跨膜逆向转运蛋白NHXFS1多克隆抗体的制备及应用

NHXFS1基因是通过DNA家族改组（DNA family shuffling）技术,以拟南芥、水稻和菊花的液泡膜Na＋/H＋逆向转运蛋白基因（NHX1）为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21（DE3）并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1：128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。     

人PD1胞外须基因的克隆、蛋白表达及抗体制备

目的：研究人PD1的生物学活性,制备人PD1胞外段区域及其特异性抗体。方法：用PCR方法扩增编码人PD1胞外段的基因序列（hPD1ecr）,将其克隆到原核表达载体pET28a（＋）中,并转化至大肠杆菌BL21（DE3）中诱导表达,表达蛋白用SDS-PAGE和Western blot鉴定。纯化目的蛋白免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附实验（ELISA）,流式细胞术检测抗体滴度及其特异性。结果：原核表达载体pET28a（＋）-PD1ecr成功构建,并可在大肠杆菌BL21（DE3）中诱导表达,得到的PD1胞外区蛋白经SDS-PAGE和Western blot鉴定正确。用纯化蛋白免疫日本大耳白兔,制备的多克隆抗体具有较强免疫特异性。结论：得到纯化的人PD1胞外蛋白,制备的多克隆抗体能够检测自然状态下人PD1蛋白,为进一步研究PD1功能奠定了实验基础。     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

家蚕GAPDH内参蛋白多克隆抗体的制备及其检测

制备家蚕GAPDH内参蛋白多克隆抗体,并对该抗体进行检测。利用PCR技术从家蚕中克隆GAPDH基因,构建其原核表达载体,转化大肠杆菌,诱导表达重组蛋白并纯化。纯化后的蛋白作为抗原免疫新西兰大白兔制备GAPDH多克隆抗体。用酶联免疫吸附法和Western blot检测抗体的效价和特异性。结果显示,成功构建GAPDH/p ET-28a原核表达载体,获得高纯度的GAPDH重组融合蛋白;经SDS-PAGE和抗His单抗检测,纯化后蛋白的分子量大小与预测的一致;以该蛋白为免疫抗原,采用4次免疫方式对新西兰大白兔进行免疫,获得GAPDH多克隆抗体血清。酶联免疫吸附检测结果表明,GAPDH抗体的效价为1:8000,并能与天然家蚕蛋白特异性结合。成功制备了家蚕内参蛋白GAPDH多克隆抗体,为深入研究家蚕中不同蛋白的生理功能和作用奠定了坚实的基础。     

MORF4L1蛋白抗体制备及生物信息学分析

[目的]构建MORF4L1原核表达载体和纯化表达产物,制备MORF4L1蛋白抗体并进行生物信息学分析。[方法]利用PCR将MORF4L1基因片段酶切、克隆、转化至大肠杆菌Bl21(DE3),经IPTG诱导表达蛋白。利用His-Tag技术纯化重组蛋白,用重组蛋白免疫新西兰白兔,采集抗血清后通过硫酸铵沉淀法将多克隆抗体从抗血清中纯化,应用Western Blotting验证多克隆抗体与内外源性MORF4L1蛋白的结合特异性。利用在线软件(GEPIA、UALCAN)进行生物信息学分析。[结果]成功构建了重组蛋白,蛋白纯化后在相对分子质量(Mr)43000位置处有单一条带,重组His-MORF4L1蛋白与His抗体发生特异性结合。制备的抗血清与MORF4L1蛋白特异性结合,纯化后仅在相对分子质量(Mr)55000和25000位置处有条带。纯化的多克隆抗体与内外源性MORF4L1蛋白发生特异性反应。MORF4L1的表达与肝癌不良预后相关。[结论]成功构建MORF4L1蛋白及抗体,MORF4L1在肝癌中高表达预后差,对进一步研究MORF4L1蛋白的结构和生物学功能具有重要意义。     

