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1.
根据GenBank中猪2型圆环病毒的核苷酸序列,设计两对引物,对来源于河南省不同地市的4株PCV2细胞培养物进行鉴定,并扩增出ORF2基因,克隆到pMD18－T载体上,获得重组质粒pMD18-T-ORF2,并对其进行测序,结果表明所克隆的ORF2基因与其它PCV2 ORF2基因核苷酸同源性在92.4%～99.6%之间,氨基酸同源性在90.6％～97.9％之间。同时采用PCR从重组质粒pMD18-T-ORF2中扩增出587bp的ORF2基因,克隆到表达载体pET-32a,成功构建了重组质粒pET-32a-ORF2,经诱导表达了ORF2基因编码的结构蛋白,表达的重组蛋白为融合蛋白,分子量为40kD,经Western-Blot检测,重组蛋白可被PCV2阳性血清识别。 相似文献
2.
根据猪圆环病毒2型（PCV2）LC株序列,设计合成3对引物,通过PCR扩增PCV2的3个不同ORF2基因片段,分别将其克隆到pET-32a载体中,将重组表达质粒转化大肠杆菌BL21,表达并纯化了重组蛋白。Western-blot分析表明,重组蛋白可以与PCV2阳性血清反应,表明该蛋白具有良好的抗原性。以纯化的重组蛋白初步建立了间接酶联免疫吸附实验（ELISA）方法,经方阵滴定确定最佳包被浓度为0．24μg／mL,血清最佳的稀释度为1：40。 相似文献
3.
根据GenBank上猪TNNC2（Fast skeletal muscle troponin C2）基因序列（GenBank accession No. DQ629177）设计一对引物,采用RT-PCR方法克隆得到617 bp TNNC2 cDNA片段（GenBank accession No. EF673726）,包括完整的开放阅读框（ORF）,与GenBank上公布的猪TNNC2基因（GenBank accession No. AY575058）的ORF核苷酸序列同源性达99 %,并发现开放阅读框内的5个点突变,319位点T→C,320 位点G→A,321位点C→T,导致氨基酸107位Ala（丙氨酸）→Met（蛋氨酸）,322位点A→G为同义突变,433位点A→T,导致氨基酸144位Glu（谷氨酸）→Asp（天冬氨酸）。根据已获得的TNNC2基因开放阅读框序列,重新设计引物扩增得到包含BamH I和EcoR I酶切位点的完整阅读框,将其首先克隆到pMD18-T载体中,经菌液PCR筛选和酶切鉴定后,用BamH I和EcoR I将目的片段切下,再克隆到原核表达载体pRSET A中构建重组表达质粒pRSET A-TNNC2。将重组质粒转化大肠杆菌BL21（DE3）,IPTG不同诱导条件诱导表达,经SDS-PAGE电泳和Western blot分析证实重组表达质粒pRSET A-TNNC2表达出24 kD左右的融合蛋白,最佳诱导时间为4 h,最佳的IPTG诱导浓度为0.6 mmol/L,表达产物以可溶性蛋白的形式存在。 相似文献
4.
参考GenBank上发表的猪圆环病毒Ⅱ型(porcine circovirus2,PCV2)全基因组序列,设计一对引物,通过反向PCR技术扩增出9株PCV2流行株的全基因,并进行了序列测定与分析,结果表明:9株PCV2的基因组全长为1768bp或1767bp,同源性比较发现,本研究的9株PCV2之间的核苷酸同源性为95.6%~100%,与GenBank上已发表的PCV2分离株之间的同源性为95.1%~99.8%,与法国和新西兰代表株的亲缘关系较近,与美国、加拿大和澳大利亚代表株的亲缘关系较远。所得9株PCV2的ORF2核苷酸及其所推导的氨基酸序列同源性分别为93.1%~100%和91.8%~99.6%,存在较大的变异。 相似文献
5.
猪圆环病毒2型BF株ORF2基因在大肠杆菌中的表达 总被引：6，自引：0，他引：6
采用PCR从构建的猪圆环病毒2型／(PCV2)ORF2重组质粒(pGEM-T-ORF2）中扩增出大小为593bp的ORb2基因，克隆到表达载体pET-32a，经异丙基硫代半乳糖苷诱导，成功表达了ORF2基因编码的结构蛋白。表达的重组蛋白为融合蛋白，分子量40kD，表达量20％左右。经Western blot检测，重组蛋白可被PCV2阳性血清识别。 相似文献
6.
