抗人黑色素瘤单链抗体基因的克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞ＨＢ８７６０中克隆了抗体轻、重链可变区基因,然后用（Ｇｌｙ４Ｓｅｒ）３连接肽基因将ＶＨ、ＶＬ连接成ＳｃＦｖ基因,并进行了序列测定．计算机分析表明ＶＨ,ＶＬ均符合小鼠抗体可变区的特征,为功能性重排的抗体可变区基因．ＶＨ、ＶＬ、ｌｉｎｋｅｒ拼接正确．ＳｃＦｖ基因全长７２９ｂｐ,其中ＶＨ基因长３６０ｂｐ,编码１２０个氨基酸,ＶＬ基因长３２４ｂｐ,编码１０８个氨基酸．在噬菌粒表达载体ｐＣＡＮＴＡＢ５Ｅ中表达了可溶性的ＳｃＦｖ蛋白,表达产物经流式细胞仪检测可特异地与黑色素瘤细胞结合,不与肝癌、胃癌及良性黑痣细胞结合     

Overexpression, Purification, and Refolding of a Porphyromonas gingivalis Cysteine Protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.     

香蕉枯萎病拮抗放线菌Da08006的筛选与鉴定

以尖孢镰刀菌4号小种为指示菌株,对海南尖峰岭原始森林和发病香蕉园健康植株根际土壤的放线菌进行筛选,获得一株具有较强拮抗作用、遗传稳定的放线菌Da08006.通过形态特征、生理生化特征、16S rDNA序列及其系统发育分析研究,鉴定菌株Da08006为Streptomyces morookaense.     

太阳辐射减弱对冬小麦旗叶光合速率的影响

以冬小麦扬麦13号为供试材料,设计了15%(T₁₅)、20%(T₂₀)、40%(T₄₀)、60%(T₆₀)和100%（CK）自然光5个处理,在大田条件下研究了模拟太阳辐射减弱对
冬小麦旗叶光合速率日变化及其影响因素的影响.结果表明：太阳辐射减弱显著提高了冬小麦叶绿素和叶黄素含量,降低了光合速率（P_n）.不同辐射减弱条件下冬小麦P_n日变化差异较大,日最高值表现为CK＞60%自然光＞40%自然光＞20%自然光＞15%自然光,其中CK呈双峰曲线变化,有明显的“午休”现象,其他各处理均呈单峰型曲线变化,“午休”现象不明显,但峰值出现时间滞后.相关分析表明,太阳辐射减弱是影响P_n日变化的主导因子,但其他因子也显著影响P_n.与CK相比,60%和40%自然光处理中光合有效辐射（PAR）、叶温（T_l）、气孔导度（G_s）和蒸腾速率（T_r）与P_n均呈显著正相关,表明上述因子对P_n有正效应;冬小麦叶片胞间二氧化碳浓度（C_i）和气孔导度限制值（L_s）在60%和40%自然光处理中与P_n呈显著负相关,但在20%和15%自然光处理中与P_n呈显著正相关,说明太阳辐射强度高于40%自然光时C_i和L_s对P_n有负效应,太阳辐射强度低于40%自然光时则为正效应.     

抗乙型肝炎病毒表面抗原“y”亚型及“a”组抗原决定簇杂交瘤细胞株的建立与应用

用部分纯化的HBsAg/adr和ayw亚型免疫Balb/c小鼠的脾细胞,在PEG作用下与Sp2/o骨髓瘤细胞进行融合,经ELISA及RIA法筛选出了分泌抗HBsAg/a(SH 1 D9)、抗HBsAg/y(SG 3 B10)亚型决定簇的杂交瘤细胞系,在组织培养上清液中它们的滴度分别     

不同pH模拟酸雨对冬小麦籽粒营养品质的影响

在大田试验条件下研究了不同pH模拟酸雨对冬小麦籽粒氨基酸、蛋白质、可溶性糖、还原糖、淀粉以及总酸度的胁迫效应,以期为防治我国农作物的酸雨灾害提供基础理论依据。结果表明,pH≤4.5酸雨处理会抑制总游离氨基酸的合成,且酸度越强,抑制越明显。蛋白质(可溶性蛋白质、总蛋白)含量随着酸雨酸度的增强逐步下降,且在极酸性条件下降低较明显。pH≤4.5酸雨处理对可溶性糖的合成会有较强的抑制作用。pH≤2.5时还原糖的合成明显受抑。随着酸雨酸度的增强,总酸度和酸糖比均表现出先增加后下降的变化趋势,且在pH=3.5处达到最大值。pH≤3.5酸雨处理显著抑制淀粉的合成,且对支链淀粉的影响较明显,致使淀粉支/直比下降。