抗丙肝病毒核心抗原单克隆抗体的研制与初步鉴定

用基因工程重组技术获得的丙肝病毒（ＨＣＶ）核心蛋白抗原与鼠血清白蛋白交联后免疫Ｂａｌｂ／ｃ小鼠,用杂交瘤技术成功地建立了４株稳定分泌抗核心抗原单克隆抗体的杂交瘤细胞,试验结果表明,该４株ＭｃＡｂｓ与免疫抗原及核心区Ｃ３３肽、ＣＰ９、ＣＰ１０抗原有较强的抗原－抗体反应,与ＨＣＶ ＮＳ３、ＮＳ４、ＮＳ５无反应,在竞争ＥＬＩＳＡ中,对ＨＣＶ－ＩｇＧ阳性血清有较好的抑制作用。４株ＭｃＡｂｓ中３株为ＩｇＧ２     

野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…     

丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究

用ＰＣＲ 方法从丙型肝炎病毒（ＨＣＶ） ｃＤＮＡ 文库中克隆了两段ＤＮＡ 片段,即ＨＣＶ 基因组非结构ＮＳ３区抗原基因（约０．７ ｋｂ）和核心抗原Ｃ区抗原基因（约０．６ ｋｂ）的ｃＤＮＡ 片段。在两段ｃＤＮＡ 间加入连接肽Ｓｅｒ－ Ｐｒｏ－ Ｇｌｙ－ Ｓｅｒ 的密码子序列,构建成融合抗原基因ＮＳ３－ Ｃ。将该融合基因与衣藻叶绿体基因ａｔｐＡ 的启动子和ｒｂｃＬ 基因的３′末端连接,得到丙肝病毒融合抗原基因ＮＳ３－ Ｃ表达盒,再将该表达盒与选择标记基因ａａｄＡ 表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体ｐＳＳ６。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交分析表明,融合抗原基因ＮＳ３－ Ｃ已整合到衣藻叶绿体基因组中。     

从睡眠剥夺树鼩（TBC）尿液提取内源性“睡眠因子”（sleep factor）的研究

本研究报道从睡眠剥夺（ＳＤ）４８—７２ｈ的灵长类原宗（ｐｒｉｍｉｔｉｖｅｓｔｏｃｋ）Ｔｕｐａｉａｂｅｌａｎｇｅｒｉｃｈｉｎｅｎｓｉｓ（ＴＢＣ）提取内源性“睡眠因子”（ｓｌｅｅｐｆａｃｔｏｒ）Ｓ２Ｃ和Ｓ４Ｂ。收集的尿液经超滤,清液冻干经ＳｅｐｈａｄｅｘＧ１５分离得到ＦｒａｃｔｉｏｎⅠ－Ⅴ。活性测定发现Ｆｒａｃｔｉｏｎ－Ⅲ（Ｓ２Ｃ）呈现显著δ－增强促眠效应。经ＳｅｐｈａｄｅｘＧ２５和ＳｅｐｈａｄｅｘＬＨ２０进一步净化的Ｓ４Ｂ也呈现显著δ－增强促眠效应。     

新型HCV EIA诊断试剂盒的研制

丙型肝炎病毒（ＨＣＶ）基因组结构区核壳蛋白（Ｃ）区抗原、膜蛋白Ｅ１和Ｅ２区抗原,以及非结构区ＮＳ３－ＮＳ５区抗原的区段,已经在原核细胞中获得有效的表达。同时,相应区段中的优势抗原表位肽也经化学合成法大规模地制备。ＨＣＶ基因组上各区段抗原性的分析发现,由Ｃ区和ＮＳ３区分别编码的Ｃ抗原和Ｃ３３ｃ抗原是ＨＣＶ基因组上两个优势抗原区段。其相应的抗体出现早（感染后６周可检出抗Ｃ３３ｃ抗体）,阳转率高（约９９％阳性检出率）,特异性和重复性均优于其它区段抗原。以中国人ＨＣＶ的Ｃ３３ｃ重组蛋白和分支状合成肽ＭＡＰ－Ｃ－１９为复合抗原,研制了适合我国抗ＨＣＶ抗体检测的新型丙型肝炎病毒酶免疫测定（ＨＣＶＥＬＡ）诊断试剂盒。它同当代美国Ａｂｂｏｔｔ／ＵＢＩＨＣＶＥＬＡ诊断试剂的符合率约９８％,同加拿大ＹＥＳ公司ＨＣＶＥＩＡ诊断试剂的符合率约９７．８％,阳性检出率提高了约２％,３次重复性达１００％,表明其特异性、敏感性和重复性均达到了当代第二代ＪＣＶＥＬＡ诊断试剂的水平。我国人群中抗ＨＣＶ抗体的分布情况为：正常人群的检出率１％－２％;外科类住院病人检出率约２８．８％;肝炎患者抗ＨＣＶ阳性率为３４．４％,慢活肝、肝硬化和重症肝炎患者     