小鼠Prune蛋白DHH结构域的原核表达及多克隆抗体的制备

[目的]表达、纯化小鼠Prune蛋白DHH结构域(m-Prune D),并制备多克隆抗体。[方法]生物信息学方法分析m-Prune D氨基酸序列;PCR扩增目的基因m-Prune D,克隆入原核表达载体p ET28a(+);IPTG诱导目的基因表达,SDS-PAGE和Western Blot鉴定蛋白表达,亲和层析法纯化蛋白;用纯化的重组m-Prune D免疫小鼠制备多克隆抗体;Western Blot检测多克隆抗体特异性。[结果]PCR成功扩增m-Prune D基因,双酶切及测序结果表明成功构建m-Prune D原核表达载体,SDS-PAGE和Western Blot鉴定表明成功表达约25 k Da的重组蛋白。纯化蛋白免疫小鼠后抗体滴度最高可达1∶25 600,所制备的多克隆抗体可特异性识别原核和真核细胞中DHH结构域蛋白。[结论]在E.coli中成功表达小鼠Prune蛋白DHH结构域,制备了多克隆抗体血清,可用于Prune蛋白生物学功能的进一步研究。     

人源核受体hLRH-1 的表达纯化及多克隆抗体制备

目的：制备抗人源核受体hLRH-1 的多克隆抗体,为进一步研究其功能奠定基础。方法：构建含有hLRH-1基因全长克隆的 原核表达载体pET507a-hLRH-1 并用IPTG 诱导其在Rosseta2 菌株中表达重组蛋白His-hLRH-1,经亲和层析纯化后按常规方法 免疫新西兰兔制备多克隆抗体,并用Western Blot 对其特异性进行鉴定。结果：原核表达载体pET507a-hLRH-1经测序证实构建 成功,将其转化大肠杆菌Rosseta2菌株后成功诱导表达重组蛋白His-hLRH-1,经纯化免疫新西兰兔后得到抗hLRH-1 多克隆抗 体,Western blot 证实抗体具有高度特异性。结论：成功表达His-hLRH-1 重组蛋白并制备出多克隆抗体,为进一步用于hLRH-1 的 免疫学检测及其功能研究奠定了基础。     

神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

DC-SIGN胞外区基因的克隆、蛋白表达及抗体制备

旨在构建DC-SIGN胞外区基因原核表达质粒,诱导蛋白表达并制备多克隆抗体。用PCR的方法扩增编码DC-SIGN胞外区的基因序列,将其克隆到原核表达载体pET-28a(+)中,利用大肠杆菌表达系统表达DC-SIGN胞外区蛋白,用H is抗体做W estern Blotting鉴定目的蛋白的免疫原性。用纯化的DC-SIGN胞外区蛋白免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,免疫荧光法检测其特异性。结果显示,原核表达载体pET-28 a(+)-DC-SIGN胞外区基因成功构建,可在大肠杆菌BL21(DE3)中高效表达,获得相对分子质量约20 kD的DC-SIGN胞外区蛋白,经Westernb lotting鉴定为正确。纯化后的蛋白免疫大耳白兔,制备的多克隆抗体具有较强免疫原性和特异性。本研究得到了纯化的DC-SIGN胞外区蛋白,并制备了具有特异性和高效价的抗体,为研究DC-SIGN生物学功能提供试验基础。     

香蕉MaROP1蛋白抗体的制备

ROP(Rho-related GTPases from plants)蛋白是一类在植物信号转导方面有重要作用的蛋白质.将香蕉中的MaROP1基因连接在pET-30a载体上,并在大肠杆菌BL21(DE3)中诱导表达,获得了融合表达蛋白.将融合蛋白免疫新西兰大白兔制备了MaROP1的高效价、特异的多克隆抗体,为进一步深入研究MaROP1的生物学特性及其在香蕉果实成熟过程中的功能奠定了基础.     

An apple sucrose transporter MdSUT2.2 is a phosphorylation target for protein kinase MdCIPK22 in response to drought

