黄芩醇提液对甲型流感病毒核蛋白基因表达的影响

目的：探讨黄芩醇提液对甲型流感病毒核蛋白（NP）的作用。方法本实验设HeLa细胞组、pcDNA3.1（＋）/empty组、pcDNA3.1（＋）/NP 组、TFSB 组,其中pcDNA3.1（＋）/empty 组、pcDNA3.1（＋）/NP 组分别通过瞬时转染将重组质粒 pcDNA3.1（＋）/empty、pcDNA3.1（＋）/NP转染到HeLa细胞中;黄芩醇提液组在将重组质粒pcDNA3.1（＋）/NP转染到HeLa细胞的同时使用黄芩醇提液进行药物干预。结果与真核重组质粒pcDNA3.1（＋）/NP组比较,黄芩醇提液组NP基因起始拷贝数具有统计学意义,P＜0.05。结论黄芩醇提液能够下调甲型流感病毒NP基因的表达。     

副黏病毒Tianjin株NP蛋白的稳定表达及其检测

目的:构建副黏病毒Tianjin株NP基因的真核表达载体,转染HEK293细胞,经G418筛选出稳定表达NP蛋白的细胞.方法:应用RT-PCR技术克隆副黏病毒Tianjin株NP基因,插入真核表达载体pcDNA3.1(+).经PCR、酶切和测序鉴定后,将构建的pcDNA3.1(+)-NP用LipofectaminenTM2000转染试剂转染HEK293细胞.应用免疫荧光法及Western Blot检测NP蛋白的瞬时和稳定表达.结果:RT-PCR扩增得到NP基因,重组质粒pcDNA3.1(+)-NP转染HEK293细胞后,经免疫荧光法检测到目的蛋白的瞬时和稳定表达.Western Blot法可见NP蛋白瞬时和稳定表达的条带.结论:副黏病毒Tianjin株NP基因在HEK293细胞可以瞬时和稳定表达,为研究副黏病毒Tianjin株NP蛋白功能与病毒致病性、宿主亲嗜性奠定了基础.     

鱼腥草及三丫苦对甲型流感病毒NPAG表达的影响

目的：探讨鱼腥草及三丫苦对甲型流感病毒（nucleo protein antigen,NPAG）表达的影响,了解鱼腥草及三丫苦抗甲型流感病毒的分子机制。方法以胶体金法测定NPAG的表达。瞬间转染,实验分为：Hela细胞组、空质粒组[PCDNA3.1（＋）/empty]、重组质粒组[PCDNA3.1（＋）/NP）]、脂质体组、三丫苦醇提物组和鱼腥草油组。药物实验加入重组质粒同时将药物加入培养细胞孔中进行干预。转染48h后,测定其上清液NPAG的表达。结果胶体金法测试显示：Hela细胞组、PCDNA3.1（＋）/empty组、脂质体组、鱼腥草油组为阴性;PCDNA3.1（＋）/NP组、三丫苦醇提物为阳性,NPAG浓度1：64。结论鱼腥草油组可抑制甲型流感病毒NPAG的表达,能抗甲型流感病毒。三丫苦醇提物组不影响NPAG的表达。     

真核表达质粒pcDNA3．1（＋）-rmCGβ的构建和鉴定

目的构建恒河猴绒毛膜促性腺激素β亚基macacam ulattac horionicg onadotrophin β subunit,rmCGβ）基因真核表达质粒pcDNA3．1（＋）-rmCGβ,并了解该质粒能否在真核细胞中表达。方法通过PCR扩增rmCGβ的全段基因cDNA,应用基因工程技术将扩增的cDNA克隆至PGM-Teasy,测序后插入pcDNA3．1（＋）真核表达质粒,构建重组真核表达质粒pcDNA3．1（＋）-rmCGβ,经限制内切酶酶切分析及测序鉴定正确后,用脂质体转染技术转染B16细胞,通过RT-PCR扩增出B16／pcDNA3．1（＋）-rmCGβ细胞株中rmCGβ全段基因cDNA。结果经4轮PCR,成功扩增出rmCGβ的cDNA全长基因,酶切和测序证明正确构建了真核表达质粒pcDNA3．1（＋）-rmCGβ,通过RT-PCR方法证实该质粒能在B16真核细胞中正确表达。建立了稳定的细胞株B16／pcDNA3．1（＋）-rmCGβ。结论成功克隆和构建了rmCGβ的真核表达质粒载体pcDNA3．1（＋）-rmCGβ,为进一步研究rmCGβ的新功能和免疫治疗奠定了基础。     

