Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

BACKGROUND: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. METHODS: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. RESULTS: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. CONCLUSION: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.     

不同产量潜力小麦品种氮素积累与转运的差异

为探明不同产量潜力小麦品种氮素积累与转运的规律,于2019-2020年度小麦生长季,以3个产量潜力不同的小麦品种烟农1212、济麦22和良星99为供试材料,分析了3个小麦品种氮素积累、转运和籽粒产量的差异。结果表明,烟农1212在小麦拔节至开花期和开花至成熟期植株氮素积累速率显著高于济麦22和良星99,开花期和成熟期植株氮素积累量也显著高于其他两个品种;在开花后0~7 d,籽粒氮素积累量和积累速率在3个品种间无显著差异,花后7~14 d,两个指标在烟农1212和济麦22间无显著差异,但均显著高于良星99,花后21~35 d,烟农1212的籽粒氮素积累量和积累速率显著高于其他两个品种。烟农1212花前氮素转运量和开花后氮素积累量均最高。相关分析表明,籽粒产量与开花期和成熟期的氮素积累量呈极显著正相关,烟农1212较济麦22和良星99分别增产9.32%和14.10%,获得最高的氮素吸收效率、氮素收获指数和氮肥偏生产力。在本试验条件下,烟农1212是开花期和成熟期氮素积累量、花前氮素转运量和产量最高的小麦品种。     

Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2)

Background: RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. Methods: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. Results: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. Conclusion: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Key Words: Protein isoforms, Spermatogenesis, Male infertility     

DNA and the chromosome – varied targets for chemotherapy

The nucleus of the cell serves to maintain, regulate, and replicate the critical genetic information encoded by the genome. Genomic DNA is highly associated with proteins that enable simple nuclear structures such as nucleosomes to form higher-order organisation such as chromatin fibres. The temporal association of regulatory proteins with DNA creates a dynamic environment capable of quickly responding to cellular requirements and distress. The response is often mediated through alterations in the chromatin structure, resulting in changed accessibility of specific DNA sequences that are then recognized by specific proteins. Anti-cancer drugs that target cellular DNA have been used clinically for over four decades, but it is only recently that nuclease specific drugs have been developed to not only target the DNA but also other components of the nuclear structure and its regulation. In this review, we discuss some of the new drugs aimed at primary DNA sequences, DNA secondary structures, and associated proteins, keeping in mind that these agents are not only important from a clinical perspective but also as tools for understanding the nuclear environment in normal and cancer cells.     

西卡柱花草受精作用和胚及胚乳发育的研究

应用常规石蜡切片与压片方法,用铁矾苏木精、汞溴酚蓝和ＰＡＳ反应等进行染色观 察西卡柱花草（Ｓｔｙｌｏｓａｎｔｈｅｓｓｃａｂｒａ ｃｖ． ｓｅｃａ）受精作用和胚及胚乳发育过程。开花后 ３～６ ｈ, 发生双受精作用。卵核的受精作用属有丝分裂前配子融合类型。极核受精过程有２种类型： （１）两极核融合成次生核,然后次生核与精核融合;（２）精核先与上极核融合,受精极核与 另一极核直到有丝分裂开始时才融合。花后２２～２３ ｈ,合子分裂为二细胞原胚,经历单列细 胞胚、长棒形胚、球形胚、心形胚、鱼雷形胚、幼胚、扩大生长胚阶段,花后 ２５～３０ｄ胚发育成 熟。胚发育属于茄型。在扩大生长压时期胚体开始积累淀粉和蛋白质。花后８～９ｈ初生胚乳 核分裂,胚乳发育为核型。至胚长大,充满整个胶囊时胚乳开始退化,胚发育成熟时胚乳仅余 残迹。      

基于iTRAQ技术的干旱胁迫下玉米苗期蛋白质组学研究

采用同位素标记相对定量(iTRAQ)技术,对干旱胁迫条件下苗期玉米的蛋白质组学变化进行分析。结果表明,共检测到玉米幼苗中的207个蛋白在干旱胁迫后发生了显著的丰度变化。根据蛋白注释情况可将这些蛋白归入信号传导、渗透调节、蛋白合成与折叠、ROS清除、膜运输、转录相关、细胞结构与细胞周期、脂肪酸代谢、碳水化合物与能量代谢、光合作用与光呼吸等代谢途径。干旱胁迫后,涉及光反应和呼气作用的差异蛋白多表现为丰度上升;涉及碳水化合物及蛋白质合成差异蛋白多表现为丰度下降;与渗透调节相关的脱水蛋白、脯氨酸代谢和渗透胁迫相关的蛋白酶则显示为丰度上升。干旱胁迫还能导致植物体内活性氧大量产生,活性氧清除相关的酶类也会发生明显的丰度上升。根据研究结果推测,玉米苗期主要通过降低植株生长速率、减少水分散失、清除自由基等多种方式维持其在干旱胁迫条件下的生长发育过程。     

