gcm蛋白的表达、纯化及多克隆抗体的制备

gcm(glial cells missing)是调控神经元细胞和神经胶质细胞相互转化的一个基因开关.在gcm功能缺损的突变体中,预期的神经胶质细胞发育成神经元细胞;而在gcm过表达的突变体中,预期的神经元细胞转化为神经胶质细胞.此外,gcm还调控血浆细胞发育.为了进一步研究gcm在发育中的功能,需要获得gcm蛋白并制备其抗体.根据已报道的gcm基因序列,以果蝇cDNA文库为模板进行PCR扩增得到gcm部分编码区序列,然后将其连接到pET-28a载体以获得原核表达载体.重组载体经酶切测序鉴定确认后,转化大肠杆菌(E.coli)BL21,并用IPTG诱导融合蛋白表达.采用Ni-IDA凝胶柱亲和纯化蛋白,将纯化的His-gcm融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体效价.获得的gcm原核表达重组融合蛋白及高效价的特异性兔抗gcm多克隆抗体,为gcm功能的进一步研究奠定了基础.     

果蝇Domeless蛋白的原核表达及多克隆抗体的制备研究

在果蝇(Drosophila melanogaster)的研究中发现Domeless接受器参与发育期间的JAK/STAT信号调节,在心脏疾病的发生机制中发挥重要作用.为了克隆Domeless,我们利用生物信息学选择果蝇Domeless基因抗原亲水区,将扩增出的PCR片段克隆到原核表达pET-28a载体中,转入E.coli(Escherichia coli)中后通过IPTG(Isopropylβ-D-thiogalactoside)诱导融合蛋白表达,Ni-IDA凝胶柱亲和纯化,纯化后的His-Domeless融合蛋白免疫新西兰大白兔制备多克隆抗体.用Western blot检测抗体的效价和特异性.获得了Domeless原核表达重组融合蛋白以及高效价的、特异性兔抗Domeless多克隆抗体,为后续Domeless功能研究奠定了基础.     

Notch信号途径中的Su(H)和E(spl)基因对果蝇天然免疫的影响

天然免疫系统是多细胞生物抵抗各种入侵微生物的第一道防线.Notch途径介导相邻细胞之间的相互作用,调节细胞、组织、器官的分化和发育.为了进一步探索Notch信号途径在果蝇天然免疫中的功能,利用Notch途径下游基因Su(H)和E(spl)的低表达突变体果蝇,通过体外注射病原体分析了生存率、血细胞的噬菌功能和抗菌肽的表达量以及突变体的血细胞数量.结果表明,革兰氏阴性细菌和真菌感染后果蝇E(spl)突变体的生存率、噬菌能力及抗菌肽的表达量明显降低,而且幼虫期血细胞出现异常增殖;Su(H)突变体只对真菌表现出敏感性,抗菌肽的表达量降低,但是对真菌的噬菌能力正常.此结果表明,Notch途径不仅影响个体的生长发育,而且在果蝇天然免疫中也起重要的调节作用.     

果蝇心脏发育标记基因lbe的多克隆抗体制备

lbe是已经证明的心脏标记基因。为了深入研究lbe在心脏发育中的功能,需要获得lbe蛋白并制备其抗体。首先提取野生型成体果蝇的总RNA,反转录获得其cDNA文库,通过PCR克隆出lbe编码区序列,将其连接到pET-28a原核表达载体上。经酶切及测序鉴定后,质粒构建成功。将重组质粒（pET-28a-lbe）转化大肠杆菌菌株Rosseta,用IPTG诱导表达出融合蛋白,经Ni-IDA凝胶柱纯化后,最后将纯化的融合蛋白免疫新西兰大白兔制备lbe多克隆抗体,并用Western blotting检测抗体的效价和特异性。结果显示获得了lbe原核表达重组融合蛋白及高效价的特异性兔抗lbe多克隆抗体,为lbe功能的进一步研究奠定了基础。     

