口服sST2质粒DNA在炎症性肠病小鼠中的免疫水平

目的制备口服可溶性ST2(Soluble ST2,sST2)质粒,观察其对于小鼠炎症性结肠黏膜免疫应答水平。方法提取小鼠脾脏总RNA,扩增sST2基因,并克隆至pcDNA3.1/myc-His A载体,构建pcDNA3.1-sST2-myc-His A质粒,借此转染COS-7细胞,使之表达sST2-myc-His A融合蛋白。利用脂质体LipofectamineTM2000包裹,制备口服sST2质粒,经灌胃给予用葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱生溃疡性结肠炎的C57BL/6实验小鼠,并用组织病理学方法观察结肠黏膜组织形态变化。用Real-time PCR检测sST2基因表达,用免疫印迹检测sST2质粒融合蛋白的表达;用ELISA检测小鼠肠固有层淋巴细胞培养上清中细胞因子(IL-4、IL-5和IL-13)的分泌水平。结果Real-time PCR检测到sST2基因的正确表达,经双酶切及测序鉴定,证明质粒pcDNA3.1-sST2-myc-His A构建成功,免疫印迹试验在转染的COS-7细胞检测到sST2-myc-His A融合蛋白的表达,其相对分子质量与预期的相符;观察到结肠黏膜组织形态发生的变化;Real-time PCR结果显示,sST2质粒组在结肠黏膜组织内sST2的表达水平明显高于pcDNA3.1组和生理盐水组(P0.01);ELISA结果显示sST2质粒组结肠固有层淋巴细胞培养上清中IL-4、IL-5和IL-13的分泌水平高于pcDNA3.1组和生理盐水组(P0.05)。结论实验成功制备口服sST2质粒;该质粒可抑制小鼠炎症性结肠黏膜组织内Th2型免疫应答。     

小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达

目的:构建小鼠白介素27(Interleukin 27,IL-27)单链融合基因的真核表达载体并检验其在RAW264.7细胞中的表达情况。方法:提取小鼠脾细胞总RNA,通过RT-PCR扩增出小鼠EBI3和p28 c DNA。采用重叠延伸PCR(splicing by overlap extension PCR,SOE PCR)通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3和p28基因片段,构建小鼠IL-27单链融合基因(mouse single chain IL-27,msc IL-27),并将其克隆至pc DNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体,将重组质粒pc DNA3.1-IL-27通过脂质体转染法转染小鼠巨噬细胞株RAW264.7,通过RT-PCR方法检测目的基因的表达。结果:测序分析表明,小鼠IL-27单链融合基因中EBI3、linker和p28的连接顺序、方向及碱基序列与预期相符。在转染后的RAW264.7细胞中检测到了小鼠IL-27 m RNA的表达。结论:成功构建了小鼠IL-27单链融合基因及其真核表达载体,并在RAW264.7细胞中实现表达,为进一步探讨IL-27的生物学功能奠定了基础。     

信号肽和辅助性T细胞表位增强HBV核心抗原DNA疫苗诱导的免疫应答

为增强HBVDNA疫苗的免疫效率 ,于HBV核心抗原 (HBcAg)基因 5′末端引入人IL 2信号肽和一个通用型辅助性T淋巴细胞表位基因 ,并构建成DNA疫苗 ,转染COS7细胞后经ELISA检测出分泌型HBcAg。通过肌肉注射途径分别将这种DNA疫苗和编码天然HBcAg的DNA疫苗免疫BALB/c小鼠 ,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应 ,结果表明前者诱导细胞和体液免疫应答的强度均明显超过后者 ,且更趋向于T辅助细胞 1(Th1)型免疫应答 ,故其对慢性HBV感染的治疗可能有潜在的应用价值     

大肠杆菌 LT B亚基基因表达载体对犬细小病毒VP2 DNA疫苗免疫应答的增强作用

【目的】通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素(LT)B亚基基因对犬细小病毒VP2DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA,通过PCR方法从基因组DNA中扩增LTB基因,同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因(VP2-70,编码70个氨基酸)。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上,分别构建成它们的分泌型真核表达载体,pcDNA-CD5sp-LTB和pcDNA-CD5sp-VP2-70。再利用酶切连接的方法构建LTB与VP2-70融合的真核表达载体pcDNACD5sp-LTB-VP2-70。然后用pcDNACD5sp-VP2-70(VP2-70组)、pcDNACD5sp-LTB-VP2-70(VP2-LTB融合组)、pcDNA-CD5sp-LTB/pcDNACD5sp-VP2-70(VP2-LTB共免疫组)和pcDNA3.1A(空载体对照组)分别免疫小鼠。免疫后用间接ELISA检测不同时间小鼠血清的抗体水平,用MTT方法检测小鼠免疫5周后脾脏淋巴细胞的增殖活性。【结果】经过测序表明本研究扩增的LTB和VP2基因序列和构建的相关表达载体结构正确。通过Western-blot检测证明构建的表达载体均能介导相应基因在真核细胞进行分泌表达。ELISA检测结果表明,3组实验组小鼠接受VP2DNA疫苗免疫后均能产生特异的体液免疫应答反应,特别是VP2-LTB基因融合组小鼠的抗体水平在第5周时高达1:5120,明显高于其它两组(P<0.01)。3组免疫小鼠抗体的亚型均表现IgG1抗体水平明显高于IgG2a抗体水平(P<0.01)。淋巴细胞增殖实验结果表明,在ConA的刺激下,3组免疫小鼠的淋巴细胞刺激指数均明显高于对照组(P<0.01),说明VP2DNA疫苗能够引起淋巴细胞的增殖。但3组免疫小鼠之间的刺激指数没有明显差异(P>0.05)。【结论】在小鼠体内,LTB基因表达载体可明显提高CPVVP2DNA疫苗的体液免疫应答水平。     

