口蹄疫三价重组鸡痘病毒疫苗分子设计及其构建

针对我国口蹄疫(FMD)流行情况,利用计算机软件模拟分析,构建了口蹄疫病毒(FMDV)复合多表位基因工程疫苗表达盒OAAT.该表达盒以O型、A型FMDV结构蛋白VP1全基因和AsiaI型FMDV 2个基因拓扑型的结构蛋白VP1基因上的5个抗原表位基因(其中2个抗原表位来源于FMDVIND63/7株,该毒株与我国新疆分离株属于同一基因拓扑型,其余3个抗原表位来源于FMDVYNBS/58株)作为主要免疫原基因,以非结构蛋白上的2个Th2细胞表位基因及结构蛋白上的1个Th2细胞表位基因作为辅助基因.将构建好的表达盒OAAT和猪IL-18基因分别插入到鸡痘病毒(FPV)表达载体pUTA-16-LacZ复合启动子(ATI-P7.5×20)和单一启动子(P7.5)下游,构建了携带OAAT表达金和猪IL-18基因的重组鸡痘病毒转移载体质粒pUTAL-OAAT-IL18.应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E4株FPV共转染鸡胚成纤维细胞(CEF),经BrdU进行3次加压蚀斑筛选后,以不同代次的细胞mRNA为模板,利用FMDV VP1基因和猪IL-18基因特异引物进行RT-PCR和IFA检测,筛选出OAAT和猪IL-18基因共表达的重组鸡痘病毒rF-PV-OAAT-IL18,该重组鸡痘病毒的构建为新型FMDV活载体疫苗的研究奠定了基础.     

O139霍乱弧菌LPS基因和O抗原基因在大肠杆菌中的克隆表达及O抗原基因结构的初步研究

本研究采用构建基因文库的方法,构建了O139霍乱弧菌的粘粒基因组文库及重组粘粒的亚克隆文库.另外构建了质粒基因组文库.从文库中筛选到可以表达O139霍乱弧菌LPS和O抗原的重组克隆,经鉴定重组克隆表达的LPS和O抗原不但具有与O139霍乱弧菌的LPS和O抗原具有相同的反应原性,而且还具有相同的免疫原性.O抗原基因的部分测序分析表明,O139霍乱弧菌的抗原基因与O1群霍乱弧菌的O抗原基因不同源.     

共表达FMDV P1-2A和3C重组鸡痘病毒疫苗与核酸疫苗的实验免疫研究

将口蹄疫病毒(FMDV)衣壳蛋白前体P1-2A基因和蛋白酶3C基因分别插入到鸡痘病毒表达载体质粒pUTA-16Lac Z的复合启动子和单一启动子的下游,构建共表达重组鸡痘病毒转移载体质粒pUTAL3CP1,与282E4株FPV共转染鸡胚成纤维细胞,经RT-PCR、IFA及Western blotting检测,成功获得能正确表达目的蛋白的重组鸡痘病毒株vU-TAL3CP1。并与FMDV重组核酸疫苗免疫小鼠实验表明,各实验免疫组均能诱导小鼠产生特异性的细胞免疫和体液免疫反应,其中以重组鸡痘病毒与核酸疫苗联合免疫效果好于其它实验各组。     

PPV VP2和PCV2 ORF2重组病毒样颗粒的获得及其免疫原性研究

将PCV2 ORF2基因插入PPV VP2基因HindⅢ酶切位点(即PPV VLPs的N端1/3处)获得重组基因VP2.ORF2,并将其克隆到真核表达载体pEGFP-C1中得到重组质粒pEGFP-VP2.ORF2,经转染及电镜观察表明成功获得VP2.ORF2重组病毒样颗粒。重组病毒样颗粒经超速离心纯化后免疫小鼠,同时设立PPV普通疫苗、PCV2弱毒株及空白对照组,并在不同时期检测小鼠CD_3~+、CD_4~+、CD_8~+T淋巴细胞动态变化情况及PPV和PCV2抗体水平。结果显示:VP2.ORF2重组病毒样颗粒免疫小鼠后能诱导较高水平的细胞免疫和高于同等条件普通疫苗和弱毒株的体液免疫水平。此研究结果为进一步探索VP2.ORF2重组病毒样颗粒的形成机制提供了依据,也为PPV-PCV2二联疫苗的研发打下了基础。     