HCG抗原决定簇与乙肝病毒核心抗原的融合表达

利用ＰＣＲ方法获得编码人绒毛膜促性腺激素β链羧端３７肽基因片段（ＨＣＧ－β－３７－ＣＴＰ）,并将其分别和乙肝核心抗原的氨端（第１位）、羧端（第４５４位）、中间（第７５～８３位）以及中间和羧端同时融合,构建Ｔ４个重组融合表达克隆：ｐＣｎ－ＨＣＧ、ｐＣｃ－ＨＣＧ、ｐＣｍ－ＨＣＧ和ｐＣ－ＨＣＧ２,在大肠杆菌中实现了表达。对表达产物中的乙肝核心抗原和ＨＣＧ抗原的抗原性和表达水平、融合蛋白的颗粒性及其免疫原性进行了分析。其中ｐＣｍ－ＨＣＧ和ｐＣｃ－ＨＣＧ能形成颗粒。用ｐＣｍ－ＨＣＧ免疫小鼠能产生高滴度的抗ＨＣＧ抗体,说明核心抗原的中间部位是合适的融合位点。     

从睡眠剥夺树qu（TBC）尿液提取内源性“睡眠因子”的研究

本研究报道从睡眠剥夺（ＳＤ）４８－７２ｈ的灵长类原宗Ｔｕｐａｉａ ｂｅｌａｎｇｅｒｉ ｃｈｉｎｅｎ－ｓｉｓ（ＴＢＣ）提取内源性“睡眠因子”Ｓ２Ｃ和Ｓ４Ｂ。收集的尿液经超滤,清液冻干经Ｓｅｐｈａｄｅｘ Ｇ１５分离内源性“睡眠因子”Ｓ２Ｃ和Ｓ４Ｂ。收集的尿液经滤,清液冻干经ＳｅｐｈａｄｅｘＧ１５分离得到Ｆｒａｃｔｉｏｎ Ｉ－Ｖ。活性测定发现Ｆｒａｃｔｉｏｎ－Ⅲ（Ｓ２Ｃ）呈现显著δ－增强促眠效应。经Ｓｅ     

乙肝表面抗原专一的单链嵌合抗体在大肠杆菌中的表达

利用重组ＰＣＲ技术将乙肝表面抗原专一单抗的Ｖｋ与人源的Ｃ基因拼接成轻链嵌合抗体Ｖ－Ｃ,再通过编码柔性肽段（Ｇｌｙ４Ｓｅｒ）的Ｌｉｎｋｅｒ与Ｖ连接成单链嵌合抗体ＳｃＦＶ－Ｃ,并分别在大肠杆菌的热敏与分泌型表达系统中进行表达。表达产物的Ｗｅｓｔｅｒｎ－ｂｌｏｔ与间接ＥＬＩＳＡ检测结果表明,ＳＣＦｖ－Ｃ产两种系统中的表达产物均具有抗原结合活性,并已在分泌肽的指导下ＳｃＦｖ－Ｃｋ能被Ｅ．ｃｏｌｉ分泌出胞外。     

