Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

扩展青霉碱性脂肪酶cDNA的克隆和表达

采用TRIzol试剂一步法所抽提的总RNA的D（260）／D（280）值为1.82,甲醛变性电泳呈现真核微生物所特有的28S rRNA和18S rRNA条带。根据扩展青霉所产碱性脂肪酶（Lip PE)N末端20个氨基酸残基序列和真核生物mRNA3‘端具有poly(A)等所提供的生物信息,采用RT－PCR技术和3‘－RACE法扩增了Lip PE成熟肽编码区和3‘非编码区的cDNA,直接将该PCR产物克隆至pUCm-T载体中。序列分析表明,该碱性脂肪酶含有258个氨基酸,其中保守的五肽序列为Gly-His-Ser-Leu-Gly。进一步采用Clot Tech公司的SMART^TM PCR cDNA文库构建试剂盒,扩增、克隆和测定了自转录起始点至编码区的cDNA片段,从而完成了Lip EP完整cDNA的分析测定。最后将编码完整脂肪酶蛋白的cDNA克隆至pGEX-5X-3表达载体中,在大肠杆菌BL21中进行IPTG诱导表达,SDS－PAGE检测结果表明,所表达的GST－Lip EP融合蛋白分子质量约为53ku;免疫印迹（Western Blotting)技术证明了所克隆的cDNA确为编码扩展青霉WMC20718脂肪酶的基因。     

Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达

【目的】了解海洋细菌Shewanella haliotis BP-1中海藻酸裂解酶降解海藻酸钠的生物活性。【方法】应用基因克隆和大肠杆菌异源表达技术,过量表达海藻酸裂解酶,将粗酶液通过DEAE Sepharose FF柱分离纯化后检测其酶活性。【结果】从S.haliotis BP-1菌株的基因组DNA中克隆得到一个大小为2 157bp的海藻酸裂解酶基因Alg17S,该基因编码的海藻酸裂解酶Alg17S属于PL17家族的蛋白,大小为79 726Da,其中包括N端26个氨基酸的信号肽,与Saccharophagus degradans 2-40菌株产生的海藻酸裂解酶Alg17C具有高度同源性,相似性为52%。经纯化后获得的重组酶Alg17S和△snAlg17S(N端不含26个氨基酸的信号肽)均具有降解海藻酸钠的活性,但△snAlg17S对海藻酸钠的催化活性比Alg17S高,其酶比活力高达9 635U/mg。【结论】重组海藻酸裂解酶△snAlg17S兼具高表达水平及高酶活性,是进一步研究海藻酸盐糖化和生物燃料生产的潜在的优势酶。     

大熊猫肌红蛋白的纯化及一级结构的研究

本文从大熊猫骨骼肌分离纯化了肌红蛋白,鉴定了纯度,测定了分子量及氨基酸组成。确定了该蛋白的N端及C端氨基酸。采用CNBr裂解的方法,用HPLC分离得到4个肽段,分别测定了大熊猫肌红蛋白1—52位,56—77位及132—153位氨基酸的排列顺序。大熊猫肌红蛋白共含153个氨基酸,本文已完成了96个氨基酸的序列测定。用此结果与食肉目有关动物已知肌红蛋白的氨基酸顺序进行比较,获得了有意义的结论。     

中国白兔IL-6基因的分子克隆及其表达研究

运用RT-PCR技术对用ConA刺激的中国白兔外周血淋巴细胞(PMBCr)进行了扩增,将纯化后的PCR产物克隆入pMD18-T中进行核苷酸序列测定.结果中国白兔IL-6基因全长726 bp,编码242个氨基酸,其中前26个氨基酸残基构成信号肽序列.与不同物种IL-6基因相比,核苷酸和推导的氨基酸序列有一定的差异.在推导的中国白兔IL-6氨基酸序列中,在108-110位存在1个潜在的N-联糖基化位点,同时存在9个Cys残基.将pTIL-6双酶切,回收目的基因片段克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-6,转化大肠杆菌BL21(DE3),并用IPTG进行了诱导.结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量为29.5kDa的重组目的蛋白.经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的16.2%.     