2002—2003年流感流行季节使用流感疫苗成分的建议

2001年10月至2002年2月，全球分离到的甲型流感病毒主要为甲型流感（H3N2）病毒，与乙型流感病毒共同传播，在一些国家也分离到甲型流感（H1N1）和甲型流感（H1N2）病毒。对流感病毒分离株的抗原性分析表明，甲型流感（H3N2）病毒分离株的抗原性与A／莫斯科／10／99／和A／巴拿马／2007／99参考病毒密切相关；甲型流感（H1N1）病毒和甲型流感（H1N2）病毒分离株的抗原性与A／新喀里多尼亚／20／99密切相关；乙型流感病毒分离株的抗原性与B／四川／379／99和B／香港／330／2001参考病毒密切相关。     

pcDNA3.1/eIF5A真核表达载体的构建及其在真核细胞CCRF-CEM中的表达

目的：摸索真核细胞翻译启动因子5A（eIF5A）编码基因的合成、pcDNA3．1／eIF5A真核表达载体的构建及其在真核细胞CCRF-CEM中的表达。方法：用PCR合成eIF5A基因，将基因分别接在克隆载体pMD18-T的EcoR V多克隆位点上和真核表达载体pcDNA3．1的Hind Ⅲ和EcoR Ⅰ的多克隆位点之间，构建eIF5A基因的克隆载体和真核表达载体，分别在大肠杆菌E．coli DH5α中转化并提取质粒，将真核表达载体pcDNA3．1／eIF5A的质粒提取后，用脂质体包合并转染真核细胞CCRF-CEM，用流式细胞仪对eIF5A的表达水平进行检测。结果：eIF5A在CCRF-CEM细胞中的表达水平为107．03，eIF5A在转染细胞CCRF-CEM／trans中的表达水平为114．27，表达升高。结论：本实验构建了pcDNA3．1／eIF5A真核表达载体，发现其在CCRF-CEM细胞中高表达。本实验结果将有助于探讨肿瘤治疗过程中的新靶标。     

转染p21基因对人宫颈癌Hela细胞的影响

目的研究转染p21基因对人宫颈癌Hela细胞的抑制及凋亡影响。方法用Lipofectamine2000脂质体介导,将质粒pcDNA3．1（＋）-p21基因转入人宫颈癌Hela细胞株;免疫组化法检测转染021基因后,其编码蛋白在Hela细胞的表达情况;描绘p21基因转染后Hela细胞的增殖曲线;WST-1法测定OD值,计算细胞生长抑制率及流式细胞仪检测p21基因转染对Hela细胞的凋亡影响。结果免疫组化法显示p21基因转染后在Hela细胞的胞核内高表达;p21基因的表达可抑制Hela细胞的体外生长,且Hela／021细胞的生长曲线较对照组明显降低;WST-1法显示Hela／p21的细胞活力与对照组相比有显著性差异（P〈0．05）;流式细胞仪检测显示转染p21基因的Hela细胞凋亡率显著高于对照组,约20％以上。结论转染p21基因对Hela细胞具有明显的抑制和诱导凋亡作用。     

人乳腺癌转移抑制基因真核表达载体的构建及鉴定

目的构建乳腺癌转移抑制基因（BRMS1）的真核表达载体pcDNA3．1（-）B／myc-BRMSI,为进一步研究恶性肿瘤的转移机制及基因治疗提供物质基础。方法常规方法培养人乳腺癌细胞株MCF-7,Trizd法提取细胞株总RNA;设计一对特异性引物,经过逆转录反应获得BIIMS1 cDNA的CDS序列,连接到质粒pcDNA3．1／mye—His（-）B上,构建BRMS1的真核表达载体peDNA3．1（-）B／myc．BRMS1,行基因测序鉴定正确后转染人胚肾细胞HEK-293,行Westernblot验证其是否表达。结果重组质粒pcDNA3．1（-）B／myc-BRMS1经双酶切及基因测序分析,验证了克隆的人BRMS1基因cDNA序列与GenBank[AF159141]公布的人BRMS1基因的cDNA序列吻合,重组体peDNA3．1（-）B／myc—BRMS1中插入的目的基因BRMS1是正向、单倍插入。结论成功构建了BRMS1真核表达载体pcDNA3．1（-）B／myc—BRMS1,为深入研究BRMS1基因功能和BRMS1基因治疗奠定了物质基础。     