果蝇唾液腺特异基因CG8419多克隆抗体的制备及检测

果蝇CG8419基因与脊椎动物中TRIM45基因为同源基因.以果蝇cDNA为模板通过PCR扩增出亲水性和特异性好的果蝇CG8419基因片段,将其克隆入表达载体PET-28a,构建出重组表达质粒PET-28a-CG8419.将重组质粒转化大肠杆茵Rosetta,经IPTG诱导出带His标签的重组融合蛋白.通过尿素洗涤包涵体并切胶回收纯化融合蛋白,然后再免疫新西兰大白兔制备多克隆抗体.Western blot实验分别验证抗体的效价和特异性.果蝇胚胎抗体染色显示该基因在果蝇唾液腺中表达.     

抗果蝇zip基因产物抗体的制备和胚胎zip蛋白分布的研究

zip基因为果蝇晚期神经发生所必需。分子生物学研究表明,zip基因产物是一个膜上整合糖蛋白,可能作为神经细胞的识别或粘联分子参与神经系统的发生。通过基因工程的方法,我们提取了lacZ-zip融合蛋白,继而免疫兔子制备了抗lacZ-zip融合蛋白抗体。该抗体在经过蛋白质印迹鉴定后,用于整幅果蝇胚胎的标记。结果显示zip基因产物主要在胚带缩短后表达,表明zip基因可能参与了晚期神经的发生。抗zip抗体除了识别中枢神经系统(CNS)中的个别神经元外,还标记了侧神经纤维,证实了以前的推测,即在CNS中表达的zip基因可能参与神经纤维束化的建立和维持。     

Nodal蛋白的表达、纯化及多克隆抗体制备

斑马鱼心脏发育模型中Nodal编码转录因子调节心脏的左右不对称发育,为了进一步研究Nodal信号途径在心脏发育中的调控作用和心脏疾病发生的分子机制,需要获得斑马鱼Nodal蛋白并制备其抗体.采用从斑马鱼心脏组织中提取RNA,通过反转录得到心脏组织各种表达基因的cDNA为模板,PCR扩增得到Nodal部分编码区序列,然后将其连接到pET-28a载体上获得原核表达.经酶切及测序鉴定后,转化Rosseta细菌,并用IPTG诱导表达融合蛋白,Ni-IDA凝胶柱亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blotting检测抗体.获得了Nodal原核表达重组融合蛋白及高效价的特异性兔抗Nodal多克隆抗体,为Nodal功能的进一步研究奠定了基础.     

黑胸大蠊浓核病毒NS2蛋白的表达、抗体制备及亚细胞定位

ns2是黑胸大蠊浓核病毒的一个非结构基因, 所编码的蛋白质大小为30 kD, 是一个功能未知的基因。为了对该基因进行深入的功能研究, 从感染了黑胸大蠊浓核病毒的蟑螂的后肠组织中通过RT-PCR得到ns2基因编码序列, 将其构建于原核表达载体pET-28a, 转化大肠杆菌BL21(DE3) 获得融合表达产物。此融合蛋白经分离纯化后, 免疫新西兰大白兔, 制备其多克隆抗体。采用Western印迹技术, 用该抗体检测ns2基因的真核表达产物, 证明该抗体有较好的针对NS2蛋白的专一性, 可用于对NS2结构和功能的研究。同时, 将此编码序列克隆至果蝇细胞表达载体pAC, 得到重组质粒后转染果蝇S2细胞表达重组蛋白, 通过共聚焦显微镜用该抗体检测该蛋白在S2细胞中的亚细胞定位, 发现NS2蛋白主要定位于细胞质, 核内仅有少量分布。     

