内切酶SCaI对不同纯度质粒的酶解效率探讨

限制性内切酶ＳＣａＩ适用于多种质粒载体,尤其是融合蛋白表达载体,ｐＧＥＸ系统,因它具有二个ＳＣａＩ酶切位点,因此用ＳＣａＩ酶解图谱鉴定以ｐＧＥＸ系统为载体的重组子甚为便捷。简便碱裂解法提取重组ＤＮＡ省时,省耗。本文探讨了ＳＣａＩ对用常规和简便两种碱裂解法提取的重组ＤＮＡ的酶解效率。结果表明：（１）简便碱裂解法抽提的重组ＤＮＡ,用ＥｃｏＲＩ、ＥｃｏＲＶ、ＰｓｔＩ、ＢａｍＨＩ等限制性内切酶酶解,可以获     

自发性高血压大鼠原癌基因表达和限制性内切酶片段长度多态性分析

观察自发性高血压大鼠（ＳＨＲ）血管平滑肌细胞（ＶＳＭＣｓ）的生长曲线、ｃ－ｆｏｓ原癌基因表达和ｃ－ｆｏｓ基因酶切图谱的情况,与对照组京都维斯特大鼠（ＷＫＹ）进行对比。结果显示,在小牛血清作用下ＳＨＲ的ＶＳＭＣｓ生长速率和ｃ－ｆｏｓ原癌基因表达明显大于ＷＫＹ;ｃ－ｆｏｓ原癌基因限制性内切酶片段长度多态性（ＲＦＬＰ）分析表明,经ＢａｍＨ Ｉ或ＥｃｏＲ Ｉ酶切后的ＳＨＲ－ＷＫＹ的酶切图谱一致,同时也未发     

转BmK IT4基因烟草的抗虫性

用酶切方法从「ｐＢＳ－ＢｍＫ ＩＴ４质粒中获得ＢｍＫ ＩＴ４基因片段,并构建ＣａＭＶ ３５Ｓ启动子下的ＢｍＫ ＩＴ４基因表达质粒ｐＥ３－ＢｍＫ ＩＴ４,以根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｒｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶盘法转化云烟（Ｎｉｃｏ－ｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）Ｋ３２６叶片,获得４５株抗卡那霉素的再生植株。用这些再生植株进行抗虫     

自发性高血压大鼠原癌基因表达和限制性内切酶片段长度多态性分析

观察自发性高血压大鼠（ＳＨＲ）血管平滑肌细胞（ＶＳＭＣｓ）的生长曲线、ｃ－ｆｏｓ原癌基因表达和ｃ－ｆｏｓ基因酶切图谱的情况,与对照组京都维斯特大鼠（ＷＫＹ）进行对比．结果显示,在小牛血清作用下ＳＨＲ的ＶＳＭＣｓ生长速率和ｃ－ｆｏｓ原癌基因表达明显大于ＷＫＹ;ｃ－ｆｏｓ原癌基因限制性内切酶片段长度多态性（ＲＦＬＰ）分析表明,经ＢａｍＨⅠ或ＥｃｏＲⅠ酶切后的ＳＨＲ和ＷＫＹ的酶切图谱一致,同时也未发现ＳＨＲ的ｆｏｓ基因扩增现象．提示,ＶＳＭＣｓ的异常增殖和ｃ－ｆｏｓ原癌基因的表达异常与高血压的形成有关,而ｃ－ｆｏｓ原癌基因的过度表达可能是由于某些与基因转录调控有关的因素异常所致．     

生物技术在植物病害诊断中的的应用

１免疫技术１．ｌ酶联免疫法Ｖｏｌｌｅｒ"'和Ｃｌａｒｋ"'等早在７０年代中期就将现代血清学技术--酶联免疫法（ｅｎｚｙｍｅ-ｌｉｎｋｅｄｌｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙ,ＥＬＩＳＡ）应用于植物病毒的检测。ＥＬＩＳＡ与血清学技术相比具有明显的优越性,可灵?..     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Ｂａｌｂ／ｃ鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为１：２０８４（４Ｈ８）、１：２５６（６Ｈ９、４Ｈ３、２Ｈ５、２Ｃ８）１：１６（３Ｆ１０）;腹水抗体效价为１０＾９（４Ｈ３、４Ｈ８）、１０＾８（２Ｈ５）、１０＾７（６Ｈ９）、１０＾５（３Ｈ１０）。竞争间接酶联免疫吸附试验测定六     

