Cloning and characterization of 37 kDa laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063 bp cDNA clone encoding a putative 37 kDa laminin receptor precursor (37 kDa LRP) is isolated from the mantle tissue of pearl oyster, Pinctadafucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kDa. RT-PCR analysis shows that 37 kDa LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveals that 37 kDa LRP is expressed in the outer epithelial cells of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. Fucata. The identification and characterization of 37 kDa LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

Cloning and characterization of 37 kD laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063bp cDNA clone encoding a putative 37 kD laminin receptor precursor (37 kD LRP) is isolated from the mantle tissue of pearl oyster, Pinctada fucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kD. RT-PCR analysis shows that 37 kD LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveales that 37 kD LRP is expressed in the outer epithelial cells of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kD LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.     

育珠蚌外套膜组织培养适宜条件的研究

对育珠蚌外套膜细胞在组织培养过程中分泌珠质的适宜条件进行了研究。经过几年的实验和比较,筛选出了适宜于河蚌外套膜细胞生长、分泌的培养基及一套完善的操作方法,能使育珠蚌外套膜的细胞在体外人工培养条件下较旺盛地生长、繁殖、分泌,并产生大量的珍珠质.     

马氏珠母贝外套膜组织培养

用本实验室已建立的贝类组织培养技术对马氏珠母贝外套膜组织成功地进行了体外培养,在外套膜组织中最先迁出的是颗粒细胞,紧随其后为透明细胞,在培养到２０ｈ时,圆形的上皮细胞开始迁出,上皮细胞很快在组织块圆形形成生长晕,继而铺满整个培养瓶底面,培养４ｄ以后,上皮细胞开始分泌颗粒状的物质,这时的上皮细胞从形态上发为Ａ型和Ｂ型两类,Ｂ型上皮细胞含有许多颗粒物质,而Ａ型上皮细胞不含或含少量颗粒物质,反映了其合成     

Molecular Cloning and Distribution of a Plasma Membrane Calcium ATPase Homolog from the Pearl Oyster Pinctada fucata

Plasma membrane calcium ATPaso (PMCA) plays a critical role in transporting Ca2 out of the cy- tosol across the plasma membrane which is essential both in keeping intracellular Ca2+ homeostasis and in biomineralization.In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata.This PMCA shares similarity with other published PMCAs within the functional domains.Reverse transcdption-polymerase chain reaction analysis shows that it is expressed ubiquitously.Furthermore,in situ hybridization reveals that it is expressed in the inner epithelial calls of the outer fold and in the outer epithelial calls of the middle fold,as well as the edge near the shell,which suggests that PMCA may be involved in calcified layer formation.The identification and characterization of oyster PMCA can help to further under-stand the structural and functional properties of molluscan PMCA,as well as the mechanism of maintaining Ca2+ homeostasis and the mechanism of mineralization in pead oyster.     

背角无齿蚌组织培养细胞分泌的珍珠质的药理作用研究

利用背角无齿蚌的外套膜,进行离体组织培养,所得培养液及组织水解液,作用于大鼠离体子宫、兔离体小肠,其药理作用与珍珠质有效成分之一的牛磺酸相同,结果证明,背角无齿蚌外套膜培养细胞能分泌珍珠质,具有天然珍珠的药用有效成分及相同的活性.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum.A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pead oyster.This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata,namely PCRT.PCRT encodes a deduced 414-amino acid protein,which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL.The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species.Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle.In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold,the inner side of middle fold,and the inner side of outer fold of the mantle epithelium.All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.     