果蝇Hsp83多克隆抗体的制备及鉴定

目的制备和鉴定抗Hsp83蛋白的多克隆抗体。方法利用PCR技术从果蝇cDNA中获得hsp83基因片段,构建重组质粒;将其转化到BL21(DE3)菌株中诱导蛋白表达,利用Ni-NTA亲和法纯化重组蛋白;再将纯化的蛋白免疫BALB/C小鼠制备多克隆抗体;利用免疫印迹法(Western blot)和免疫荧光染色法检测多克隆抗体的特异性。结果构建的pET28ahsp83质粒在大肠杆菌中成功表达了Hsp83融合蛋白,蛋白纯化后作为抗原免疫小鼠,获得了抗Hsp83的多克隆抗体。免疫印迹法和免疫荧光染色法检测显示,抗果蝇Hsp83多克隆抗体具有较高的特异性,并能检测出内源性Hsp83蛋白。果蝇卵巢免疫荧光染色显示,Hsp83蛋白定位在卵巢细胞的细胞质中。结论成功制备了小鼠抗Hsp83蛋白的特异性抗体,此工作为深入研究Hsp83蛋白的功能奠定了基础。     

果蝇心脏发育标记基因Hand多克隆抗体的制备

制备果蝇心脏标记基因Hand抗体对研究果蝇心脏发育具有重要意义。从果蝇体内提取出总RNA,反转录得到果蝇的cDNA,将其作为模板PCR得到Hand基因部分片段,将片段连接到pET-28a上,构建重组质粒pET一28a—Hand,将重组质粒转化rosetta受体菌,IPTG诱导表达,表达产物经镍柱纯化,SDS—PAGE电泳分析,结果表明Hand基因在大肠杆菌中成功表达,表达的Hand融合蛋白分子量大约为24kD,经镍柱纯化后获得了高纯度可溶性的Hand蛋白。     

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

高流动性蛋白基因过量表达对果蝇发育的影响

HmgD基因编码果蝇高流动性蛋白（High mobility group proteins, HMG）的同源物,它可以参与染色质的组装。目前关于HMGD蛋白在果蝇胚胎发育过程中的作用尚无定论。我们采用UAS-Gal4系统,通过功能获得性突变的方法研究了HmgD基因过量表达对果蝇发育的影响。结果表明：HmgD过量表达对果蝇胚胎期发育的影响较弱,而对后期幼虫的发育具有很大的影响;HmgD过量表达的果蝇胚胎死亡率增高,但这种影响不是很大,因为一部分胚胎仍然能够发育至成体;但是当HmgD在广泛表达的Gal4驱动子（ActGal4）的控制下过量表达时导致子代大量死亡,特别是用4个拷贝的转基因果蝇进行杂交时,后代中的突变型在三龄幼虫末期全部死亡;部分突变型幼虫体内长有黑色素瘤,其血淋巴中的血细胞数量极显著地高于野生型。RT-PCR分析表明,突变幼虫中与血细胞增殖有关的Ras-MAPK途径和Toll途径被异常激活。这些结果显示：HmgD过量表达可能引起染色质结构疏松,激活了特定的转录因子,从而引发了三龄幼虫期异常的转录调控,并导致幼虫死亡。     

Developmental control of blood cell migration by the Drosophila VEGF pathway.

We show that a vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and we speculate that the ancestral function of VEGF pathways was to guide blood cell movement.     

split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells

Signaling by DER, the Drosophila epidermal growth factor receptor tyrosine kinase (RTK), is essential for proper migration and survival of midline glial cells (MGCs) in the embryonic central nervous system (CNS) [1-4]. We recently isolated a gene called split ends (spen) in a screen designed to identify new components of the RTK/Ras pathway [5]. Drosophila Spen and its orthologs are characterized by a distinct set of RNA recognition motifs (RRMs) and a SPOC domain, a highly conserved carboxy-terminal domain of unknown function [5-7]. To investigate spen function in the context of RTK signaling, we examined the consequences of spen loss-of-function mutations on embryonic CNS development. We found that spen was required for normal migration and survival of MGCs and that embryos lacking spen had CNS defects strikingly reminiscent of those seen in mutants of several known components of the DER signaling pathway. In addition, spen interacted synergistically with the RTK effector pointed. Using MGC-targeted expression, we found that increased Ras signaling rescued the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the DER/Ras signaling pathway.