犬白细胞介素-2 基因(IL-2 )的克隆与表达 总被引：4，自引：1，他引：3
根据GenBank上发表的犬(Canis familiaris)IL-2基因序列，设计了1对引物。采用RT-PCR技术，以ConA刺激的犬外周血淋巴细胞为材料，从总RNA中扩增出犬IL-2基因。琼脂糖凝胶电泳显示扩增片段约为550bp。分离纯化片段，克隆入pMD18-T载体，经酶切鉴定，测序结果显示，克隆的犬IL-2基因与GenBank上发表的序列一致，生物软件分析结果表明该序列与牛、羊和鹿的亲源性更近。构建表达质粒pET28-CaIL-2，转化大肠杆菌(Escherichia coli)BL21，IPTG诱导表达，SDS—PAGE电泳显示21．8kD左右的表达条带。MTT比色法测定活性结果表明：表达的重组犬IL-2蛋白具有极显著的体外增殖活化淋巴细胞的活性。 相似文献
7.
猪细小病毒VLPs供外源蛋白插入的候选位点发现及其重组PCV2-ORF2的病毒样颗粒构建 总被引：2，自引：0，他引：2
先将猪细小病毒(PPV)SC-1株VP2基因扩增产物克隆到pMD18-T载体构建质粒pMD-VP2;设计两对引物(分别引入Hind Ⅲ和Sac Ⅰ两个酶切位点)扩增猪圆环病毒二型(PCV2)SC株ORF2基因,构建了pMD-ORF2.A和pMD-ORF2.B两质粒;经相应内切酶酶切后回收目的片段,插入PPV SC-1株VP2基因的Hind Ⅲ和Sac Ⅰ两个特异限制酶切位点处(分别对应于PPV VP2蛋白N端和C端1/3处),得到重组质粒pPVP2-ORF2.A和pPVP2-ORF2.B.经鉴定后的质粒pPVP2-ORF2.A和pPVP2-ORF2.B用Kpn Ⅰ、BamH Ⅰ和ApaL Ⅰ三酶切,回收含VP2和ORF2基因的目的片段克隆至真核表达载体(pEGFP-C1),得到重组质粒pEGFP.VO.A和pEGFP.VO.B.脂质体法转染重组质粒于Cos7细胞后,采用荧光显微镜和电镜观察基因表达情况,结果仅在转染pEGFP.VO.A的样品中观察到了病毒样颗粒(VLPs).纯化VLPs免疫小鼠,结果显不能产生较好的细胞免疫和针对PPV和PCV2的特异性体液免疫,该结果表明研究中获得了PCV VP1-PCV2ORF2重组VLPs,同时也揭示PPV VPLs的N端1/3适于外源蛋白插入构建重组VLPs 相似文献
8.
猪链球菌2型纤连蛋白/血纤蛋白原结合蛋白
全基因的克隆及融合表达 总被引：4，自引：0，他引：4
采用发表的引物对,以猪链球菌2型（Streptococcus suis type 2）江苏分离株HA9801的基因组DNA为模板,采用PCR方法扩增纤连蛋白/血纤蛋白原结合蛋白基因fbps（GenBank登录号为AY565303）克隆于pMD-T18载体构建成载体pMD-T-fbps.经内切酶酶切和测序鉴定后,将由pMD-T-fbps内切酶切下的片段定向克隆于表达载体pET-32a（＋）,得到重组质粒pfbps.将重组质粒pfbps转化大肠杆菌（Escherichia coli）BL21株,经IPTG诱导,可高水平地表达相对分子量为83 kD的融合蛋白.配体印迹结合试验表明,表达的融合蛋白可与人纤连蛋白结合. 相似文献
9.
用猫白介素18(interleukin,IL-18)基因特异性引物对刀豆蛋白(ConA)刺激后猫外周血单核细胞(PBMCf)总RNA进行了RT-PCR扩增,并将扩增产物纯化后克隆入pMD18-T中进行核苷酸序列测定。结果该基因全长579bp,编码192个氨基酸(GenBank登录号:DQ100372)。在推导的猫IL-18氨基酸序列中,无信号肽序列和潜在的N-联糖基化位点,但存在4个Cys残基。与不同物种IL-18相比,猫IL-18与犬、羊、牛和猪IL-18核苷酸序列有较高的同源性,分别为89.8%、88.6%、88.4%和88.1%,但与小鼠和鸡IL-18有明显的种属差异。将目的基因片段进一步亚克隆到大肠杆菌(Escherichia coli)表达载体pET28a中构建了重组质粒pETIL-18,转化大肠杆菌BL21(DE3),并用IPTG诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到分子量为27.5kD的重组目的蛋白。经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的13.6%。 相似文献
10.
WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)菌株,经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267,被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-,得到的工程菌能正常表达70 kD蛋白,形成方形晶体。生物测定结果表明,cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。 相似文献
11.