漂白粉对三角帆蚌外套膜粘液细胞及珍珠囊细胞的影响

研究了不同浓度的漂白粉（0.5mg/L,1.0mg/L,1.5mg/L)处理后对三角帆蚌的影响,结果表明各浓度漂白粉对外套膜和珍珠囊细胞的超微结构均有不同程序损害,表现为粗面内质网脱颗粒,网腔扩张,线粒体基质电子密度改变等,其中1．5mg/L漂白粉的影响最为严重,除发生上述变化外,外套膜内表皮粘液细胞中硫酸化粘液减少,酸性粘液增加,外表皮细胞中性粘液几乎消失,该浓度组的结缔组织中钙球体的数量也明显高于其它各组。     

可溶性人干细胞因子的原核表达和纯化

用Trizol提取人骨髓总RNA,通过RT-PCR扩增人干细胞因子DNA,克隆到pMD18-T载体中并测序．将其定向连入原核表达载体pET32a( ),获得了重组表达载体pET32a( )／hSCF,其cDNA长度为504bp,测序结果与已知序列吻合．然后在大肠杆菌BL21中经IPTG诱导表达,获得37kD的融合蛋白,约占菌体总蛋白量的30％．经Ni^2 -NTA树脂亲和层析柱纯化获得纯度大约90％的重组蛋白．     

育珠河蚌分泌珍珠质的外表皮细胞的扫描电镜观察

用石蜡切片和酶消化的育珠河蚌外套膜的外表皮细胞,作了扫描电镜观察,发现有4类表面结构不同的细胞。细胞间有细丝相连结。研究表明,外表皮细胞除了有分泌珍珠质的功能外,还有物质吸收、保护的功能。     

Cloning, prokaryotic expression,purification and sequence analysis of 20S proteasome subunit gene from T.harzianum

The gene encoding the 20S proteasome subunit（PR29） was cloned from cDNA library of Trichoderma harzianum and expressed in Escherichia coli BL21 （D3） using a pET-28a expression system. The molecular weight of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on gels. The target protein was insoluble when induced at 22℃ with 0.4 mmol/L IPTG, while dissoluble if induced at 37℃ with 0.8mmoL/L IPTG. The expressed product was purified through Ni-magnetic beads His Bind. The purity of the fusion protein reached above 80%. The entire eDNA sequence consisted of 1094 bp with 173 and 135 bp in 5＇ and 3＇ untranslated regions respectively. The gene encoding 261 amino acids has no signal peptide sequence. These results could provide a basis for validating the func-tions of PR29. It also provided a preliminary indication for further study of the mechanism and function of proteasome, and more information of proteasome mechanism in T.harzianum could be obtained.     

定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA, 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列, 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo, 转化大肠杆菌BL21(DE3), 筛选表达菌株BL21-Mute, 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明, 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%, 失去抑制人脐静脉内皮细胞增殖的活性.     

青岛文昌鱼核糖体蛋白AmphiL 37a基因的克隆和同源性分析

对青岛文昌鱼神经胚cDNA进行测序,获得了3个AmphiL 37a的EST,经拼接得到编码文昌鱼核糖体蛋白的AmphiL37a基因全长cDNA序列并演绎出AmphiL37a的氨基酸序列.通过对AmphiL37a蛋白的结构分析及其与多种脊椎动物和无脊椎动物中同种蛋白的同源性进行比较,发现AmphiL37a具有典型的四个半胱氨酸的锌指结构,与人、鼠、鸡的L37a,尤其与果蝇、隐板石鳖等高等无脊椎动物核糖体L37a具有较高的同源性,说明文昌鱼在进化上更接近于高等无脊椎动物;同时说明核糖体蛋白L37a基因在进化上具有较高保守性.     

人Rab蛋白cDNA的克隆和表达

从人胎脑cDNA文库中克隆到一种新的Rab cDNA,全长920bp,以编码213个氨基酸残基,该蛋白预测的分子质量为24567u,等电点7．34,经同源比较,该cDNA与GenBank数据库中登录号为X14964的Rab蛋白有83％的相似性和76％的相同性,将该cDNA克隆到经改造的PBV220表达质粒,转化DH5a菌株诱导表达出该蛋白,取24种不同组织的总cDNA各100ng,用该基因序列设计引物作PCR,结果在胎肝组织中检测到有明显条带,表明该Rab基因相对在胎肝有高表达。