平颏海蛇CRVP基因全长cDNA片段的克隆和序列分析

通过对平颏海蛇毒腺cDNA文库的随机测序和PCR筛选,克隆得到两种分别长为1309和1307bp的编码半胱氨酸丰富毒蛋白（cysteine-rich venom protein,CRVP）的全长cDNA序列。这两种crvp基因同源性为97％,分别编码238和199个氨基酸残基,其中包括一段相同的由19个氨基酸残基组成的信号肽。氨基酸序列分析表明,这两种CRVP蛋白与蜥蜴helothermine毒素蛋白以及台湾饭铲倩CRVP毒蛋白高度同源,而且具有半胱氨酸丰富分泌蛋白（cysteine-rich secretory protein,CRISP)家族的典型结构特征。     

Human nectin-like 1, a novel membrane protein interacting with protein 4.1 R

Human nectin-like 1 (NECL1) full-length cDNA was cloned by bioinformatics method when searching for candidate membrane proteins interacting with members of protein 4.1 family. The cytoplasmic and extracellular regions of NECL1 were expressed in and purified from E. coli, and the polyclonal antibody was produced. Interaction between the cytoplasmic region of NECL1 and the 30 kD membrane binding domain of protein 4.1 on red blood cell (4. 1R) was demonstrated by IAsys-biosensor system and GST pull-down experiment. Results of biotin-labeled peptide ELISA further demonstrated the key amino acids for the binding. The interaction research of NECL1's cytoplasmic domain provides basis for further study of the functions of NECL1 in nervous system.     

基于全基因组序列的尖孢镰刀菌分泌蛋白预测及其特征分析

尖孢镰刀菌是一种以土壤习居为主要特征的植物病原菌,广泛分布于世界各地,且寄主广泛,能引起豆科、茄科、葫芦科等科100多种植物根腐、茎腐、茎基腐等病害,严重时造成植株萎蔫死亡,严重影响着植物的产量和品质.同时,目前生产上对于该菌的防治多使用甲霜灵·锰锌、多菌灵、百菌清等化学药剂,然而,防治效果不佳,急需开发针对该菌新的作用靶标的药剂.前人已经明确植物病原真菌分泌蛋白在侵染、操控植物等过程中发挥着重要功能,然而,学术界尚未见有关该菌中分泌蛋白的报道.本研究以尖孢镰刀菌菌株(Fusarium oxysporumf.sp.lycopersici 4287)的蛋白序列为基础数据,以分泌蛋白所具有的N-端含有信号肽、不含有跨膜结构域、没有GPI锚定位点、将蛋白分泌在胞外4大特征为依据,利用生物信息学在线分析程序明确该菌中含有778个分泌蛋白,并对上述蛋白开展特征分析,明确上述蛋白的氨基酸长度主要集中在101～400aa、氨基酸组成中以G,T,S,A较多,信号肽长度主要集中在16～21aa、信号肽氨基酸组成中以P,S,A,T,G较多,信号肽切割位点为T-X-T类型.为今后进一步开展尖孢镰刀菌的分泌蛋白功能解析和开发新型药剂靶标提供了重要的理论基础.     

Characterization,gene cloning and expression of new xylanase XYNB with high specific activity

Extracellular xylanase XYNB from Streptomyces olivaeeoviridis A1 has been purified and characterized.The optimal pH value and temperature of XYNB for its activity are 5.2 and 60℃, respectively. The specific activity of XYNB is as high as 2869.78 U/mg. Metal cations, EDTA and SDS have no effects on enzyme activity of XYNB. The gene xynB coding mature protein of XYNB has been cloned by PCR. The forward oligonucleotide primer used in the PCR reaction was synthesized based on the N-terminal amino acid sequence of XYNB mature protein, and the reverse oligonucleotide primers are random oligonucleotide. The cloned gene xynB is 576 bp long and its G C content is 64.3%. The xynB encodes 191 amino acid residues, and the putative molecular weight of XYNB is 20.839 kD. The xynB has been expressed in E. coli, and the expressed xylanase has normal bioactivity.     

