Effects of NF-κB decoy oligonucleotides on apoptosis in lung cancer cell A549

AIM: To investigate the effects of NF-κB decoy oligodeoxynucleotides (ODNs) on apoptosis in lung cancer cell A549. METHODS: The treatments of lung cancer cells (A549) were divided into three groups: group A (control group); group B (decoy ODN group) and group C (scramble decoy ODN group). FITC-labeled NF-κB decoy ODNs was transfected into A549 with LipofectAMINE^TM2000. The activation was observed by electrophoretic mobility shift assays (EMSA). The proliferation was observed by growth curve. The apoptosis of cells were observed by flow cytometry and TdT mediated dUTP-biotin Nick End Labeling (TUNEL). The expression of Bcl-2 and Fas were observed by Western blot. RESULTS: After FITC-labeled decoy ODNs was transfected for 1 hour, the decoy ODNs was detected in the nuclei of A549 cells. EMSA performed the depression of the NF-κB binding to the nucleus. The growth curve showed the inhibition of the A549 cell growth and the percentage of apoptosis was increased compare with control group by flow cytometry and TUNEL. The amount of apoptosis inhibitor (Bcl-2) in group A and group C were 2.0 times and 2.1 times more than that in group B, respectively. The level of apoptosis accelerator (Fas) in group B were 2.6 times and 2.3 times more than that in group A and group C, respectively via Western blot. CONCLUSION: The NF-κB decoy ODNs accelerate the apoptosis of lung cancer cell A549 and the mechanism may be due to its inhibiting the expression of Bcl-2 and increasing the level of Fas.     

鹅细小病毒VP2基因在大肠杆菌中的表达及纯化

将鹅细小病毒 (GPV) VP2基因插入原核表达载体 p PROEX- HTb,获得重组表达质粒 p PROEX- HTb- VP2。将其转化大肠杆菌 DH5α,用 IPTG诱导表达 ,表达菌体蛋白中可产生与预期大小相符的约 72 0 0 0的蛋白。以光密度扫描对该表达产物进行定量分析 ,表达产物约占菌体总蛋白的 14 %。经 His- tag金属螯合层析纯化 ,获得纯度较高的 6 His- VP2融合蛋白。 Western- blot结果表明 ,所得蛋白与鹅抗 GPV高免血清有较好的免疫反应性 ,说明在 VP2的 N端融合 6个组氨酸不影响其与特异抗体结合的活性。     

丝状霉形体丝状亚种SC型脂蛋白LppQ N末端基因克隆与序列分析

脂蛋白LPPQ是丝状霉形体丝状亚种SC型(MmmSC)非洲株、欧洲株和疫苗株所特有的。LPPQ N末端域具有良好的免疫原性，在牛体内可诱导产生强大、特异、早期、持续的免疫反应。本研究根据已发表的LPPQ基因序列设计引物，用Pyrobest^TM高保真DNA聚合酶从MmmSC HVRI X株中扩增出了LPPQ N末端基因序列，并进行了克隆与序列测定。核苷酸序列比较结果显示，HVRI X株的LPPQ N末端基因序列与国外发表的序列同源性为99．7％，由其推导的氨基酸序列同源性为99，1％，为脂蛋白LPPQ N末端基因体外表达奠定了基础。     

猪瘟病毒E2(gp55)基因在昆虫细胞中的表达

将除去3'端跨膜区的猪瘟病毒E2基因亚克隆到杆状病毒转移载体pFastBacHTb,获得重组质粒pFBHT-E2,转化进含穿梭载体Bacmid的感受态细胞DH10Bac,发生转座作用,经抗性及蓝白斑筛选得到含E2基因的重组载体质粒rBacmid-E2,以脂质体介导的方法将此重组载体质粒转染sf9昆虫细胞,获得重组病毒,命名为rvBac-E2.经SDS-PAGE和Western-blot及ELISA等方法检测,结果表明,此E2基因在昆虫细胞中正确表达,表达的蛋白能与猪瘟阳性血清发生特异性反应.     

