姜花挥发性成分的固相微萃取一气相色谱质谱分析

分别采取 1g新鲜黄姜花 (HedychiumflavumRoxb .)和白姜花 (H .coronariumKoen .)花瓣和花柱 ,置 4mL螺口玻璃瓶中 ,用聚四氟乙烯衬里的硅橡胶垫密封 ,插入 10 0 μm聚二甲基硅氧烷 (PMDS)萃取纤维头 ,于室温下顶空取样 6 0min。采用FINNIGANTRACEMS气质联用仪 (美国 )进行分析。色谱条件为DB 5石英毛细管柱长 30m ,内径 0 2 5mm ,液膜厚 0 2 5 μm ,载气He ,柱头压力 6 8 974kPa ,程序分流 /不分流 (LSS)进样口温度 2 5 0℃。程序升温 :5 0℃ ,保持 2min ;以 3℃ /min的升温速度升至 2 5 0℃ ,保持 30min。在SPME分析中 ,…     

气相色谱法分析柱状田头菇子实体中农药残留量

本研究以柱状田头菇(Agrocybe cylindracea,俗称杨树菇)为食用菌子实体样本,以有代表性的毒死蜱、氯氰菊酯和溴氰菊酯3种农药为检测目标,建立1种利用气相色谱对食用菌子实体中多种农药残留量进行检测分析的方法.所建方法的色谱条件为:Agilent 6890N气相色谱仪,63Ni-ECD检测器;HP-5毛细管柱(30 m×0.32 mm×0.25 μm);高纯氮气(纯度＞99.999％)为载气,流速1.5 mL/min;进样口温度为250℃;检测器温度为280℃;柱前压为72 psi,尾吹60 mL/min;柱升温程序为初始柱温150℃,恒温3 min后以6℃/min的速率升温至270℃,恒温25 min后,不分流进样,进样量为1.0 μL.方法学考察结果表明,该方法灵敏度和准确度高、加样回收率和变异系数符合《农药残留试验准则》相关要求;利用该法对柱状田头菇子实体样品农药残留进行检测的数据稳定、误差小.初步表明,建立的气相色谱方法适合用于柱状田头菇子实体中多种农药残留量的检测分析.     

梅花香气成分初探

1　材料与方法 　采用顶空固相微萃取 (HS SPME)和气质联用 (GC/MS)技术分析了 6个梅花品种 (表 1)的香气组成。手动SPME进样器和 75 μmCarboxen/PDMS萃取头为美国Supelco公司产品。FinniganTraceMS气相色谱—质谱联用仪为美国Finnigan公司产品。 3月 4日 8:30～ 11:30在无锡梅园摘取枝端已开放的梅花 5～ 8朵置于 15mL样品瓶中盖上盖子立即带回实验室 ,于室温 (12℃ )下将固相微萃取头通过瓶盖的橡皮垫插入到样品中吸附 4 0min后抽回插入气质联用仪 ,于 2 5 0℃解吸 2min ,采集数据。采用NIST谱库和Willey谱库对各物质进行检…     

HPLC法测定香菇中香菇嘌呤含量

建立1种运用高效液相色谱(HPLC)测定香菇(Lentinula edodes)中香菇嘌呤含量的方法。优化后的色谱条件为:Ultimate AQ-C18柱(5μm,4.6 mm×250 mm),流动相为甲醇-磷酸盐缓冲液(7∶93,pH 4.67)等度洗脱,流速1 mL/min,柱温30℃,检测波长259 nm,进样量10μL。研究结果发现该方法准确、灵敏、重现性好,适用于香菇嘌呤的定量分析。     

固相微萃取气质联用测定番茄香气成分条件优化

以番茄为试材,利用固相微萃取(SPME)气相色谱-质谱(GC/MS)联用技术分析番茄香气组分,探讨了萃取头、萃取温度、萃取时间、装样率、盐离子浓度(NaCl)和解析时间等条件对分析结果的影响。结果表明:番茄的SPME-GC/MS最优萃取方法为装样率50%,NaCl质量浓度为0.30g·mL~(-1),CAR/PDMS(75μm)萃取头在50℃下萃取40min后,高温解析3min,在此试验条件下,利用顶空固相微萃取可以从试验用的番茄中萃取到75种香气成分(匹配率800以上),其中包括醇类21种、醛类24种、酮类11种、酯类10种、呋喃类3种、酚类2种、其它类4种。     