Cloning and expression of ophc2, a new organphosphorus hydrolase gene

The amino acid sequences of N-terminal and internal peptide of OPHC2, purified from Pseudomonas pseudoalcaligenes strain C2-1 in our lab, are determined. The full-length organphosphorus hydrolase gene ophc2 is cloned by PCR using the degenerate primers designed according to the sequences and future inverse PCR. The ophc2 gene is 975 bp long with G C content of 63%, comprising one open reading frame encoding a polypeptide of 324 amino acids with a molecular weight of 36 kD. The nucleotide sequence of ophc2 shows low homologies with those organphosphorus hydrolase genes deposited in GenBank, one of which exhibits the highest homology of 46.4% with ophc2. The organphosphorus hydrolase protein expressed in E. coli bears normal bioactivity.     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.     

暗纹东方纯CD8α基因克隆、原核表达与多克隆抗体制备

CD8是与Ⅰ型主要组织相容性复合体（MHCI）结合，是T细胞表面受体（TCR）的共受体．也是T淋巴细胞表面的重要标志物。该研究报道了暗纹东方纯胸腺等组织中克隆获得CD8αcDNA序列及其相应的基因组序列。克隆到的cDNA序列全长1061bp，包含1个657bp的开放读码框（ORF），编码218个氨基酸；其对应的基因组序列为1533bp，包含5个内含子和6个外显子。生物信息学分析表明，D8d蛋白质序列由信号肽、胞外可变区、铰链区、跨膜区和胞内区5部分组成。胞外区的可变区和铰链区部分各有两个高度保守的半胱氨酸残基，可能参与链内和链间二硫键的形成。氨基酸序列多重比对表明，暗纹东方鲍与其他鱼类的CD80α，特别是与鲽形目鱼类具有较高的同源性。为进一步研究暗纹东方纯CD8的生物学功能，构建了表达CD8α成熟肽胞外区的重组质粒，诱导表达出重组蛋白。以纯化的重组蛋白为抗原免疫大白兔，制备了抗血清。经间接ELISA法检测抗体效价表明。获得了高效价的特异性暗纹东方纯CD8c~抗体，为进一步研究CD8在鱼类淋巴细胞进化和适应性免疫中的作用奠定了基础。     

东亚钳蝎抗神经兴奋肽在大肠杆菌中的表达

自从 1 96 4年以来Ｍｉｒａｎｄａ等人[1] 报道Ａｎｄｒｏｔｏｎｕｓａｕｓｔｒａｌｉｓ和Ｂｕｔｈｕｓｏｃｃｉｔａｎｕ中的神经毒素 ,蝎毒中一些神经毒素尤其是哺乳动物毒素和昆虫毒素已被广泛研究 .从新鲜蝎毒中制备和纯化天然神经毒素困难重重 ,1 988年Ｃａｒｂｏｎｅｌｌ等人[2 ] 首次由Ｂｕｔｈｕｓｅｕｐｅｕｓ蝎中昆虫神经毒素氨基酸序列推导并化学合成其基因序列 ,且在杆状病毒载体中得到表达 ,自此人们广泛试验将各种属的系列蝎毒素基因重组到大肠杆菌、酵母、杆状病毒、植物等表达载体进行表达 .在中国 ,蝎子及其组织或抽提物被…     

中国明对虾CrusFc-2基因的重组表达及抗血清制备

利用PCR和DNA重组技术克隆了中国明对虾CrusFc-2基因的成熟肽编码序列并构建了pET-DsbA-CrusFc-2原核表达载体,转化E.coli BL21(DE3)后,经异丙基-β-D硫代半乳糖苷(IPTG)诱导得到了重组蛋白.重组蛋白经纯化后免疫新西兰大白兔制备了抗血清,Western Blot分析表明Crus...     