骨形成蛋白2基因转染对犬牙髓细胞生物学特性的影响

目的构建骨形态发生蛋白2（bone morphogenetic proteins 2,BMP2）绿色荧融合蛋白pEGFP-N1-BMP2真核表达质粒,然后再用其在体外转染犬牙髓细胞（DDPCs）形成BMP2-DDPCs细胞,观察BMP2基因转染对牙髓细胞生物学特性的影响。方法构建pEGFP-N1-BMP2真核表达质粒,采用阳离子脂质体转染法将BMP2基因转染体外培养的DDPCs,检测BMP2-DDPCs碱性磷酸酶（ALP）活性,骨钙素（OC）产量和Ⅰ型胶原合成量等指标的测定,研究BMP2基因转染对牙髓细胞生物学特性的影响。结果成功构建pEGFP-N1-BMP2真核表达质粒,对构建的BMP2真核重组质粒用XhoI、HindIII进行双酶切,其产物进行琼脂糖凝胶电泳后,在1.2、4.7kb可见两条特异条带;并进行全基因序列测序,报告100%符合,证明pEGFP-N1-BMP2重组质粒构建成功。BMP2-DDPC中ALP活性,OC产量和Ⅰ型胶原合成量增加,与对照组DDPCs、N1-DDPCs相比差异有统计学意义（P〈0.05）,通过增强ALP、OC和Ⅰ型胶原等成骨标志物的表达,促进牙髓细胞向成牙本质细胞分化。结论 pEGFP-N1-BMP2真核表达质粒转染后的牙髓细胞,具有成牙本质细胞的特征。     

2001—2002年流感流行季节使用流感疫苗成分的建议

2000年10月至2001年2月,全球分离到的甲型流感病毒主要为甲型流感（H1N1）病原,而在一些国家则与乙型流感病毒一起传播,对流感病毒分离株的抗原性分析表明,甲型流感（H1N1）病毒分离株的抗原性与A／新 里多尼亚／20／99密切相关;甲型流感H3N2）病毒分离株的抗原性与A／莫斯科／10／99和A／巴拿马／2007／99密切相关;乙型流感病毒分离株的抗原性与B／四川／379／99和B／山东／7／97密切相关。     

双黄连抑制甲1型流感病毒诱导细胞凋亡的机制研究

目的:研究双黄连抑制甲1型流感病毒诱导细胞凋亡的机制。方法:将宫颈癌(Hela)细胞分成4组:病毒对照组、实验组、细胞对照组、药物对照组,采用流式细胞术计算细胞凋亡的百分率。结果:实验组细胞凋亡的百分率与病毒对照组比较有非常显著性差异(P<0.01)。结论:双黄连可抑制甲1型流感病毒诱导Hela细胞凋亡。     

Pro370Leu突变型MYOC基因真核表达质粒的构建及其表达特点

目的：构建Pro370Leu突变型MYOC基因真核表达质粒，并在Hela细胞中表达以研究蛋白的定位和分泌特点。方法：以pGEM—T—MYOC质粒中MYOC为模板，用PCR介导的定点突变技术，得到Pro370Leu突变型MYOC基因（mMYOC），并定向亚克隆到真核表达质粒pDsRed2-N1上，得到pDsRed2-N1—mMYOC重组表达质粒，再用限制性内切酶消化和DNA测序鉴定，最后用脂质体包埋转染法转染Hela细胞，进行荧光显微镜观察，并用Western Blot分析蛋白分泌情况。结果：经酶切和DNA序列测定，证实重组质粒构建成功，mMYOC基因能在Hela细胞中表达，并且蛋白只定位在细胞质中，经Western Blot分析，mMYOC蛋白不能分泌到培养液中。结论：成功构建Pro370Leu突变型MYOC基因真核表达质粒pDsRed2-N1—mMYOC，并能有效表达，其表达的蛋白定位在细胞质，并且不能分泌。     