人粒细胞集落刺激因子（hG—CSF）cDNA3‘—UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ（）ｃＤＮＡ３＇端非翻译区（３＇ＵＴＲ）中存在的ＤｒａＩ酶切位点,通过部分酶切与完全酶切,删除３＇－ＵＴＲ不同长度构建了四种ｈＧ－ＣＳＦ ｃＤＮＡ瞬时重组表达质粒,转染ＣＯＳ－７细胞手,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３＇－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３＇－ＵＴＲ对ｈＧ－ＣＳＦ ｃＤＮＡ表达的     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段,插入质粒ｐＵＣ１９的ＰｓｔＩ位点,转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）,利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长３０ｋｂ,其中各有一个ＫｐｎＩ,ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组,且以单拷贝形式存在,它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。     

酶联免疫检测（ELISA）药盒的开发应用现状及其展望

酶联免疫检测（ＥＬＩＳＡ）药盒的开发应用现状及其展望王益民张万泰（安徽省生物研究所合肥２３００３１）（合肥市曙光医院合肥２３００１１）各种酶联免疫检测药盒的技术都是构筑在由瑞典学者Ｅｎｇｖａｌ和荷兰学者ＶａｎＷｅｅｍｅｎ和Ｓｃｈｕｕｒｓ等于７０年代早...     

青霉素酰化酶活性中心的定点突变

对Ｅ．ｃｏｌｅ ＡＴＣＣ １１１０５的青霉素酰化酶（ｐｅｎｉｃｉｌｌｉｎ Ｇ ａｃｙｌａｓｅ,ＰＧＡ）活性中心的Ｓｅｒ２９０分别进行了定点突变和和化学修饰,将活性中心的Ｓｅｒ２９０改变成Ｃｙｓ或Ｓｅｃｙｓ,ＰＧＡ水解活力均大为下降,但仍保留部分活性。其对底物６－硝基（３－苯乙酰氨基）苯甲酸（６－ｎｉｔｒｏ－３－ｐｈｅｎｙｌａｃｅｔａｍｉｄｏ ｂｅｎｚｏｉｃ ａｃｉｄ,ＮＩＰＡＢ）的ｋｃａｔ分别从１     

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) uninucleate microspores

The objective of this study was to correlate the time that DNA synthesis first occurs in haploid microspores of barley with cell cycle and plant morphological stages and to subsequently assess the influence of pretreatments on DNA synthesis at different stages of microspore development. Spikes with microspores in early, mid, and late uninucleate stages of the two-rowed barley cultivars Manley and Igri were subjected to two commonly used pretreatments. First, during cold pretreatment for 28 days there was a slow increase in relative DNA values as well as asymmetric nuclear divisions in some microspores. Second, during a 4-day cold plus 0.3 M mannitol pretreatment, there was very little change in the microspore stage or DNA values indicating that for the duration of this pretreatment the progression of the cell cycle was essentially suspended at all stages, both in Igri and Manley. The results are discussed relative to the potential for genetic transformation of microspores.     

Study of the backcross progeny of barley-wheat hybrids by the RAPD and RAMPO methods

The genomes of alloplasmic wheat lines were analyzed by PCR-based methods: random amplified polymorphic DNA (RAPD) and random amplified microsatellite polymorphism (RAMPO). Lines L-16(1) and L-17(2) were obtained by three backcrosses and line L-79(10), by four backcrosses of the barely-wheat hybrid Hordeum vulgare (2n = 14) (variety Nepolegayuschii) x Triticum aestivum (2n = 42) (variety Saratovskaya 29) with different common wheat varieties. These lines proved to be euploid (2n = 42). The aneuploid line L-9 (2n = 43 + t) was obtained after a second backcross of the hybrid H. geniculatum All. (2n = 28) x T. aestivum (2n = 42) (Pyrotrix 28) with the variety Pyrotrix 28. The RAPD patterns of L-16(1) and L-17(1) contained fragments present only in the patterns of the parental wheat varieties and, in addition, fragments absent from the latter. This fragment from the pattern of L-16(1) was cloned. Analysis of its primary structure showed that the difference between L-16(1) and the parental wheat genotypes may be related to a mutation that had occurred during the development of the alloplasmic line at the binding site of an arbitrary primer. The genomes of plants of the lines L-79(10) and L-9 contain, in addition to the RAPD fragments of wheat, those characteristic of barley. RAMPO revealed higher polymorphism level among wheat varieties than that detected by RAPD. The hybridization patterns of the lines L-16(1), L-17(1), and L-79(10) contained fragments specific for wheat, and the patterns of L-9 contained both wheat and barley fragments.     

Mechanical Transmission of Barley Mild Mosaic Virus (BaMMV) to Rye (Secale cereale L.)

After mechanical spraygun inoculation barley mild mosaic virus (BaMMV) was detected in barley cv. ‘Gerbel’ (control) as well as in rye cv. ‘Somro’, but not in wheat cv. ‘Kanzler’ and oat cv. ‘Alfred’. ELISA values of infected barley and rye were similar. Furthermore, infected rye plants developed symptoms typical for barley yellow mosaic virus infection.     