WSSV-VAP1基因克隆及在毕赤酵母中的表达

根据WSSV-黏附蛋白(VAP1)基因序列设计了一对引物,在5‘引物和3‘引物中分别引入EcoRI和XbaI酶切位点。经PCR扩增,将VAP1基因克隆至穿梭载体pGAPZαA之GAP启动子下游位点,构建成含α-factor信号肽的重组表达载体,获得的重组质粒pGAPZαA-VAP1经线性化后转入毕赤氏酵母SMD1168菌株,构建成分泌型表达VAP1的酵母工程菌株。SDS-PAGE和Western-blot及结合活性检测分析表明,VAP1基因在该体系中得到成功表达,表达产物与对虾细胞膜具明显的结合活性。     

抗CD20嵌合抗体Fab'片段在大肠杆菌中高效表达

利用ＰＣＲ方法从抗ＣＤ２０ＳｃＦｖ表达载体上扩增重链可变区、轻链可变区基因,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ’表达载体中,构建成抗ＣＤ２０嵌合抗体Ｆａｂ’片段表达载体ｐＹＺＦｃｄ２０,用ｐＹＺＦｃｄ２０转化大肠杆菌１６Ｃ９,在１６Ｃ９菌中分泌表达可溶性抗ＣＤ２０Ｆａｂ’片段,经分离纯化获得具有ＣＤ２０抗原特异结合活性的Ｆａｂ’片段,竞争性竞争荧光抑制实验表明,抗ＣＤ２０Ｆａｂ’片段竞争性抑亲本属源     

抗乙肝病毒表面抗原PreS1(20-47)的单链抗体在转基因烟草根分泌物中的初步表达

为探索利用植物根分泌表达重组蛋白的可行性，构建了含有抗乙肝病毒表面抗原PreS1(20—47)单链抗体(ScFv)基因的表达载体。该ScFv基因转化烟草后在烟草根部细胞的细胞质和内质网中获得表达。实验结果表明，5’端融合ER导向信号肽的重组ScFv可通过根分泌表达。     

抗HIV-1 gp120单链抗体导向SEAD227A融合蛋白原核温控型表达载体构建及表达

人工合成了能广泛识别HIV—1 gp120的单链抗体(SeFv120)基因和金黄色葡萄菌外毒素A(SEA)基因,将SEA基因第227位编码Asp(D)的密码子GAT转换成编码Ala(A)的密码子GCT,并对两基因进行密码子优化。构建原核温控型表达质粒pBV—120和pBV—SL120,经条件优化,pBV—120和pBV—SL120分别在E．coli BL21(DE3)plys中44℃诱导6h、42℃诱导7h获得最佳表达,表达量分别占菌体总蛋白的27．673％和32．519％。目的蛋白经包涵体洗脱、分子筛层析折叠复性后可与HIV—1抗原条发生良好的结合反应。     

共表达VP3与hIL-18基因真核重组质粒的构建及对人结肠癌细胞的影响

先将鸡贫血病毒VP3基因插入真核表达载体pVAXl的多克隆位点，再将pVAX1载体上的IRES启动子及新霉素基因一同切下插入pVAXl的VP3基因下游，然后切下新霉素基因，将pIRES1基因插入其中，构建成pVVP3IL-18，将pVVP3IL-18转染Heda细胞。间接免疫荧光表明，在细胞膜和细胞浆观察到了特异的黄绿色荧光，证明表达了hIL-18。pVVP3IL-18转染人结肠癌细胞HCY-116后，经AO／EB染色及电镜观察，可见大量细胞凋亡。说明真核重组质粒pVVP3IL-18可在HeLa细胞中表达，并可在体外诱导人结肠癌细胞HCT-116凋亡。     

新型基因工程抗CD20抗体片段F（ab′）2的构建、表达和活性测定

从抗CD20Fab′表达载体上利用PCR方法扩增并修饰其重键恒定区CH1C－末端的DNA序列,然后将修饰后的CH1DNA序列重组到原Fab′表达载体中,构建成抗CD20抗体片段F(ab′）2表达载体。将该载体转化缩主大肠杆菌16C9,实现了抗CD20抗体片段F(ab′）在工程菌中的可溶性分泌表达。经分离纯化获得具有与抗原CD20特异结合的F（ab′）2在工程菌中的可溶性分泌表达。经分离纯化获得具有与抗原CD20特异结合的F（ab′）2活性片段。竞争性免疫荧光实验的结果表明：抗CD20F(ab′）2片段具有比Fab′更强的竞争性抑制亲本鼠源性单克隆抗体H147与Daudi细胞表面CD20相结合的能力;用MTT法检测所得到的数据说明：F(ab′）2与Fab′更为有效地抑制在体外培养的Daudi细胞的生长。     

DNA vaccine against Taenia solium cysticercosis expressed as a modified hepatitis B virus core particle containing three epitopes shared by Taenia crassiceps and Taenia solium