水分亏缺对不同小麦品种N、P、K分布与产量的影响

为明确华北地区不同小麦品种在正常和缺水条件下N、P、K的吸收利用特点,进一步实现资源高效利用,选用3个不同生态类型品种(沧麦6001、邯麦9、济麦22),设置相对含水量分别为60%~80%(正常)和40%~60%(亏缺)两个浇灌水平,进行人工气候室箱体栽培试验,测定各小麦品种不同器官N、P、K含量和分配、干物质积累以及对籽粒产量构成要素的影响。结果表明,N、P、K含量和分配具有明显的器官特性,其中籽粒N和P含量显著高于其他部位,K含量最低。水分亏缺限制了沧麦6001籽粒N和K的吸收,同时促进了N和P向籽粒的转运;限制了邯麦9茎秆K的吸收,促进了P向颖壳的转运;限制了济麦22叶片、籽粒P的吸收,同时促进了N向颖壳和籽粒的转运,从而改变了小麦不同部位养分比例平衡以及干物质积累。N、P含量与干物质积累呈显著正相关,K含量与其呈显著负相关,但均未直接影响产量及其构成要素。亏缺灌溉下(亏缺量为24.39mm),邯麦9穗数、产量及济麦22穗数显著降低,而沧麦6001穗数、穗粒数和产量显著提高。因此,适度控制水分并提高营养元素向籽粒的转运效率是提高小麦产量和品质的重要途径。     

减少灌水量对强筋小麦花后干物质和氮素积累、转运及产量的影响

为探究灌水量对强筋小麦花后干物质和氮素积累、转运及产量的影响,选用强筋小麦品种中麦998和中麦1062,在防雨棚池栽条件下,春季于拔节期和开花期灌水,每时期设600（W₆₀₀）、300（W₃₀₀）和0 m·hm^-2（W₀）3个灌水量处理,研究了减少灌水量对强筋小麦花后干物质含量、氮素积累和转运、籽粒产量、籽粒蛋白质含量和产量的影响。结果表明,随春季灌水量的减少,强筋小麦植株干物质积累量、氮素积累量、粒重比、叶重比、籽粒产量和蛋白质产量均表现为下降趋势,而蛋白质含量和水分利用效率呈上升趋势。两品种叶片氮素转运量和氮素转运效率以W₃₀₀处理下最高,且叶片氮素转运效率在W₃₀₀和W₆₀₀处理之间均无显著差异。中麦1062在W₀和W₃₀₀处理下水分利用效率无显著差异,中麦998在W₃₀₀和W₆₀₀处理下蛋白质含量无显著差异。综上可见,W₃₀₀处理既能有效提高强筋小麦花后干物质转运量,维持较高的产量和水分利用效率,同时又能提高氮素转运量和籽粒蛋白质含量,达到节水高产的目的。     

大、小麦对镉的吸收、运输及在籽粒中的积累

大、小麦是全球最重要的粮食作物 ,生产镉低积累量的大、小麦对人们的健康至关重要 ,因为镉易被大、小麦吸收并在其体内积累。大、小麦籽粒中镉的含量与众多因素有关。根部特性是决定植株吸收镉的关键所在 ,根部特性主要决定于品种特性。根系细胞壁对镉的固定、液泡对镉的钝化、木质部液中镉的向外运输以及镉在穗部韧皮部中再运输共同决定籽粒中镉的含量。本文阐述了镉在植物体内的吸收、运输以及在籽粒中的积累规律 ,并讨论了降低大、小麦籽粒中镉积累的育种与栽培途径     

水稻胚乳组织的结构观察

【目的】阐明水稻糊粉层细胞、亚糊粉层细胞与中心胚乳贮藏细胞的结构特性。【方法】采用光镜、透射电镜与扫描电镜对水稻胚乳组织进行观察研究。【结果】糊粉层细胞分化过程中,大液泡变成小体积蛋白贮存液泡,蛋白贮存液泡又转变成糊粉粒。颖果背部比腹部有更多层糊粉层,但背部糊粉层细胞内糊粉粒的形成与积累速度却较慢。亚糊粉层细胞起初含有一些脂质体,后来脂质体消失,而其内部淀粉体与蛋白体逐渐增多。中心胚乳贮藏细胞含有淀粉体与蛋白体,蛋白体以液泡型蛋白体为主,它们可以相互融合而变大。中心胚乳贮藏细胞内的淀粉积累速度明显快于亚糊粉层细胞内的。成熟颖果的中心胚乳贮藏细胞内淀粉体最为密集,背部和侧部的亚糊粉层细胞内淀粉体排列较疏松,腹部的亚糊粉层细胞内淀粉体最为稀疏。【结论】水稻颖果背部与腹部的糊粉层细胞和亚糊粉层细胞的结构差异可能与养分吸收与转运有关;中心胚乳贮藏细胞内淀粉体发育速度快于亚糊粉层细胞。     