IL-12对巨细胞病毒DNA疫苗免疫增强作用的研究

白介素12(interleukin 12,IL-12)主要和细胞免疫应答有关,是免疫过程中重要的调节因子。本研究探讨IL-12对编码巨细胞病毒(cytomegalovirus,CMV)即刻早期基因IE1的DNA疫苗的免疫增强作用。将CMVIE1质粒DNA单独或与编码IL-12的质粒DNA共同免疫小鼠,然后用致死量病毒攻击小鼠。通过检测小鼠体内诱导的细胞免疫应答、小鼠的存活率、体重丢失率、器官中的病毒滴度等来评价IL-12对疫苗免疫的佐剂效果。结果显示,与单独疫苗免疫组相比,IE1 DNA联合IL-12 DNA免疫组能够在小鼠体内诱导更高的细胞免疫应答水平,同时能够降低器官中的病毒滴度,显著提高保护率,从而更好地抵抗病毒攻击。实验证明,IL-12能够作为巨细胞病毒IE1 DNA疫苗的佐剂,提高免疫保护效果。     

共表达PRRSV GP5蛋白和CSFV E2蛋白的“自杀性”DNA疫苗在小鼠体内诱导的免疫应答

为了获得新型双价"自杀性"DNA疫苗,将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratorysyndrome virus,PRRSV)GP5基因克隆于此前构建的表达猪瘟病毒(Classical swine fever virus,CSFV)E2基因的甲病毒复制子载体疫苗pSFV1CS-E2中.为了增强免疫效果,在密码子优化的GP5基因中插入了泛DR表位(PADRE),在CSFV E2基因后融合伪狂犬病病毒(PrV)UL49基因,获得了6种重组质粒.间接免疫荧光试验显示,PRRSV GP5和CSFV E2基因在瞬时转染的293T细胞中得到同时表达,将6种重组质粒和空载体pSFV1CS分别免疫BALB/c小鼠,用间接ELISA方法检测血清抗体水平,通过基于CSFE/WST-8的淋巴细胞增殖试验和细胞因子ELISA评价疫苗诱导的细胞免疫.结果显示,除pSFV1CS组外,从各疫苗组小鼠血清中均可检测到低水平的针对GP5和E2蛋白的抗体;各疫苗组小鼠脾细胞经CSFV和PRRSV刺激后均能诱导特异性的淋巴细胞增殖:部分疫苗组小鼠脾细胞经CSFV和PRRSV刺激后可分泌较高水平的IFN-γ和IL-4;引入UL49的疫苗组细胞免疫应答显著高于其它疫苗组.结果表明,这些共表达GP5和E2蛋白的自杀性DNA疫苗可以诱导体液免疫和细胞免疫,PrV UL49可以增强其细胞免疫应答.     

卡介苗菌基因组DNA对小鼠腹腔巨噬细胞激活效应的初步研究

为了比较研究卡介苗菌基因组DNA和卡介苗菌激活小鼠腹腔巨噬细胞的抗结核免疫应答过程及免疫作用,用卡介苗菌基因组DNA、卡介苗菌和生理盐水分别皮内注射小鼠第30、60 d后,分别采用Griess法、化学法、ELISA法检测小鼠腹腔巨噬细胞产生的NO、H2O2以及IL-12、TNF-α的表达.研究结果,卡介苗菌基因组DNA和卡介苗菌皮内接种小鼠后能显著诱导小鼠腹腔巨噬细胞分泌表达IL-12和TNF-α.卡介苗菌基因组DNA组和卡介苗菌组相比较,无明显差异,与生理盐水组比较,其差异均有统计学意义(P<0,05);卡介苗菌基因组DNA和卡介苗菌皮内接种小鼠后也能诱导小鼠腹腔巨噬细胞产生NO、H2O2,卡介苗菌基因组DNA组和卡介苗菌组相比较,无明显差异,与未免疫组比较均具有统计学意义(P<0.05).研究结果表明,卡介苗菌基因组DNA皮内接种小鼠后能诱导小鼠腹腔巨噬细胞活化并产生较强的激活效应,且该效应于卡介苗菌无明显差异.     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