2-Alkyl-(2H)-thiapyrans and 2-alkylthiophenes have been identified in the volatiles of cooked beef and lamb. The quantities of both groups of compounds were higher in the meat of animals fed lipid supplements high in n-3 polyunsaturated fatty acids. 2-Alkyl-(2H)-thiapyrans were formed when (E,E)-2,4-dienals (C(6)-C(11)) and hydrogen sulfide were heated at 140 degrees C for 30 min. This confirmed their proposed route of formation in cooked meat from lipid-derived aldehydes and hydrogen sulfide; the latter was produced from the degradation of cysteine, via the Maillard reaction. The mass spectra and NMR spectra of these thiapyrans are reported for the first time. Although 2-alkyl-(2H)-thiapyrans were found to have only low odor potency, the reactions by which they are formed may have important implications for meat flavor. These reactions may remove potent aroma compounds and their intermediates from meat, thus modifying the overall aroma profile. 相似文献
12.
13.
A number of 4-methyl-6-alkyl-alpha-pyrones were synthesized and characterized on the basis of 1H NMR and mass spectroscopy. These compounds were tested in vitro against pathogenic fungi, namely, Sclerotium rolfsii Saccardo, Rhizoctonia bataticola (Taub.) Butler, Pythium aphanidermatum (Edson) Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum (Hesse), and Rhizoctonia solani Nees. Lower homologues were less effective, whereas compounds such as 4-methyl-6-butyl-alpha-pyrone, 4-methyl-6-pentyl-alpha-pyrone, 4-methyl-6-hexyl-alpha-pyrone, and 4-methyl-6-heptyl-alpha-pyrone were found effective against all of the test fungi. They inhibited mycelial growth by approximately 50% (ED50) at 15-50 microg/mL. 4-Methyl-6-hexyl-alpha-pyrone, which was found most effective, was tested against S. rolfsii in a greenhouse at 1, 5, and 10% concentrations. The 10% aqueous emulsion of 4-methyl-6-hexyl-alpha-pyrone suppressed disease development in tomato by 90-93% as compared with the untreated infested soil in the greenhouse after 35 days of treatment. 相似文献
14.
This study investigated the antioxidative activity of green tea extract, and a green tea tannin mixture and its components, under conditions of radical generation using the hydrophilic azo compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals at a constant and measurable rate in the cultured renal epithelial cell line, LLC-PK(1), which is susceptible to oxidative damage. Treatment with AAPH decreased cell viability and increased the formation of thiobarbituric acid-reactive substances. However, green tea extract, and the tannin mixture and its components, comprising (-)-epigallocatechin 3-O-gallate (EGCg), (-)-gallocatechin 3-O-gallate (GCg), (-)-epicatechin 3-O-gallate (ECg), (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)-epicatechin (EC), and (+)-catechin (C), showed protective activity against AAPH-induced cellular damage. The tannin mixture and its components exhibited higher antioxidative activity than the green tea extract. Furthermore, EGCg and GCg had higher activity than EGC and GC, respectively. In particular, EGCg exerted the most significant cellular protective activity against AAPH. These results indicate that green tea tannin may inhibit cellular loss and lipid peroxidation resulting from the peroxyl radical generated by AAPH, and that the chemical structure of tannin is also involved in the activity, suggesting that the O-dihydroxy structure in the B ring and the galloyl groups are important determinants for radical scavenging and antioxidative potential. 相似文献
15.
Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) (CBF) is a widely used insecticide. Traditional methods like hydrolysis and direct photolysis cannot remove CBF effectively. In this study, the photodecay of 0.1 mM CBF in UV/H2O2, UV/S2O8(2-), and UV/H2O2/S2O8(2-) and sequential addition of a second oxidant were studied under UV light at 254 nm. The degradations of CBF follow pseudo-first-order decay kinetics. Direct photolysis was slow, but the corresponding degradation rate was increased with the addition of hydrogen peroxide (H2O2) or potassium peroxydisulfate (K2S2O8). In the UV/H2O2 reaction, the optimum reaction rate was 0.9841 min-1 at 10 mM H2O2 (pH 7); however, retardation is observed if H2O2 is overdosed. Such retardation is not observed in the UV/S2O8(2-) system, but a nonlinear increment of removal efficiency is identified. The UV/H2O2/S2O8(2-) process on the other hand shows the best performance in CBF degradation, but it has a less effective mineralization than that of the sole UV/S2O8(2-) reaction. 相似文献
16.