Many studies have provided evidence that the hepatitis B core antigen particle is useful as a vaccine carrier for foreign epitopes. Epitopes KETc1, KETc12, and GK-1 are three promising candidates for designing a vaccine against Taenia solium cysticercosis. In the present study, epitopes KETc1 and KETc12 were inserted into the immunodominant loop of the truncated HBc149, and epitope GK-1 was fused to its C-terminus. The fused protein deltaC-3n was expressed and purified successfully. The polymeric character was tested by SDS-PAGE. After inoculation of BALB/c mice with deltaC-3n, antibody titers were assayed by ELISA, and the antibody specification was analyzed by Western blot. Dot ELISA was performed to verify the protection of the three epitopes. Results showed that the purified polymeric protein was formed, high antibody titers were induced in immunized mice and three antibodies different in molecular weight were induced, serum specific antibody recognized the native peptide localized mainly in cyst wall cells, and there was no specific antibody toward the three epitopes in sera of infected pig and humans. All these revealed that the protein deltaC-3n was a potential candidate for vaccine against cysticercosis. So the deltaC-3n sequence and the signal peptide sequence of IL-2 were cloned to a vector pVAX3.0 to construct pVAX-S-deltaC-3n. Pigs were immunized with pVAX-S-deltaC-3n. Two weeks after the immunization booster, pigs were introduced to infectious T. solium eggs. The relative protective rate induced in pigs immunized with the DNA vaccine pVAX-S-deltaC-3n was 83%.     

HIV-1中国流行株gp120基因在痘苗病毒中的表达及其与核酸疫苗的实验免疫研究

将HIV－1中国流行株gp120基因在痘苗病毒中进行表达,以期获得重组痘苗病毒,与核酸疫苗混合免疫,评价免疫效果,为艾滋病疫苗开发研制打下基础。将HIV－1中国流行株gp120基因片段插入到pJ38载体启动子下游,经同源重组和血凝素阴性空斑筛选重组痘苗病毒,SDS－PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫BALB／c小鼠,进行淋巴细胞转化实验、CTL、CD4＋CD8＋T细胞数目以及血清抗体等细胞免疫和体液免疫指标检测。结果获得的重组痘苗病毒vJ38pg120能够表达Gp120蛋白并诱导机体产生细胞免疫和体液免疫,具有良好的免疫原性,其免疫效果以2rVV-DNA混合方式为最好。重组痘苗病毒vJ38gp120。     

外源抗原表位在大肠杆菌CS3菌毛上的呈现

为了探讨ＣＳ３菌毛多位点用于外源表位呈现以及重组蛋白的免疫原性,通过对ＣＳ３亚基蛋白二级结构、抗原表位、亲水性及柔韧性的预测分析,找出了两个新的插入位点７２位和１３６位,将口蹄疫病毒ＶＰ１、脊髓灰质炎病毒Ｃ３等多种表位插入到ＣＳ３中,全细胞ＥＬＩＳＡ、免疫荧光检测和电镜观察等均表明外源表位在大肠杆菌表达得到了表达,而且７２位表达水平明显高于１３６位。用重组菌腹腔注射免疫小鼠,可诱发机体产生针对ＣＳ     

聚乙烯亚胺改性的双介孔氧化硅基因载体构建

为了开发一种新型的非病毒无机基因载体, 采用3-氨丙基三乙氧基硅烷(APTMS)和聚乙烯亚胺(PEI)对嵌入型双介孔氧化硅球(EDMSNs)进行改性, 分别得到EDMSNs-NH₂和EDMSNs-PEI, 并比较了两种载体结合和保护pCMV-EGFP-N1质粒(pDNA)的能力及细胞转染性能。利用透射电镜、动态光散射及氮气吸附-脱附实验对材料的颗粒形态, 动力学粒径, Zeta电位及孔结构参数进行表征。结果显示, EDMSNs-NH₂和EDMSNs-PEI均表现出明显的双介孔结构, 形貌为规整的球形且平均动力学粒径分别为343.2和338.9 nm, 表面电位分别为+18和+43 mV。琼脂糖凝胶电泳、CCK-8法及荧光显微镜结果表明, EDMSNs-PEI对pDNA的担载量为8%, 远高于EDMSNs- NH₂(1%)。与PEI和lipofectamine2000相比, EDMSNs-PEI载体展示出更低的细胞毒性。EDMSNs-PEI/pDNA质量比为33 : 1时, EDMSNs-PEI/pDNA对293T细胞的转染效率在72 h达到最大值。因此, 与EDMSNs-NH₂相比, EDMSNs-PEI具有更高的正电位及pDNA担载量, 可作为一类有前景的非病毒无机类基因载体用于重大疾病如恶性胶质瘤的治疗。     