不同质地土壤对高油玉米碳氮运转的影响

在池栽试验条件下,研究了壤土、粘土、砂土三种质地土壤对高油玉米碳氮积累和运转的影响。结果表明,高油玉米叶、茎、鞘等营养器官贮藏物质再运转量和再运转率及营养器官物质总运转量和运转率以砂土最高,成熟期子粒重及光合同化物输入子粒量和光合同化量对子粒重的贡献率均表现为粘土>壤土>砂土。成熟期子粒氮素含量主要来源于灌浆期子粒氮素吸收,子粒氮素总量和子粒氮素吸收量表现为粘土>壤土>砂土,各营养器官贮藏氮素转移率叶>鞘>茎。三种质地土壤对高油玉米子粒产量、淀粉、蛋白质和油分产量的影响均表现为粘土>壤土>砂土。     

橡胶树HbEBP1蛋白亚细胞定位初步研究

利用融合PCR法构建HbEBP1基因与绿色荧光蛋白基因的融合表达载体,成功转化拟南芥,借助激光共聚焦显微镜检测HbEBP1-EGFP融合蛋白在转基因植株根细胞中的表达及分布.结果显示:在转基因植株根中观察到强烈荧光信号,并分布于细胞质与细胞核中.结果证实了HbEBP1蛋白同时定位于细胞质与细胞核中.     

Structural changes in root tips of wheat (Triticum aestivum L.) in response to olive oil mill wastewater

Toxic effects of the wastewater were investigated ultrastructurally in root tips of Triticum aestivum. As a result, wall and nuclear degradations, disruptions in all cytoplasmic membranes, irregular nucleus shapes and cellular organization defects were densely detected. Besides, germination ratio, total protein contents, DNA contents and root-shoot growth were found to be decreased significantly when compared to the control group. Results were compared with those of recent studies regarding excessive Na+, Fe+2, P, polyphenols and acidic pH toxicity.     

Cloning,Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A (exo A) and killed whole cell. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin (fliC) fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections. Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Key Words: Pseudomonas aeruginosa, Exotoxin A (exoA), Flagellin (fliC), Vaccines     

Kinase Gene Expression and Subcellular Protein Expression Pattern of Protein Kinase C Isoforms in Curcumin-treated Human Hepatocellular Carcinoma Hep 3B Cells

Curcumin, a yellow component of turmeric or curry powder, has been demonstrated to exhibit anti-carcinogenic effects in vitro, in vivo, and in human clinical trials. One of its molecular targets is protein kinase C (PKC) which has been reported to play essential roles in apoptosis, cell proliferation, and carcinogenesis. In this study, PKC mRNA expression was significantly inhibited in curcumin-treated human hepatocellular carcinoma (HCC) Hep 3B cells identified using a kinase cDNA microarray. Furthermore, curcumin decreased total protein expression of all PKCs in a time-related manner by immunoblotting of whole cell lysates, nuclear, membrane, and cytosolic fractions. In cytosolic fraction, the expression of PKC-α was totally inhibited by curcumin. In contrast, the expression levels of PKC-ζ and -μ were dramatically increased. Increases in expression of PKC-δ and PKC-ζ in the membrane and nucleus, and PKC-ι in the membrane were detected. In summary, the changes in expression and distribution of subcellular PKC isoforms in curcumin-treated Hep 3B cells suggest possible PKC-associated anti-tumor mechanisms of curcumin and provide alternative therapies for human HCC.     

Root Morphology and Anatomy Affect Cadmium Translocation and Accumulation in Rice