Lipase-assisted generation of 2-methyl-3-furanthiol and 2-furfurylthiol from thioacetates 总被引：2，自引：0，他引：2
Bel Rhlid R Matthey-Doret W Blank I Fay LB Juillerat MA 《Journal of agricultural and food chemistry》2002,50(14):4087-4090
Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate and S-2-furfuryl thioacetate using lipase from Candida rugosa produced 2-methyl-3-furanthiol and 2-furfurylthiol, respectively. When reactions were carried out at room temperature and pH 5.8, 2-methyl-3-furanthiol was produced in a optimal yield of 88% after 15 min of reaction, whereas 2-furfurylthiol was obtained in a yield of 80% after 1 h of reaction time. Enzymatic hydrolysis was also performed in n-hexane, n-pentane, and water/propylene glycol mixture. The reaction rates in these media were slower as compared to those in aqueous medium; however, the reaction yields were quite similar. As expected, the stability of the generated 2-methyl-3-furanthiol and 2-furfurylthiol was better in n-hexane, n-pentane, and the water/propylene glycol mixture as compared to that in water or phosphate buffer. 相似文献
17.
Bastos EL Romoff P Eckert CR Baader WJ 《Journal of agricultural and food chemistry》2003,51(25):7481-7488
This paper describes a screening method for antioxidant potential determination based on luminol/hemin/hydrogen peroxide chemiluminescence. The emission depletion, caused by an antiradical compound added during the chemiluminescence decay, is proportional to the number of reactive species trapped. Therefore, the difference between the areas of the emission decay curves, obtained in the absence and in the presence of the potential antioxidant, is a measure for the antiradical capacity of the sample. The technique has been applied to measure the antiradical capacity of pure compounds and complex mixtures from natural origin, providing reliable results that indicate the method's feasibility. 相似文献
18.
乌拉草纤维的超声波辅助碱氧-浴法提取工艺优化 总被引：3，自引：3，他引：0
为了提取一种新型植物纤维-乌拉草纤维并研究其性能,采用超声波-碱氧-浴法从乌拉草秸秆中提取乌拉草纤维,用多指标正交试验方法对超声波时间,碱煮时间,碱用量和双氧水用量进行优化设计,并通过对直径,断裂强度,回潮率3个指标的综合分析评价确定最佳提取工艺,对处理后的乌拉草纤维进行基本性能测试及保暖、抗菌效果测试.结果表明:最佳工艺参数为:超声波时间为70 min;碱氧-浴时间为100 min;0.75 L溶液中碱质量浓度为8 g/L;双氧水质量浓度为12 g/L.采用最优工艺所提取的乌拉草纤维平均直径、长度、断裂强度及回潮率分别为47.19μm、152.59 mm、2.03×10?4MPa和15.33%,与苎麻纤维性能相似,可满足可纺性要求.并有一定的保暖性和优良的抑菌效果. 相似文献
19.
2ZGF-2型甘薯复式栽植机的设计与试验 总被引：4，自引：4，他引：0
为提高甘薯机栽水平,解决传统薯苗机械栽植中下田作业机具多、主要栽插方式短缺、易压垄伤垄、作业质量不高等难题,该文研制出一款与55.13~80.85 k W大型拖拉机配套的2ZGF-2型甘薯裸苗复式栽植机,该机可一次完成两垄甘薯的旋耕、起垄、破压茬、开沟、栽插、镇压、修垄等作业。该机采用非零速栽插原理,链夹运动轨迹为余摆线,满足了甘薯种植中广泛应用的"斜插法"栽插方式作业需求,研究并确定开沟器开沟、链夹放苗与覆土压实三者间协调一致工作的重要结构参数,明确了栽插株距的主要影响因素和调整方法。该文对甘薯栽苗作业质量评价影响最重要的2个指标进行参数优化试验,得出优选参数组合为喂苗露出长度140 mm、开沟深度80 mm、前行速度0.3 m/s,此时立苗角度合格率为97.9%,栽插深度合格率为98.2%,较好的满足了甘薯机械栽插要求。该研究不仅为甘薯栽插市场提供了实用机具,也为甘薯栽插机械创新研发或优化提供了参考。 相似文献
20.
Anaerobic reoxidation of reduced products in paddy soils was investigated. Ferrous iron (Fe2+) and monosulfide ion (S2–) added to the soil chemically reduced MnO2 to Mn2+, and MnO2 and Fe(OH)3 to Mn2+ and Fe2+, respectively, where Fe2+ and S2– were considered to be oxidized to Fe3+ and S0. Elemental sulfur was oxidized to sulfate by anaerobic incubation with NO3
– MnO2 and Fe(OH)3. A new conceptual model for the reduction processes in submerged paddy soil including the reoxidation processes of reduced
products, in which soil heterogeneity in paddy fields was taken into consideration, was proposed based on the results.
Received: 20 October 1996 相似文献