Sugars increase with drought stress in plants and accumulate in the vacuole. However, the exact molecular mechanism underlying this process is not clear yet. In this study, protein interaction and phosphorylation experiments were conducted for sucrose transporter and CIPK kinase in apple. The specific phosphorylation site of sucrose transporter was identified with mass spectrometry. Transgenic analyses were performed to characterize their biological function. It was found that overexpression of sucrose transporter gene MdSUT2.2 in apple plants promoted sugar accumulation and drought tolerance. MdSUT2.2 protein was phosphorylated at Ser³⁸¹ site in response to drought. A DUALmembrane system using MdSUT2.2 as bait through an apple cDNA library got a protein kinase MdCIPK22. Bimolecular fluorescence complementary (BiFC), pull‐down and co‐immunoprecipitation (Co‐IP) assays further demonstrated that MdCIPK22 interacted with MdSUT2.2. A series of transgenic analysis showed that MdCIPK22 was required for the drought‐induced phosphylation at Ser³⁸¹ site of MdSUT2.2 protein, and that it enhanced the stability and transport activity of MdSUT2.2 protein. Finally, it was found that MdCIPK22 overexpression promoted sugar accumulation and improved drought tolerance in an MdSUT2.2‐dependent manner in transgenic apple plants. MdCIPK22‐MdSUT2.2 regulatory module shed light on the molecular mechanism by which plant accumulates sugars and enhances tolerance in response to drought stress.     

A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance

Soil salinity is one of the major abiotic stressors that negatively affect crop growth and yield. Salt stress can regulate antioxidants and the accumulation of osmoprotectants. In the study, a sucrose transporter MdSUT2.2 was identified in apple. Overexpression of MdSUT2.2 gene increased salt tolerance in the transgenic apple, compared with the WT control “Gala.” In addition, it was found that protein MdSUT2.2 was phosphorylated at Ser²⁵⁴ site in response to salt. A DUAL membrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13 interacted with MdSUT2.2. A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt‐induced phosphorylation of MdSUT2.2 protein and enhanced its stability and transport activity. Finally, it was found that MdCIPK13 improved salt resistance in an MdSUT2.2‐dependent manner. These findings had enriched our understanding of the molecular mechanisms underlying abiotic stress.     

A test of fruit varieties on entry rate and development by neonate larvae of the codling moth,Cydia pomonella

The rate of entry by neonate larvae of the frugivorous codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), into fruit material was investigated. We used no‐choice bioassays in climate‐controlled rooms to assay larval entry across four host plant species (apple, pear, quince, walnut) and three varieties within a single fruit species (apple). Larvae successfully entering apples were reared to adulthood, and we collected tissue samples from apples which were successfully colonized in order to determine sucrose concentrations. This information was used to evaluate differences in adult moth size, development time, and pulp sucrose concentration due to apple variety. Four important findings emerged: (1) neonate larvae had the highest frequency of entry (86% of larvae) into apple fruits, compared with pear (78%), quince (56%), and walnut (32%); (2) the frequency of larval entry into immature apples differed across apple varieties, and larval entry rate was highest in variety Golden Delicious (72%), compared with Granny Smith (46%) and Red Delicious (64%); (3) on average, adult moths were larger and development times were shorter on the variety with the highest entry frequency (Golden Delicious); and (4) apple pulp sucrose concentrations were higher for Golden Delicious (17.5 μg mg^?1) than for either Granny Smith (15.9 μg mg^?1) or Red Delicious (15.1 μg mg^?1) varieties, which correlates positively with entry and development data. We conclude that host fruit species and varietals within a species affect the entry rate of neonate codling moth larvae in no‐choice assays. We hypothesize that larval development is influenced by mean sucrose concentrations or other phytochemical differences associated with host fruit varieties.     

Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

Aminoethoxyvinylglycine Effects on Late-Season Apple Fruit Maturation

Aminoethoxyvinylglycine (AVG) inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and thus blocks ethylene synthesis. Preharvest foliar application of AVG to apple (Malus domestica Borkh.) fruit retards several key events of maturation including climacteric ethylene production, starch conversion to sugars, fruit softening, and abscission zone development. Although the impact of AVG on apple fruit maturation is well known, the biochemical basis of these effects is not well understood. The effects of AVG application on Redchief Delicious apple fruit maturation were studied. AVG applied four weeks prior to harvest significantly reduced internal ethylene levels, amylose degradation, and accumulation of sucrose, glucose, and sorbitol. Because AVG application coincidentally inhibited starch degradation and the increase in internal ethylene, we investigated the enzymatic basis of starch mobilization in apple fruit. Amylase activity was somewhat reduced in AVG-treated fruit. Amylase activity was less in AVG-treated fruit during the early stages of starch mobilization. Starch phosphorylase activity increased dramatically during the later stages of starch mobilization, but was not affected by AVG treatment. Soluble starch synthase activity was also unaffected by AVG treatment and remained constant throughout the eight-week harvest period. Moreover, AVG did not affect the levels of amylopectin, fructose, malate, ascorbate, citrate, or anthocyanin. These results suggest that apple fruit ripening has both ethylene-dependent and -independent processes occurring simultaneously.     