Paddy fields contaminated with cadmium (Cd) present decreased grain yield and produce Cd-contaminated grains. Screening for low-Cd-accumulating cultivars is a useful method to reduce the amount of Cd in the grains. The present study aimed to examine the roles of the root morphology and anatomy in Cd translocation and accumulation in rice plants. Twenty-two rice cultivars were used in the first experiment, after which two cultivars [Zixiangnuo (ZXN) and Jinyou T36 (JYT36)] were selected and used in subsequent experiments under hydroponic conditions. The results showed that there were significant differences in Cd concentrations in the shoots (ranging from 4 to 100 mg/kg) and the Cd translocation rates (shoot/root) (from 7% to 102%) among the 22 cultivars, and the shoot Cd concentration was significantly correlated with the Cd translocation rate of the 22 cultivars under 0.1 mg/L Cd treatment. Compared with cultivar ZXN, JYT36 had greater root Cd uptake and accumulation but lower shoot Cd accumulation and Cd translocation rate. The number of root tips per surface area of cultivar ZXN was greater than that of JYT36, while the average root diameter was lower than that of JYT36. Compared with ZXN, JYT36 had stronger apoplastic barriers, and the Casparian bands and suberin lamellae in the root endodermis and exodermis were closer to the root apex in both the control and Cd treatments, especially for suberin lamellae in the root exodermis with Cd treatments, with a difference of 25 mm. The results also showed that, compared with ZXN, JYT36 had greater percentages of Cd bound in cell walls and intracellular Cd but lower Cd concentrations in the apoplastic fluid under the Cd treatment. The results suggested that Cd translocation, rather than root Cd uptake, is a key process that determines Cd accumulation in the rice shoots. The root morphological and anatomical characteristics evidently affect Cd accumulation in the shoots by inhibiting Cd translocation, especially via the apoplastic pathway. It was possible to pre-screen low-Cd-accumulating rice cultivars on the basis of their root morphology, anatomical characteristics and Cd translocation rate at the seedling stage.     

花生iso-ARah3基因克隆及多克隆抗体制备

类花生致敏原3(iso-ARah3)是花生致敏原3的同系物,其表达活性与干旱及黄曲霉侵染相关。本研究通过RT-PCR技术从花生种子cDNA文库中克隆获得iso-ARah3的开放阅读框(ORF),全长1533bp,编码510个氨基酸,N端预测有20个氨基酸的信号肽序列。通过序列比对发现,该克隆序列有1处缺失突变,其N端210个氨基酸序列与胰蛋白酶抑制因子的同源性较高,达83.60%。通过构建原核表达载体pET28a-isoARah3,转化进大肠杆菌BL21,经IPTG诱导表达,利用SDS-PAGE检测表明,融合蛋白His-isoARah3获得高效表达,分子量约54kD。His-isoARah3经纯化和富集,对新西兰兔进行4次免疫,纯化获得的iso-ARah3多克隆抗血清,通过间接ELISA检测,表明获得了效价比(1∶512000)很好的抗体。通过对融合蛋白的诱导前和纯化后样品进行Western Blot分析,结果显示仅在纯化样品相应位置(54kD)有明显信号,表明所制备的抗体具有很高灵敏度和特异性。本研究结果为深入研究花生iso-ARah3基因的功能奠定了基础。     

鹿茸多肽对冈田酸诱导的小鼠海马神经元HT22细胞损伤模型中PI3K、AKT、Caspase-9表达的影响

目的观察鹿茸多肽对冈田酸(OA)诱导的小鼠海马神经元HT22细胞损伤模型中磷脂酰肌醇-3激酶(PI3K)、蛋白激酶B(AKT)、半胱氨酸蛋白酶-9(Caspase-9)表达的影响,探讨鹿茸多肽对HT22细胞损伤模型的保护作用机制。方法采用含10%胎牛血清(FBS)培养液(DMEM/F12)传代培养HT22细胞7d后,分为正常对照组、二甲基亚砜(DMSO)对照组、OA细胞损伤模型组、鹿茸多肽高、中、低剂量组。正常对照组给予含10%FBS的DMEM/F12,DMSO对照组给予DMSO终浓度<0.01%的DMEM/F12,OA细胞损伤模型组给予10nmol OA的DMEM/F12,鹿茸多肽高、中、低剂量组分别给予50、500、1000μg/ml的DMEM/F12,于37℃、5%CO2条件下孵育24h。利用噻唑蓝(MTT)比色法检测细胞存活率,酶联免疫法(ELISA)检测各组实验细胞内PI3K、AKT含量,蛋白质印迹法(Western Blot)检测各组实验细胞内PI3K、AKT、Caspase-9表达水平。结果MTT比色法检测结果表明,与OA细胞损伤模型组比较,鹿茸多肽能够明显提高细胞存活率(P<0.05);ELISA检测结果表明,与OA细胞损伤模型组比较,鹿茸多肽能够明显提高受损HT22细胞内PI3K、AKT含量(P<0.05或P<0.01);Western Blot检测结果表明,与OA细胞损伤模型组比较,鹿茸多肽能够明显提高受损HT22细胞内PI3K、AKT、Caspase-9表达水平(P<0.05或P<0.01)。结论鹿茸多肽对OA诱导的HT22细胞损伤模型具有保护作用,作用机制可能与调节受损HT22细胞内PI3K、AKT、Caspase-9表达水平相关。     

Anti-Diabetic Effects of CTB-APSL Fusion Protein in Type 2 Diabetic Mice

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.