Mycotoxins (trichothecenes, zearalenone and fumonisins) in cereals associated with human red-mold intoxications stored since 1989 and 1991 in China

Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.     

Screening for Barley Yellow Dwarf Luteovirus Resistance in Barley on the Basis of Virus Movement

The movement of barley yellow dwarf luteovirus (BYDV) was evaluated in susceptible and resistant barley and bread wheat genotypes. After leaf inoculation, the virus infected the root system and the growing point of susceptible earlier than resistant, barley genotypes. No difference in virus movement occurred in resistant and susceptible wheat genotypes. It was possible to reliably differentiate susceptible from resistant genotypes when root extracts of 41 barley genotypes were tested by DAS-ELISA 3 or 4 days after inoculation at the oneleaf stage. When barley plants inoculated at the two- or three-leaf stage were assayed by tissue-blot ELISA on nitrocellulose membrane, virus was detected in the phloem vessels of the growing points of the susceptible, but not of the resistant genotype, 4–6 days after inoculation. Our results thus suggest that screening for BYDV resistance in barley could be done quickly and cheaply especially when assays are made by the tissue-blot test.     

Differential Interaction Between PAV-like Isolates of Barley Yellow Dwarf Virus and Barley (Hordeum vulgare L.) genotypes

Four PAV-like isolates of barley yellow dwarf virus (BYDV) were identified as causing very severe (RG), severe (2t), moderately severe (3b) and mild symptoms (13t) in barley (Hordeum vulgare) cultivar Plaisant in a growth chamber at 25 days after inoculation. These isolates had different effects on a range of barley genotypes. Cultivar Vixen, which contains the Yd2 resistance gene, and 80-81 BQCB10 were not affected by any isolate. Five other genotypes were significantly affected by at, least one of the isolates. Line Ea52 (which is a mutant of the Japanese cultivar Chikurine Ibaraki) was more susceptible to BYDV-PAV than Chikurin Ibaraki 1. No serological differences were detected between the four isolates using monoclonal or polyclonal antibodies. Virus antigen concentration, estimated by enzyme-linked immunosorbent assay (ELISA), was correlated with the decrease in the shoot fresh weight for all isolates and all genotypes except for Vixen and 80-81BQCB10. In field tests, the severity of symptoms induced by the BYDV-PAV isolates was in accordance with that estimated in the growth chamber. However isolate 2t was more severe on cultivar Vixen and overcame the partial resistance of Chikurin Ibaraki 1 to the three other isolates. The results show that virus antigen concentration not only contributes to characterizing the resistance levels of barley genotypes but also the severity of BYDV-PAV isolates.     

miRNA-877-5p inhibits malignant progression of prostate cancer by directly targeting SSFA2

In this study, we aimed to investigate the role of miR-877-5p in the malignant phenotypes of prostate cancer (PCa) cells and its underlying mechanism. RT-qPCR analysis was performed to examine the expression of miR- 877-5p and sperm-specific antigen 2 (SSFA2) in PCa tissues and cells. Cell counting kit-8 (CCK-8) assay, 5- ethynyl-20-deoxyuridine (EdU) assay, flow cytometry, wound-healing assay, and Transwell invasion assay were performed to determine the functional roles of miR-877-5p in PCa cells. The association of miR-877-5p with SSFA2 was determined by luciferase reporter and RNA pull-down assays. In this study, we found that the expression level of miR-877-5p was decreased in PCa tissues and cells. Functionally, overexpression of miR- 877-5p exerted tumor suppressor properties in PCa cells. Mechanistically, SSFA2 was identified as a target gene of miR-877-5p, while overexpression of SSFA2 could abrogate the anti-tumor effects of miR-877-5p in PCa cells. These findings demonstrated that miR-877-5p/SSFA2 axis functioned as a potential target for PCa treatment.Key words: prostate cancer, miR-877-5p, proliferation, migration, invasion     

Monitoring of fusarium toxins in cereals and feed-stuffs from thuringia (1998 - 2001)

From 1998 to 2001 a total of about 1172 conventionally and organically produced samples of wheat, rye, barley and triticale were examined for the presence of deoxynivalenol (DON) and zearalenone (ZON). Furthermore, feedstuffs for pigs were included in the monitoring of Fusarium toxins. DON and ZON analyses were performed using ELISA or HPLC. The incidences and levels of toxins varied from year to year. Overall contamination levels were highest in wheat and triticale, followed by rye and barley. The highest DON contaminations were found in 1998. The probes of the years 1999-2001 showed lower incidences of Fusarium toxins. The second examined mycotoxin ZON was detected at lower levels in cereals. Similar results were observed in the monitoring of feedstuffs.