甜樱桃设施栽培技术（中）

5设施内环境调控技术 5．1升温时间升温时间根据设施条件、气候状况、预定成熟期等因素确定。一般情况下，西安地区1月20-30日扣棚升温。按照每2-3天提高1℃的速度升温，至18℃时保持到开花，确保从升温到开花的间隔期不少于35天。采用人工低温暗光促眠、具备保温加温条件的设施可提前到12月中下旬至次年1月上旬升温。日出后揭开草帘升温，日落前放下草帘保温。     

北五加皮不同时间水提物中绿原酸含量的变化研究

利用HPLC法研究了北五加皮不同时间水提物中绿原酸含量的变化规律。采用色谱柱Agilent ZORBAX SB-C18(150mm×4.6mm,5μm),流动相为0.4%磷酸溶液(A)-乙腈(B),梯度洗脱[0～8min 24%B;8～12min 50%B],检测波长278nm,流速为1.0mL/min,柱温为25℃。结果表明:绿原酸的最佳提取时间为2h,该方法简单快速、可行,含量测定准确。     

TLC和HPLC法同时测定苦豆子总碱中槐定碱和苦参碱的体系优化

采用薄层层析法(TLC),展开剂为氯仿-甲醇-氨水=8∶2∶0.1;同时采用高效液相色谱法(HPLC),色谱柱为Ultra IITMC18(5μm×250mm×4.6mm)、流动相为0.01mol/L磷酸盐缓冲液-甲醇(45∶55)、检测波长为216nm、流速为1.0mL/min、柱温为30℃、进样量为10μL;建立TLC和HPLC同时分离测定苦豆子生物碱体系的优化方法。结果表明:槐定碱和苦参碱在200~900μg/mL浓度范围内呈良好的线性关系,R2分别为0.9994和0.9996。平均回收率(n=9)分别为99.47%和99.82%;试验表明可通过TLC和HPLC法同时测定苦豆子总碱中槐定碱和苦参碱的含量,并可用该方法进行苦豆子药材及含苦豆子药用成分的质量控制。     

新疆野生樱桃李SSR体系的建立及应用

为建立适宜新疆野生樱桃李的SSR分子标记技术体系,通过对PCR反应程序、反应体系(DNA模板量、引物浓度、Mg2+浓度、dNTP浓度、Taq酶用量)以及退火温度进行了探索,建立了适宜野生樱桃李的SSR-PCR反应体系。结果表明:在25μL反应体系中,Mg2+1.0 mmol/L、引物0.5μmol/L、dNTPs 0.20 mmol/L、TaqDNA聚合酶1.0 U、模板DNA 30 ng、退火温度为60℃。SSR扩增程序:94℃预变性5 min,35个循环(94℃30 s,60℃1 min,72℃1 min),72℃延伸10 min,可筛选出稳定性好、多态性高的SSR引物。     

快速检测琯溪蜜柚中农药残留的方法

建立了同时测定琯溪蜜柚中残留的10种农药的固相萃取—气质联用色谱分析方法。蜜柚样品经绞碎后,以乙腈为提取剂,超声波反复提取,提取液经无水硫酸钠脱水后,过GCB/PSA混合小柱净化,并以乙腈-甲苯(体积比为3:1)溶液洗脱,洗脱液经浓缩后,用于气相色谱-质谱联用仪分析。采用DB-35MS毛细管柱分离,以高纯氦气为载气,梯度升温,在选择离子(SIR)监测模式下进行分析检测。分析表明,10种农药标准曲线的回归系数R2均大于等于0.98,线性范围为0.1~1.0 mg·L-1,检出限为0.01~0.05 mg·L-1,在蜜柚中添加浓度为0.2mg·kg-1的各种农药,其添加回收率为88.3%~104.0%,相对标准偏差为4.46%~10.98%。该方法具有快速、灵敏的特点,符合现行农药残留分析的要求。     