丙型肝炎病毒基因组C区和NS3区基因cDNA的融合及其在大肠杆菌中的表达与初步应用

通过逆转录（ＲＴ）－聚合酶链式反应（ＰＣＲ）,从中国人丙型肝炎病毒（ＨＣＶ）携带者的血清中扩增并克隆到２段ｃＤＮＡ片段,即ＨＣＶ基因组Ｃ区抗原基因Ｃ８３１ｃＤＮＡ片断（约５３０ｂｐ）和ＮＳ３区抗原基因Ｃ３３ｃｃＤＮＡ片段（约８６０ｂｐ）。Ｃ３３ｃｃＤＮＡ片段同Ｃ８３１ｃＤＮＡ片段经连接　　　肽Ｓｅｒ－Ｐｒｏ－Ｇｌｙ－Ｓｅｒ连接成为基因嵌合体Ｃ３３ｃ－Ｃ８３１（约１４００ｂｐ）。Ｃ３３ｃ－Ｃ８３１基因嵌合体同温控型原核表达载体ｐＢＶ２２０重组,构建成表达质粒ｐＢＶ／Ｃ３３ｃ－Ｃ８３１,并在大肠杆菌细胞中获得了重组嵌合抗原Ｃ３３ｃ－ＣＬ的表达。通过酶切分析和Ｗｅｓｔｅｒｎ免疫印迹法,对约占菌体可溶性蛋白９％的表达产物做了鉴定。采用ＴｒｉｔｏｎＸ－１００和盐析处理,获得粗提表达产物。粗提的表达产物经尿素裂解和离子交换层析纯化,得到可用于检测抗ＨＣＶ核壳蛋白和抗ＮＳ３区抗体的重组嵌合抗原Ｃ３３ｃ－ＣＬ。对Ｃ３３ｃ－ＣＬ做抗原性分析发现,它同时具有完整的Ｃ３３ｃ抗原和Ｃ２２抗原的免疫反应活性,完全能替代单纯的Ｃ３３ｃ和Ｃ２２抗原。该嵌合抗原在血清学诊断中有重要的应用价值,可望成为新一代ＨＣＶＥＩＡ诊断试剂的优选抗原。     

木兰亚纲的分子系统学研究进展：（一）叶绿体rbcL基因序列的分支分析

木兰亚纲的分子系统学研究进展（一）叶绿体ｒｂｃＬ基因序列的分支分析王艇,苏应娟,Ｃ．Ｒ．Ｐａｒｋｓ（中山大学生物系,广州５１０２７５）（美国北卡罗来纳大学生物系）ＰＲＯＧＲＥＳＳＩＮＭＯＬＥＣＵＬＡＲＳＹＳＴＥＭＡＴＩＣＳＯＦＴＨＥＭＡＧＮＯＬＩＩＤ...     

光对水稻非光合组织谷氨酰胺合成酶同工酶表达的影响

以前的研究表明,高等植物叶绿体谷氨酰胺合成酶（GS2）受光调节,但叶片胞液GS（GS1）和非光合作用组织中的GS很少受光的影响,在本报道中,笔者运用GS活性染色和Western blotting研究了光对非光合作用组织水稻根GS同工酶表达的影响,在阳光的直接照射下以及在室内不同光照强度下,可以很清楚地观察到GSra和GS rb的活性带及其蛋白质带,但是,当用尼龙网档住阳光的直接照射下,GSrb的活性带和蛋白质带消失,当阳光被尼龙网遮挡住后,其光强度仍然比室内光照强度大得多,表明光照强度不是影响GSrb表达的主要因素,当分析生长在暗处以及生长在光／暗转换下的水稻幼苗根GS同工酶变化时,仍然可以观察到GSrb的在,在所有实验条件下,GSra都未发生明显变化,这些结果提示,光对GSrb表达的影响可能是由某些光谱相互作用所产生的未知因素造成的。     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Immunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody

The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch [1989] Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific GS that does not cross-react either with a second GS isozyme found in the pedicel or with the GS isozymes from the embryo, roots, or leaves. When used as a probe for tissue printing, the antibody labeled the pedicel tissue uniformly and also labeled some of the pericarp surrounding the lower endosperm. Silver-enhanced immunogold staining of whole-kernel paraffin sections revealed the presence of GSp1 in both the vascular tissue that terminates in the pedicel and the pedicel parenchyma cells, which are located between the vascular tissue and the basal endosperm transfer cells. Light staining of the subaleurone was also noted. The tissue-specific localization of GSp1 within the pedicel is consistent with its role in the metabolism of nitrogenous transport compounds as they are unloaded from the phloem.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

