚕血液通过硫酸铵沉淀法,两次凝胶过滤与一次离子交换层析的策略纯化出单一的目的蛋白。3.不同时期高糖饲料喂养家蚕后表型和BmCP8基因表达的分析从三龄起和五龄起分别用含5%和20%葡萄糖的人工饲料喂养家蚕,于五龄三天对各处理组进行形态学分析,以及调查其脂肪体中BmCP8基因和头中BombyxinA2基因的表达情况。结果发现,加糖饲料喂养的家蚕个头小,发育迟缓。同时,长期喂食含糖饲料使得BombyxinA2和BmCP8基因表达水平也在各处理组中呈现出下降的趋势。4. BmCP8蛋白对家蚕卵巢细胞生长情况的影响用不同浓度(0、0.5、1、5、10μg／孔)的BmCP8蛋白与处于对数生长期的卵巢细胞共培养,用家蚕表皮蛋白CPR56作为蛋白对照。培养48小时后,对卵巢细胞生长情况进行检测发现BmCP8并不能促进其生长。5. BmCP8蛋白对酚氧化酶级联反应的影响对五龄三天的家蚕幼虫分别注射大肠杆菌(Eescherichia coli)、白僵菌(Beauveria bassiana)、黑胸败血芽孢杆菌(Bacillus bombyseptieus)和对照(0.85%生理盐水),选取5个时间点即1、3、6、12、24小时。通过荧光定量PCR检测BmCP8的表达情况,发现在注射黑胸败血芽孢杆菌1小时后,BmCP8基因高量表达,在随后的23个小时里表达量则逐渐减少,但整体表达水平都比对照组高。说明BmCP8能在短时间内对黑胸败血芽孢杆菌的诱导做出快速而持续的应答。BmCP8蛋白同时还能抑制黑化反应。肽聚糖和葡聚糖能诱发黑化反应,而当肽聚糖和葡聚糖与2ug/ul BmCP8共同加入到SLP试剂中时,黑化反应却受到了明显的抑制。更多还原

【Abstract】 Insulin and insulin-like growth factors (IGFs) signal system control growth and metabolism in both vertebrates and invertebrates. As an important effective factor, insulin-like growth factor binding proteins (IGFBPs) play the essential role on the life-process, such as maintaining the balance of blood glucose level, promoting cell proliferation et al. Although researchers found the insulin-like peptide in some insect species, there are still no reports about the functional homologue of IGFBPs in invertebrate.As an important part of the innate immune system, PO (Phenoloxidase) cascade system protects the host from the infection of exogenous microorganisms. This immune response will kill the pathogen through melanin or Quinones, reactive oxygen species, NO, which were the by-products produced during the melanin formation. However, the excessive.melanization would be harmful to the host too. So the negative-regulator in the PO system will need to be required to control the over melanization.Silkworm larvae own the open blood vascular system. Hemolymph protein will be involved in metabolism, mass-energy conversion, immune response, et al. During the functional analysis of BmCP8, the results could be fall into two different divisions:the insulin-like peptide signal and PO cascade system. The main results are as follows:1. Bioinformatics analysis of BmCP8Based on the B1ASTP in the UniprotKB database, the alignment of amino acid sequences of the BmCP8 with A. mylitta fungal proteinase inhibitor AmFPI-1 and N-terminal domain sequence of mammalian IGFBPs founds the sequence similarity, all of them were rich in the cysteine residues. Multiple sequence comparison demonstrated that BmCP8 also contained the reactive site of AmFPI-1 and the sequence "GCGCCXXV" from BmCP8 owns a high similarity with the "GCGCCXXCA" in the conserved region of N-terminal domain sequence of IGFBPs.2. Purification of BmCP8 protein In order to obtain more native BmCP8, we improved the original purification strategy into ammonium sulfate precipitation, twice gel filtration and cation ion-exchange chromatography, which could effectively get more products.3. Phenotype and BmCP8 gene expression pattern analysis after the high-glucose diet treated.From the third and fifth instar respectively, Silkworm larvae were fed on the artificial diet containing 5% and 20% glucose. Investigating gene expression of BmCP8 and Bombyxin A2 was conducted at the fifth instar day 3, following phenotype analysis. The result demonstrated that the high-glucose diet treated silkworm larvae was characterized by the small size and delayed growth. Compared to the control, BmCP8 gene and Bombyxin A2 gene were down-regulated with glucose-treatment.4. Effects of BmCP8 protein on the growth of silkworm ovarian cellsThe BmCP8 protein with different concentrations (0,0.5,1,5,10 ug/hole) were incubated with the silkworm ovarian cells in logarithmic growth phase. In this experiment, the silkworm cuticle protein CPR56 was taken as the protein control. After being incubated for 48 h, the growth of the ovarian cells was measured. The results showed that BmCP8 can not stimulate the growth of ovarian cells.5. Effects of BmCP8 on the PO cascade system.Injecting fifth day 3 silkworm larva with E. coli, B.bassiana and B. bomyseptieus, the gene expression of BmCP8 was analyzed by q-PCR. Results revealed that the expression of BmCP8 was suffered an acute up-regulation after infected with B. bombyseptieus one hour later. The expression of BmCP8 will be gradually reduced during the following hours. But compared with the control, the expression of BmCP8 still stayed in a high level even at the later 24 hours. The experiment data demonstrated that BmCP8 will quickly and persistently response to the infection of B. bomyseptieus. Meanwhile, the experiment proved that BmCP8 own the ability to inhibit the PO system. When 2ug/ul BmCP8 were add to the SLP reagent which were induced by PGN and BGN, the effect of melanism will be repressed by BmCP8.更多还原