The potential of a sucrose ester coating material for improving the storage and shelf-life qualities of Cox's Orange Pippin apples

A sucrose-ester coating material was tested for its potential as a storage technique and as an extender of the shelf life of apple (cv. Cox's Orange Pippin). Apples treated with 1·25% sucrose ester formulation were stored in air at 3·5°C for up to 5 months. Sucrose ester treatment did not reduce detrimental changes in terms of fruit firmness, yellowing and weight loss but did increase core flush incidence. When applied after storage, the sucrose ester reduced yellowing and loss of firmness and markedly increased internal carbon dioxide levels during a 21 day simulated marketing period. Effects were enhanced with increasing sucrose ester concentrations between 1% and 4%. Sucrose ester did not markedly reduce weight loss in the fruit, did not cause accumulation of alcohol or induce any internal physiological disorders during the simulated marketing period. Treatment of fruit with an external atmosphere containing 8% carbon dioxide, a level similar to that found in fruit treated with 3% sucrose ester, did not have the same effects as 3% sucrose ester on firmness or ground colour changes, suggesting that the effects of the sucrose ester are not solely the result of the raised carbon dioxide level.     

锌对苹果果实膨大期叶片13C光合产物合成及向果实转移分配的影响

为了探究锌对叶片光合产物向果实转移分配的影响机理,为苹果果实发育关键时期通过补锌措施提高果实品质奠定理论基础,以8年生‘寒富'/GM256/山定子为试材,采用¹³C同位素标记技术,研究苹果树叶片涂抹不同浓度锌(CK、Zn₁、Zn₂、Zn₃、Zn₄,分别代表浓度为0、0.1%、0.2%、0.3%和0.4%的ZnSO₄·H₂O溶液)对果实膨大期叶片¹³C同化能力及¹³C光合产物向果实运输的影响。结果表明: 随着锌浓度的增加,苹果树叶片Rubisco酶活性、净光合速率、山梨醇和蔗糖含量、6-磷酸山梨醇脱氢酶和蔗糖磷酸合酶活性,以及¹³C同化能力呈现先升高后降低的趋势,且均在Zn₃处理下达到最高。与其他处理相比,Zn₃处理的¹³C自留量(标记叶片+标记枝条)最低,为61.2%,而输出量最高,为38.8%。果实¹³C吸收量表现为Zn₃＞Zn₂＞Zn₄＞Zn₁＞CK。这说明叶片涂抹适宜浓度锌(0.3%ZnSO₄·H₂O溶液)处理增强了叶片的光合作用,提高了叶片光合产物的合成能力,促进了光合产物由叶片向果实的定向运输。     

Acid invertase is predominantly localized to cell walls of both the practically symplasmically isolated sieve element/companion cell complex and parenchyma cells in developing apple fruits

The acid invertase (β‐fructosidase, EC 3·2·1·26) was localized at subcellular level via immunogold electron microscopy in the phloem‐unloading zone of developing apple fruit. The enzyme (immunogold particles) was found to reside predominantly in the cell walls of the sieve element/companion cell (SE/CC) complex, phloem parenchyma cells and other parenchyma cells. There was almost no gold particle found in cytoplasm and vacuole. This distribution pattern remained unchanged throughout the growing season, but the enzyme numbers varied. The density of immunogold particles increased during fruit development. The immunoblotting of soluble and insoluble acid invertases provided a supporting proof for the assays of immunolocalization. The biochemical analysis showed a predominantly cell‐wall‐distributed activity of acid invertase that corresponds essentially with its amount distribution. The ultrastructural observations showed that there were numerous plasmodesmata between the parenchyma cells, but almost no plasmodesmium between the SE/CC complex and its surrounding parenchyma cells, practically resulting in the symplasmic isolation of the SE/CC complex. It is therefore suggested that the unloading pathway of sucrose from the SE/CC complex may be predominantly apoplasmic in the developing apple fruit, and that the unloaded sucrose may be hydrolysed by the functional acid invertase localized in the cell wall before it is loaded in sink cells.