不同氮源对小麦幼苗谷氨酰胺合成酶的影响

利用ＤＥＡＥ－纤维素柱层析、酶活性测定、Ｎｏｒｔｈｅｒｎ 分子杂交等技术,研究了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）幼苗的根、叶和离体叶在不同氮源培养条件下谷氨酰胺合成酶（ＧＳ）活性和同工酶变化, 以及不同氮源对ＧＳ基因转录－ＧＳ－ｍ ＲＮＡ 的影响． 同时与硝酸还原酶（ＮＲ）活性进行比较, 结果表明∶当以ＮＨ＋４ 作唯一氮源时,小麦幼苗根谷氨酰胺合成酶（ＧＳｒ）和叶细胞质谷氨酰胺合成酶（ＧＳ１）活性要比以ＮＯ－３ 作唯一氮源的高．当以ＮＯ－３ 为唯一氮源时, ＮＯ－３ 则促进完整叶片和离体叶片叶绿体谷氨酰胺合成酶（ＧＳ２）活性． 从转录水平上看,ＮＨ＋４ 促进根ＧＳ－ｍ ＲＮＡ 的合成,而ＮＯ－３ 促进叶ＧＳ－ｍ ＲＮＡ 的合成     

Cytosolic glutamine synthetase in higher plants. A comparative immunological study

Cytosolic glutamine synthetase (GS1) was purified to homogeneity from etiolated barley leaves by DEAE-Sephacel and hydroxyapatite chromatography, gel filtration and polyacrylamide gel electrophoresis. Specific antibodies against the purified protein were raised by the immunization of rabbits. Immunoprecipitation experiments demonstrated that cytosolic glutamine synthetases isolated from the leaves of different plant species were very similar proteins. Good recognition of other cytosolic glutamine synthetases from roots, root nodular tissue and seeds by barley GS1 antibodies was obtained, suggesting that they too are all quite similar proteins. In contrast, chloroplast glutamine synthetase (GS2) was considered to be a different protein in view of its low level of recognition by barley GS1 antibodies.     

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots

PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a cDNA library prepared from mRNAs of rice shoots utilizing the product of PCR as probe. Two cDNA clones, prxRPA and prxRPN, were isolated. They encode distinct isozymes of peroxidase. Sequence analysis indicated that the clones encode mature proteins of approximately 32 kDa, both of which possess a putative signal peptide. Comparison of the amino acid sequences of the two rice peroxidases showed that they are about 70% similar to each other but are only 40% to 50% similar to other plant peroxidases. RNA blot hybridization revealed that mRNAs that corresponded to prxRPA and prxRPN cDNAs accumulate at high levels in roots but only at low levels in stems and leaves. In various tissues of rice plants, levels of both mRNAs were stimulated by wounding and by ethephon. These results indicate that at least two isozymes of peroxidase are expressed not only in shoots but also in roots of rice plants, and that the expression of these genes is influenced by ethylene which is the simplest plant hormone.     

Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.     

蔗糖对水稻幼苗叶片谷氨酰胺合成酶和1,5-二磷酸核酮糖羧化酶/加氧酶的影响

报道了在光照和暗处培养下,不同的浓度的蔗水稻幼苗叶片GS及其同工酶、1,5－二磷酸核酮糖羧化酶／加氧酶（Rubisco）的影响。无论是在光照或在暗处,蔗糖对GS活性均有抑制作用,尤其是在较高蔗糖下作用更为明显;虽然Rubisco及可溶性蛋白的水平在光照和暗处有显著的差别,但蔗糖对其未见明显影响。NativePAGE与活性染色表明,在光照下或在暗处,蔗糖对GS2的抑制蔗糖浓度升同而加强,但对GS1未有明显影响。这些结果提示,在水稻幼苗生长中,蔗糖不能象不光一样诱导叶水GS活性及其同工酶表达。