禽副粘病毒-2型标准毒株与不同分离株HN基因的序列测定及分析

提取实验室保存禽副粘病毒-2标准毒株Yucaipa株和3株分离毒株F4、F6、F8株的RNA，经RT—PCR，获得4株毒株的HN基因，同时测得F基因与HN基因之间的序列。序列分析结果表明，HN基因的ORF全长为1743 nt，编码一个由580个氨基酸组成的蛋白。运用DNAMAN软件分析，4株毒株与GenBank上已发表毒株的HN基因的核苷酸和氨基酸之间的同源性相比，同源率分别为99．89％和99．69％。分析F基因与HN基因间序列发现两基因间序列与副粘病毒科其他病毒的基因间序列相似，含有基因起始信号和终止信号，但不含3个碱基的连接序列。通过序列分析，在分子水平上进一步证实分离于同一批进口七彩文鸟的F4、F6株是抗原性存在差异的两个毒株。毒株间的血清学关系与分子水平的核苷酸序列、氨基酸序列的比较关系一致。     

体外试验中不同粗精比对山羊瘤胃细菌氨态氮利用效率的影响

利用体外发酵法研究了4种粗精比(10∶0、8∶2、6∶4和4∶6)底物对山羊瘤胃细菌产量、15NH3-N利用效率的影响。结果表明:混合培养液的pH值与氨态氮浓度在8h内急剧下降,而后至24h呈缓慢下降;经24h培养的细菌产量在8∶2、6∶4和4∶6组中差异不显著(P>0.05),但都极显著地高于10∶0组(P<0.01);细菌体15N丰度以8∶2及4∶6组较高,显著高于10∶0和6∶4组(P<0.05);细菌体15N富集量以8∶2组最高,底物547.43μg15N中有120.44μg被细菌体富集,显著高于10∶0组(P<0.05),但6∶4、4∶6组与10∶0组间差异均不显著(P>0.05)。细菌赖氨酸的相对百分组成随精饲料比例的增加而显著增加,胱氨酸在粗饲料组(10∶0)显著高于其它组,但各精饲料添加组间差异不显著,组氨酸以8∶2组为最高,显著高于10∶0组,但与其它精饲料添加组间差异不显著(P>0.05)。     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

几种缓释肥的氮释放特性以及对草坪草生长的影响

实验室内采用水浸泡法对尿素、3种缓效肥和山西省农业科学院土壤肥料研究所研制(自研草坪肥)的缓效肥的初期溶出率和微分溶出率进行了测定.其中尿素的初期溶出率为22.75%,超出了国际上公认的缓/控释肥的初期溶出率小于15%的指标,而微分溶出率为0.22%,也小于国际上公认的缓/控释肥的微分溶出率(0.25%～2.5%)指标.其他几种缓释肥的初期溶出率为8.73%～14.42%,微分溶出率为0.30%～2.11%,符合国际上公认的缓/控释肥的初期溶出率和微分溶出率指标.田间试验比较和研究了尿素、3种缓效肥和山西省农业科学院土壤肥料研究所研制的缓效肥对草坪草的干草产量和养分含量的影响,尿素和包衣尿素处理的草坪草的生长量前期较高,后期产草量较低,呈马鞍型.其他处理的草坪草干草产量则比较平缓,肥料的氮释放特性与草坪草生长趋势相同.不同肥料处理对草坪草的中量、微量元素含量有一定的影响.     

鸭流感病毒4/2004-H6N2亚型毒株的血凝素基因全序列克隆与分析

2004初从正常鸭群中分离到一株鸭源禽流感病毒，命名为A／Duck／HN／4／2004(H6N2)。经对血凝素基因(HA)序列分析发现HA基因全长为1744bp，共编码566个氨基酸，在裂解位点仅含一个碱性氨基酸-精氨酸(R)，符合LPAIV的标准。将所得基因序列与已发表的同一亚型参考序列分析表明，与H6亚型流感HA基因同源性为89．2％-97．1％，经分子遗传演化分析表明本次分离株与香港分离株A／Duck／Hong Kong／3600／99(H6N2)、A／Duck／Hong Kong／3600／99(H6N2)最近。     

毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因在大肠杆菌的表达

根据克隆的毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因(EnMIC-2(Gd))的cDNA序列设计特异性引物，用PCR方法扩增其阅读框架(ORF)后，克隆至质粒表达载体pET-32a( )，成功构建了重组表达质粒pET-32a( )-EnMIC-2。用CaCl2法将其转化至宿主细菌E.cliBL21(DE3)，并用IPTG成功诱导了EnMIC-2重组抗原在大肠杆菌表达。表达的重组蛋白约占菌体总蛋白的10．8％，其相对分子质量约为55000。重组蛋白经SDS-AGE分析后，用E．necatrix(广东株)感染鸡的高免血清进行Western Blotting分析，结果为阳性。