Molecular cloning of cDNA for murine interleukin-3

The cDNA sequence for murine interleukin-3, one of the colony stimulating factors that regulate haematopoiesis, codes for a polypeptide of 166 amino acids including a putative signal peptide. The predicted amino acid sequence indicates that formation of mature interleukin-3 involves proteolytic removal of not only the signal peptide but additional amino-terminal amino acids.     

中华鳖抗菌肽Hepcidin成熟肽基因的克隆及同源性的初步研究

采用同源克隆的方法,以用于扩增大菱鲆hepcidin成熟肽基因的引物,从中华鳖肝脏cDNA中扩增得到1条长78 bp的基因片段.初步判断其属于Hepcidin家族,与HAMP1具有较高的同源性,分析表明其为Hepcidin成熟肽.根据基因序列推断得到的成熟肽蛋白序列如下:QSHISLCRWCCNCCKANKGCGFCCKF.与其他物种的成熟肽进行比对,发现其与大菱鲆的成熟肽序列同源性达到100 %,并构建了基于各物种Hepcidin前体肽的系统发育树,为研究物种进化提供了证据.     

史氏鲟（Acipenser schrenckii）两种促性腺激素β亚基的原核表达

 采用遗传工程方法,重组表达史氏鲟两种促性腺激素β亚基蛋白。选用原核表达载体pET－22b(+),分别插入史氏鲟GtHⅠ&;Ⅱβ亚基成熟肽cDNA序列,构建成C端含有6个组氨酸(6-His)融合蛋白标签的表达质粒;分别转化大肠杆菌表达菌株BL21（DE3）并诱导表达2个基因。SDS-聚丙烯酰胺凝胶电泳显示重组融合蛋白相对分子质量分别为：GtHⅠβ亚基大约14 000,GtHⅡβ亚基15 000左右。分别用抗6个组氨酸融合标签的单克隆抗体及兔抗鲟鱼GTH多克隆抗体对2个表达蛋白进行Western Blot分析,结果显示重组蛋白表达正确且有较高的免疫活性。GTHⅠβ重组蛋白在2 h就有明显的表达,6 h后随着时间增加表达量不再增加;25 ℃诱导重组蛋白产量比37 ℃诱导产量低。获得的重组蛋白质将可用于建立鲟鱼GtH的放射免疫测定方法。     

Cloning and expression of murine interleukin-1 cDNA in Escherichia coli

Interleukin-1 (IL-1), a peptide hormone produced by activated macrophages, possesses the ability to modulate the proliferation, maturation and functional activation of a broad spectrum of cell types and may play a major role in the initiation and amplification of immune and inflammatory responses through its action on these diverse cell populations. IL-1 exhibits microheterogeneity in terms of its relative molecular mass (Mr, 13,000-19,000) and charge properties, and although murine IL-1 has been purified and some of its basic structure-function relationships have been elucidated, it has proved difficult to prepare sufficient amounts of IL-1 for direct and detailed sequence and structural studies. Here we report the cloning, sequence analysis and expression of murine IL-1 cDNA in Escherichia coli. The IL-1 cDNA codes for a polypeptide precursor of 270 amino acids. Biologically active IL-1 was produced in E. coli by expressing the carboxy-terminal 156 amino acids of the IL-1 precursor.     

禽多杀性巴氏杆菌X-73株ompH基因的克隆和表达

以禽多杀性巴氏杆菌国际标准株X-73基因组DNA为模板,采用PCR扩增得到编码信号肽除外的成熟外膜蛋白H的ompH基因,将ompH基因克隆至pMAL-p2X表达载体中,构建重组表达质粒pMAL-p2X-ompH,转化大肠杆菌BL21,用SDS-PAGE电泳检测IPTG诱导后的表达蛋白.DNA测序结果表明ompH基因片段大小为1 002 bp,与已报道的ompH基因的核苷酸序列完全相同、而SDS-PAGE结果显示表达分子质量约为78 ku,与预期的融合蛋白MBP-OmpH分子质量相符,表明成功构建出原核表达质粒并实现了目的蛋白表达,为进一步开展禽多杀性巴氏杆菌保护抗原的研究奠定基础.