黄瓜动蛋白特异肽段的原核表达和多克隆抗体制备

 以黄瓜果实总RNA为模板,通过RT-PCR获得了动蛋白基因CsKF1和CsKF2的cDNA序列,进行测序,构建T-easy载体。SMART在线预测CsKF1和CsKF2均含有动蛋白（Kinesin）约340个氨基酸残基的马达区保守序列（moter domain）。体外表达蛋白的马达区和非马达区,将带有His标签的CsKF1-N、CsKF2-C、CsKF1-M、CsKF2-M融合蛋白通过E. coli BL21（DE3）表达,摸索不同诱导条件下目的蛋白的表达和纯化。其中His-CsKF1-N、His-CsKF2-C、His-CsKF1-M原核表达蛋白的分子量分别为25、30和40 kD,均以0.1 mmol · L^-1 IPTG,22 ℃ 6 h诱导表达效果最好。His-CsKF2-M原核表达蛋白的分子量约38 kD,以18 ℃ 8 h诱导表达效果最好。融合蛋白His-CsKF1-N和His-CsKF2-C经过亲和纯化后作为抗原,通过5轮免疫注射兔子后,取心脏血作为抗血清,通过AminoLink Plus kit试剂盒亲和纯化得到多克隆抗体anti-CsKF1-N和anti-CsKF2-C。经过Western blot分析表明多克隆抗体具有特异性,可与抗原特异结合。     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

辣椒脉斑驳病毒CP基因的原核表达及其抗血清的制备

采用RT-PCR方法克隆了辣椒脉斑驳病毒文昌分离物（ChiVMV-WC）的CP基因,并将其连接到原核表达载体pET-30b（+）上,克隆测序以确定其阅读编码框的正确性,然后将获得的重组质粒pET30b-ChiVMV CP转化大肠杆菌Rosetta（DE3）后,用IPTG进行诱导表达。SDS-PAGE分析结果表明,CP基因在大肠杆菌中获得了高效表达,获得的融合蛋白分子量约为38 kD。用Ni2+-NTA 琼脂糖亲和层析纯化的融合蛋白免疫兔子并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的ChiVMV-WC编码CP蛋白发生特异性反应。间接酶联免疫吸附法（ID-ELISA）检测抗血清效价为1/106。通过对田间20个样品的ID-ELISA检测,证实了所制备的抗血清与ChiVMV病叶具有良好的反应特异性。     

Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas

AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients.     

香蕉果实转录因子MaWRKY1基因的原核表达和多克隆抗体制备

MaWRKY1是从香蕉果实中克隆出的一个转录因子基因。为进一步研究该基因的功能,制备了MaWRKY1多克隆抗体。选取MaWRKY1基因全长中N端第168 ~ 400个氨基酸之间的包括两个WRKYGQK保守域的cDNA序列,构建了原核表达载体pET-MaWRKY1,并转化到大肠杆菌BL21中诱导表达菌体蛋白。SDS-PAGE电泳检测结果表明,His-MaWRKY1融合蛋白成功获得了高效表达,分子量在26 kD左右。His-MaWRKY1融合蛋白经过Ni-NTA琼脂糖凝胶树脂纯化,SDS-PAGE制备胶割胶富集,电洗脱法纯化后得到的纯化蛋白浓度达到0.5 mg • mL-1。经对新西兰兔进行5次免疫,获得了多克隆抗血清,采用免疫吸附方法对抗血清进行了纯化。将纯化后的抗体通过间接酶联免疫（ELISA）和蛋白质印迹（Western blot）分析,表明所制备的抗体具有很好的效价,效价比为1︰160 000,同时具有良好的灵敏度和特异性。进一步提取香蕉不同组织总蛋白,Western blot检测显示,在分子量26 kD左右处出现特异的蛋白质条带,证明所制备的抗体可以与香蕉WRKY蛋白特异性结合,并且低温可以诱导香蕉果实中MaWRKY1蛋白表达,暗示MaWRKY1蛋白表达可能与果实耐冷性有一定的关系。     

普通白菜1，5- 二磷酸核酮糖羧化/ 加氧酶小亚基基因BcrbcS 的克隆及表达分析

利用RACE 技术，从普通白菜抗霜霉病品种苏州青叶片克隆到1，5- 二磷酸核酮糖羧化/ 加氧酶小亚基（ribulose-1， 5-bisphosphate carboxylase/oxygenase small subunit，BcrbcS）基因的全长cDNA 序列。采用qRT-PCR 分析该基因在普通白菜不 同组织的表达模式。利用SDS-PAGE 技术分析了该基因的原核表达特征。序列分析结果表明，BcrbcS 基因的cDNA 序列全 长为733 bp，其中开放阅读框长度为543 bp，共编码181 个氨基酸，分子质量为20.3×10³ Da，理论等电点为8.23。氨基酸 同源系统进化分析表明，普通白菜BcrbcS 基因与同科植物的进化关系相近。实时定量分析结果表明，BcrbcS 基因在普通白 菜叶中表达最强；在SA 和NaCl 处理下，BcrbcS 基因表达量均在处理24 h 后达到峰值。原核表达载体经IPTG 诱导表达出分 子质量约为20×10³ Da 的融合蛋白。     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Prokaryotic expression of Staphylococcus aureus Panton-Valentine leukocidin and its cytolytic activities to human polymorphonuclear neutrophils