Characterization of water-soluble conjugates of polyacrylic acid and antigenic peptide of FMDV by size exclusion chromatography with quadruple detection

Intermolecular complex by electrostatic interaction and specifically coupled conjugates between polyacrylic acid (PAA) and a synthetic peptide representing 170-188 sequence from the GH loop of VP1 protein of foot-and-mouth disease virus (FMDV) were investigated as an intermolecular model system due to their importance in biotechnology and immunology. In this study, polyacrylic acid (PAA) with a synthetic peptide representing 170-188 sequence from the GH loop of VP1 protein of foot-and-mouth disease virus at a wide range of mixing ratios of components (C_Peptide/C_PAA 0.1, 0.25, 0.5, 0.75 and 1.0, respectively) were characterized by size-exclusion high performance liquid chromatography with on-line refractive index, UV, light scattering and viscometer detectors. The results revealed that two molecules are both negatively charged as a result of repulsive forces preventing complex formation at neutral pH. Therefore, these molecules bound covalently to each other by using water-soluble carbodiimide when pH levels are higher than the pI of the peptide. High performance liquid chromatography analysis showed that the amount of protein-polymer complex increased and free peptide amount decreased with the increase in molar ratio of the peptide. Also, this paper presents that number of the bound peptide molecules with one PAA molecule was expressed by a Langmuir-type equation as a function of the amount of excess synthetic peptide existing free in the solution.     

基因枪轰击小鹅瘟病毒VP3基因疫苗诱导鹅体内体液免疫的机制

将小鹅瘟病毒VP3基因疫苗(pcDNA-GPV-VP3)分别以每只1μg、3μg和6μg的基因枪轰击免疫30日龄四川白鹅,用免疫组织化学法检测pcDNA-GPV-VP3在鹅体内各组织器官表达情况;用间接ELISA检测免疫鹅血清中GPV抗体滴度.结果:①各剂量免疫组1d时能在免疫部位皮肤,3d时能在心脏/十二指肠/空肠/盲肠/肝脏,7d时在回肠/直肠检测到pcDNA-GPV-VP3的表达;7d表达最多;表达可持续至35～63d;表达产物的数量和表达持续时间总体规律为6μg组＞3μg组＞1μg组.②免疫后第7d血清抗体开始升高,21d达到最大值,免疫后第14～217d极显著(P<0.01)高于PBS和空白质粒对照;体液免疫效果存在一定的剂量依赖.研究表明,pcDNA-GPV-VP3基因枪轰击免疫雏鹅后能在免疫部位皮肤、心肌、肝脏和各肠段中表达并能够诱导雏鹅产生良好的体液免疫.     

Use of EAF dust as heterogeneous catalyst in Fenton oxidation of PCP contaminated wastewaters

In this study, chemical oxidation tests using H2O2 were performed on a solution contaminated with 100 mg l(-1) of pentachlorophenol (PCP). The effectiveness of electric arc furnace dust and hematite as heterogeneous catalysts was evaluated. Reactions were conducted at pH 2 for 24 h. Either H2O2 stabilized with KH2PO4 or un-stabilized H2O2 was used. Total organic carbon (T.O.C.) removal and chloride release from PCP molecule were monitored. Results showed that the maximum removal yields for electric arc furnace (EAF) dust (49.2% T.O.C., 56.7% Cl) were achieved when H2O2:PCP ratio was 10:1 and Fe:H2O2 = 1:5 for unstabilized H2O2 and when H2O2:PCP = 10:1 and Fe:H2O2 = 1:1 for stabilized H2O2 (48% T.O.C., 60.6% Cl). The maximum yield using hematite (45.2% T.O.C., 55.2% Cl) was obtained when H2O2:PCP ratio was 10:1 and Fe:H2O2 was 1:2. When EAF dust was used and Fe:H2O2 > 1:5, Cl release was higher than the one expected from T.O.C. removal.     

抗菌核病转基因油菜植株的获得

将携带菜豆几丁质酶基因的植物表达载体ｐＢｃｈ通过农秆菌介导法导入甘兰型油菜Ｈ１６５中。经卡那霉素对Ｔ０、Ｔ１和Ｔ２代植株进行连续的筛选和菌核病（ｓｃｌｅｒｏ－ｔｉｎｉａ ｓｃｌｅｒｏｔｉｎｏｒｉｕｍ）接种试验,共获得２２株抗菌核病的Ｔ２代植株。对其中的２２株进行ＰＣＲＳ检测,从６株扩增得到与菜豆几丁质酶基因大小对应的ＤＮＡ带型,Ｓｏｕｔｈｅｒｎ杂交证实其中２株呈现阳性结果,表明外源基因已整合到油菜