甲型H5N1禽流感病毒血凝素在sf9昆虫细胞中的表达及其鉴定

目的  利用杆状病毒-昆虫细胞表达系统表达甲型H5N1禽流感病毒血凝素（hemagglutinin,HA）并对其进行鉴定。方法  根据sf9昆虫细胞密码子偏好性,对A/Goose/Guangdong/1/96（H5N1）禽流感病毒的HA基因进行序列优化并全长合成。构建重组供体质粒pFastHA,经PCR及测序鉴定后,转化DH10Bac感受态细胞,获得重组穿梭质粒BacmidHA,用M13引物进行PCR鉴定。采用脂质体法转染sf9细胞,获得重组杆状病毒rBacHA。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）、蛋白质印迹法以及间接免疫荧光法对rBacHA表达产物进行分析。结果  供体质粒pFastHA经双酶切后产生了1条与预期大小相符的1 707 bp条带,序列测定证实目的基因无突变。穿梭质粒BacmidHA经PCR扩增,得到1条与预期大小相符的3 180 bp条带。SDS-PAGE结果显示,rBacHA表达产物的相对分子质量约为65 000。蛋白质印迹法分析表明,表达产物能与禽流感病毒阳性血清结合。在荧光显微镜下,感染rBacHA的sf9细胞呈现绿色荧光,提示HA基因得到表达。结论  利用杆状病毒-昆虫细胞表达系统成功制备了具有良好抗原性的重组HA蛋白。     

Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice

RNA interference (RNAi) is a powerful tool to silence gene expression. Small interfering RNA (siRNA)-induced RNA degradation has been recently used as an antivirus agent to inhibit specific virus replication. Here, we showed that several siRNAs specific for conserved regions of influenza virus matrix (M2) and nucleocapsid protein (NP) genes could effectively inhibit expression of the corresponding viral protein. We also evaluated the antiviral potential of these siRNAs targeting M2 and NP of H5N1 avian influenza virus (AIV), which are essential to viral replication. We investigated the inhibitory effect of M2-specific siRNAs and NP-specific siRNAs on influenza A virus (H5N1, H1N1 and H9N2) replication in Madin-Darby canine kidney (MDCK) cells and BALB/c mice. The results showed that treatment with these siRNAs could specifically inhibit influenza A virus replication in MDCK cells (0.51-1.63 TCID(50) reduction in virus titers), and delivery of pS-M48 and pS-NP1383 significantly reduced lung virus titers in the infected mice (16-50-fold reduction in lung virus titers) and partially protected the mice from lethal influenza virus challenge (a survival rate of 4/8 for H1N1 virus-infected mice and 2/8 for H5N1 virus infected mice). Moreover, the treatment of pS-M48 and pS-NP1383 could suppress replication of different subtypes of influenza A viruses, including a H5N1 highly pathogenic avian isolate strain. The results provided a basis for further development of siRNA for prophylaxis and therapy of influenza virus infection in humans and animals.     

金茵清热口服液对甲型H1N1流感病毒致细胞病变效应的影响

目的：研究金茵清热口服液（试药）抑制甲型H1N1流感病毒的作用及其对喉癌上皮细胞（Hep-2细胞）免疫机能的影响。方法：甲型H1N1流感病毒感染狗肾细胞（MDCK细胞）和Hep-2细胞后,给予不同浓度的试药。显微镜观察细胞病变效应（cytopathic effect,CPE）,MTT法检测细胞活性,荧光定量PCR（qRT-PCR）检测试药作用于Hep-2细胞后细胞因子（TNF-α,IFN-1α,IFN-1β,IL-1α和IL-6）mRNA的水平。结果：实验细胞感染病毒后用试药处理,当试药质量浓度为3 mg·mL^-1时,MDCK细胞存活率提高了61.6%;试药质量浓度为4 mg·mL^-1时,Hep-2细胞存活率提高了40.6%,显著高于药物空白组细胞的存活率（P<0.05）。同时,试药显著上调Hep-2细胞的TNF-α（2.39倍）和IFN-1α（1.98倍）水平,下调炎症因子IL-1α（90%）和IL-6（64%）水平（P<0.05）,而IFN-1β水平的差异无统计学意义（P>0.05）。结论：金茵清热口服液可以显著提高染毒细胞的存活率,并能显著上调Hep-2细胞抗病毒免疫分子,同时抑制炎症反应,推测金茵清热口服液调节细胞免疫应答可能是其抗流感病毒作用机制之一。     

Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge

Influenza virosome is one of the commercially available vaccines that have been used for a number of years. Like other influenza vaccines, the efficacy of the virosomal vaccine is significantly compromised when circulating viruses do not have a good match with vaccine strains due to antigenic drift or less frequent emergence of a pandemic virus. A major advantage of virosome over other influenza vaccine platforms is its intrinsic adjuvant activity and potential carrier capability which have been exploited in this study to broaden vaccine protectivity by incorporating a conserved component of influenza virus in seasonal vaccine formulation. Influenza nucleoprotein (NP)-encoding plasmid was adsorbed onto surface of influenza virosomes as a virosome/DNA vaccine complex. Mice were immunized with a single dose of the influenza virosome attached with the NP plasmid or NP plasmid alone where both influenza virosomes and NP gene were derived from influenza A virus H1N1 New/Caledonia strain. Analysis of the cellular immune responses showed that 5μg (10-fold reduced dose) of the NP plasmid attached to the virosomes induced T cell responses equivalent to those elicited by 50μg of NP plasmid alone as assessed by IFN-γ and granzyme B ELISPOT. Furthermore, the influenza virosome/NP plasmid complex protected mice against intra-subtypic challenge with the mouse adapted H1N1 PR8 virus, while mice immunized with the virosome alone did not survive. Results of hemagglutination inhibition test showed that the observed intra-subtypic cross-protection could not be attributed to neutralizing antibodies. These findings suggest that influenza virosomes could be equipped with an NP-encoding plasmid in a dose-sparing fashion to elicit anti-influenza cytotoxic immune responses and broaden the vaccine coverage against antigenic drift.     

Methionine enkephalin inhibits influenza A virus infection through upregulating antiviral state in RAW264.7 cells

MENK, as an immune adjuvant, has potential immune-regulatory activity on innate and adaptive immune cells. The aim of this work was to investigate the antiviral effect of MENK on influenza virus-infected murine macrophage cells (RAW264.7) and its underlying mechanisms. The results showed that MENK markedly inhibited influenza A virus (H1N1) replication in pre- and post-MENK treatment, especially in pre-MENK treatment. The mechanisms exploration revealed that MENK (10 mg/mL) significantly inhibited the nucleoprotein (NP) of influenza virus and up-regulated levels of IL-6, TNF-α and IFN-β compared with those in H1N1 control group. Further experiments confirmed that antiviral effects of MENK was associated with promotion of opioid receptor (MOR) as well as activation of NF-κB p65 inducing cellular antiviral status. The data suggest that MENK should be potential candidate for prophylactic or therapeutic treatment against H1N1 influenza virus.     

Design, synthesis, and in vitro biological evaluation of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein

The influenza virus nucleoprotein (NP) is an emerging target for anti-influenza drug development. Nucleozin (1) and its closely related derivatives had been identified as NP inhibitors displaying anti-influenza activity. Utilizing 1 as a lead molecule, we successfully designed and synthesized a series of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents. One of the most potent compounds, 3b, inhibited the replication of various H3N2 and H1N1 influenza A virus strains with IC(50) values ranging from 0.5 to 4.6 μM. Compound 3b also strongly inhibited the replication of H5N1 (RG14), amantidine-resistant A/WSN/33 (H1N1), and oseltamivir-resistant A/WSN/1933 (H1N1, 274Y) virus strains with IC(50) values in sub-μM ranges. Further computational studies and mechanism investigation suggested that 3b might directly target influenza virus A nucleoprotein to inhibit its nuclear accumulation.