牛分枝杆菌单基因及双价融合基因DNA疫苗免疫效果的研究

利用PCR技术和SOE技术扩增牛分枝杆菌ag85b、esat-6、hsp65、mpb64基因和ag85b-esat-6、hsp65-esat-6和mpb64-esat-6融合基因,连接真核表达载体pCDNA3.1(+),构建重组质粒pCA、pCE6、pCH、pCM、pCAE、pCHE和pCME。转染SP2/0细胞,检测目的基因的表达。以各重组质粒和pCDNA3.1(+)及PBS免疫BALB/c小鼠后检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN~γ分泌情况。结果表明,七种重组质粒免疫后小鼠血清抗体水平持续上升,与 pCDNA3.1(+)对照组和PBS对照组相比差异显著 （P<0.05）,其中pCA组血清抗体水平明显高于其他六种DNA疫苗免疫组 （P<0.05）;三免两周后,融合基因免疫组的刺激值（SI值）与单基因免疫组相比差异显著（P<0.05）,其中以pCME组的SI值最高;PPD刺激后融合基因DNA疫苗免疫组小鼠脾细胞分泌的IFN~γ高于单基因DNA疫苗组（P<0.05）,而两对照组则未检测到IFN~γ的产生。本试验成功构建了牛分枝杆菌ag85b、esat-6、hsp65、mpb64单基因和ag85b-esat-6、hsp65-esat-6、mpb64-esat-6双价融合基因DNA疫苗,从而为牛结核病新型疫苗的研制奠定了基础。     

表达流行性感冒病毒HA基因非复制型重组腺病毒的构建及免疫效果的研究

通过RT-PCR扩增流行性感冒(流感)病毒HA基因,克隆至腺病毒穿梭载体pAd Track-MV,该重组质粒与腺病毒DNA共转化E．coli BJ5183,通过细菌内同源重组获得重组腺病毒DNA,将其转染293细胞获得重组腺病毒。PCR证实HA基因已整合至腺病毒基因组中,Western blot结果检测到重组病毒感染293细胞中HA的表达。重组病毒经滴鼻和灌胃两种途径免疫小鼠,结果2次免疫后滴鼻组和灌胃组均产生明显的免疫应答,血清IgG抗体滴度分别为1：10000和1：1000。除血清IgG外,还在肺灌洗液中检测到分泌型IgA。滴鼻组的免疫效果强于灌胃组。经小剂量攻毒实验显示,重组腺病毒保护率为100％。该文成功构建了表达流感病毒HA基因的非复制型重组腺病毒,重组病毒免疫小鼠可产生较好的免疫效果。     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Background  

The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.     

百合LbCAT和LbGPX基因的克隆与表达分析

以切花百合(Lilium brownii var. viridulum)‘卡瓦纳’cDNA为模板,克隆了过氧化氢酶(LbCAT)和谷胱甘肽过氧化物酶(LbGPX)基因。序列分析表明,这2个基因分别包含1 479 bp和519 bp的开放阅读框(ORF),编码492个和172个氨基酸。进化分析结果表明,LbCAT蛋白与岷江百合CAT蛋白的氨基酸序列相似性最高(99.19%),且亲缘关系最近;LbGPX蛋白与油棕GPX蛋白的氨基酸序列相似性最高(78.61%),亲缘关系最近。qRT PCR结果显示,LbCAT和LbGPX在百合根、鳞茎、叶和花中都有表达。LbCAT在叶中表达量最高,LbGPX在花中表达量最高。这2个基因在百合花蕾的生长发育过程中均有表达,且表达量逐渐增加;在PEG处理后2个基因的转录水平升高,但独角金内酯(SLs)处理却显著降低了这2个基因的转录水平;该结果为百合抗逆性机理研究以及抗逆育种奠定了基础。     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

水椰八角铁甲和椰心叶甲对椰子和银海枣的寄主选择性

【目的】水椰八角铁甲和椰心叶甲均为棕榈科植物的重要入侵害虫,两者的外部形态、取食部位和危害特征相似。研究它们的寄主选择性有助于了解这2种害虫的扩散和成灾机制。【方法】在室内用椰子和银海枣2种寄主植物分别饲养水椰八角铁甲和椰心叶甲,研究在不同寄主植物上水椰八角铁甲和椰心叶甲的存活率、产卵率、发育历期等以及这2种害虫对不同寄主植物的选择性。【结果】水椰八角铁甲在2种寄主上的存活率差异显著,除了卵期和蛹期之外,幼虫期各虫态在银海枣上的存活率明显比在椰子上的存活率高;椰心叶甲在椰子上的存活率高于银海枣,各虫态平均存活率分别为95%和86%。取食银海枣的水椰八角铁甲达标准卵量概率为0.23,取食椰子不产卵,无法完成整个世代;取食椰子的椰心叶甲达标准卵量概率为0.86,取食银海枣不产卵,也无法完成整个世代;水椰八角铁甲取食银海枣完成世代的实验种群趋势指数为12.55,椰心叶甲取食椰子完成世代的实验种群趋势指数为66.55。【结论】水椰八角铁甲和椰心叶甲分别对银海枣和椰子这2种寄主植物具有明显的选择性。在海南椰子树的数量远远超过银海枣,该实验结果在一定程度上解释了椰心叶甲在海南岛广泛分布而水椰八角铁甲只是零星发生的原因。     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.