AIM:Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus epidemiologically associated with the often-lethal necrotizing pneumonia. Until now, the mechanisms of pathogenesis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult. In the present study, we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro, which provides an experimental basis for the further studies of its biological function and its toxicity in pneumonia. METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by high-fidelity PCR was cloned into prokaryotic expression vector pET22b(+), and the vector was transformed into BL21 (DE3)plysS to construct a prokaryotic expression system. The integrity of the opening-reading frame of each construct was verified by DNA sequencing. The recombinant PVL (rPVL) was induced by1.0 mmol/L IPTG. The expressed products were identified by SDS-PAGE and the fusion proteins (6His-LukS-PV and 6His-LukF-PV) were purified from lysates of transfected E. coli cells by affinity chromatography on nitrilotriacetic acid columns. The cytolytic activity was tested by incubation of rPVL with human polymorphonuclear neutrophils (PMNs) in vitro. RESULTS:The nucleotide sequence of the cloned PVL gene was the same as that of reported in GenBank. E. coli BL21 (DE3)plysS containing recombinant vectors grow at 37℃ causes some proteins to accumulate as inclusion bodies, while incubation at 30 ℃ led to a significant amount of soluble active proteins which accounted for about 31.7% of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD, and the LukF-PV protein was about 35 kD. The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated. CONCLUSION:The genes of lukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+) and expressed in E. coli respectively, which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.     

新疆红肉苹果PGIP基因的克隆及原核表达

【目的】研究克隆新疆红肉苹果[Malus sieversii f.neidzwetzkyana(Dieck)Langenf]PGIP基因并进行原核表达,探讨其抗病机制。【方法】根据Genbank中已经发表的‘金冠’苹果PGIP保守区域设计1对特异引物,以新疆红肉苹果叶片总RNA为模板,T/A克隆后进行序列测定,并对该序列进行分析。随后将该蛋白成熟肽cDNA片段连接到原核表达载体pET30a(+)中,构建融合表达质粒,转化到E.coli BL21(DE3)中进行表达。【结果】序列分析表明,新疆红肉苹果PGIP基因cDNA编码区全长993 bp,编码330个氨基酸残基,命名为MsPgip,GenBank登录号为JQ001783。MsPgip分子质量为36.6 kD,等电点为7.05,有6个潜在的N-糖基化位点,信号肽为N端24个氨基酸残基。该蛋白质还具有2个连续的24个氨基酸残基大小的LRR基序(LSQLKNLTFLDLSFNNLTGAIPSSLSQ LPNLNALHLDRN-KLTGHIPIS)。与已克隆的‘澳洲青苹’、‘金冠’、‘富士’苹果PGIP氨基酸序列同源性均高达99%。原核表达产物经SDS-PAGE分析表明,表达蛋白的分子质量与预期一致。【结论】克隆了新疆红肉苹果PGIP基因,并可在大肠杆菌中表达。     

番木瓜环斑病毒外壳蛋白基因原核表达蛋白的抗血清制备及其检测应用

 以受番木瓜环斑病毒（Papaya ring spot virus,PRSV）侵染的番木瓜（Carica papaya）叶片为供试材料,采用RT-PCR 方法克隆其外壳蛋白基因cp,并将其连接到原核表达载体pET-28b（+）上,酶切鉴定及克隆测序确定开放阅读框的正确性,将获得的重组质粒转化大肠杆菌表达宿主菌。通过摸索转化的表达宿主菌种类、IPTG 浓度及诱导时间,获得高效表达PRSV cp 的条件。SDS-PAGE 分析结果表明,CP 融合蛋白分子量为36.8 kD。以Ni2+-NTA 亲和层析柱纯化的融合蛋白为抗原,免疫注射新西兰大白兔制备得到高效价抗体,间接ELISA 测定效价为1︰16 384。Western blot 检测结果表明,制备的抗血清可与诱导表达的融合蛋白发生特异性反应。通过ID-ELISA 检测田间样品证实了制备的抗血清与PRSV 侵染病叶发生了良好的特异性反应。本试验为PRSV 的快速检测以及PRSV 编码蛋白的功能研究奠定了基础。