HPV16L1晚期基因阳性表达重组菌株的筛选

HPV16与宫颈癌的发生发展关系密切。其基因功能区包括早期区（E区）、晚期区（L区）及调节区。E区与细胞转化、致癌有关;L区分为L1和L2,L1区主要编码病毒衣壳蛋白,表达的蛋白能刺激机体产生保护性抗体,因此通过对HPV16L1基因的克隆及表达,可为制备基因工程疫苗和诊断试剂盒打基础。本实验通过甲醇诱导培养法及SDS－PAGE电泳对本室已立了HPV16L1－ρPIC3．5／GS115重组菌株进行了筛选,获得了1株HPV16L1晚期基因的GS115酵母菌重组表达株,所表达的蛋白质分子量为80KD。与预计相同,并证明第3天时L1蛋白的表达量最大。     

人乳头瘤病毒16型E7密码子优化后的原核表达及免疫原性研究

目的构建人乳头瘤病毒16型(HPV16)E7基因密码子优化后的原核表达系统,通过特异性抗体制备评价E7融合蛋白的免疫原性。方法人工合成优化后的HPV16 E7基因,利用特异引物扩增HPV16 E7基因。将E7基因连接至原核表达载体构建重组表达质粒pMAl-c2X-E7,转化至感受态细胞DE3后,经IPTG诱导,SDS-PAGE分析表达产物E7融合蛋白。纯化后的E7融合蛋白免疫动物,采用ELISA检测动物血清效价。结果 E7基因PCR产物的测序结果与优化后的目的基因序列比对一致。表达的E7融合蛋白经SDS-PAGE分析表明:在相对分子质量50 000处有特异性表达带,与预期相符。以E7融合蛋白制备的多克隆抗体其血清效价可达1∶64 000。结论成功构建的重组表达质粒pMAl-c2X-E7可有效表达MBP-E7融合蛋白,E7融合蛋白具有良好的免疫原性。     

人乳头瘤病毒16E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的实验研究

人乳头瘤病毒16型(Human papillomavirus type 16,HPV16)感染与口腔癌、宫颈癌的发病有关,HPV16 E6基因编码的蛋白是重要的癌蛋白,已经被证实能够通过增加高迁移率族蛋白B1(High mobility group box-B1,HMGB1)表达来促进宫颈癌细胞的侵袭,但是否能调控口腔癌细胞的侵袭仍未明确。为研究HPV16 E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的作用,口腔癌CAL27细胞被分为对照组、空白质粒组、HPV16 E6质粒组、NC-si RNA组(短片断干扰RNA阴性对照组)、NC-si RNA+HPV16 E6质粒组、HMGB1-si RNA+HPV16E6质粒组,检测细胞中HPV16 E6及HMGB1的表达、细胞的侵袭数目、培养基中HMGB1的含量。结果显示,HPV16 E6质粒组细胞中HPV16 E6及HMGB1的表达量、培养基中HMBG1的含量、细胞的侵袭数目均高于对照组及空白质粒组(P<0.05);HMGB1-si RNA组细胞中HMGB1的表达量明显低于对照组及NC-si RNA组(P<0.05);NC-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显高于NC-si RNA组(P<0.05),HMGB1-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显低于NC-si RNA+HPV16 E6质粒组(P<0.05)。本研究提示,HPV16 E6基因能够促进口腔癌CAL27细胞的侵袭且这一作用与增加HMGB1表达有关。     

腺病毒载体介导密码子优化型HPV 16 L1基因在哺乳动物细胞中的高效表达及病毒样颗粒的装配

为研究重组腺病毒载体作为HPV16预防性疫苗的可行性,构建了含密码子优化型HPV 16 L1基因的重组腺病毒,并对优化基因在哺乳动物细胞中的表达进行研究。首先按照哺乳动物密码子偏好对野生型HPV16 L1基因进行改造并合成优化基因,命名为mod.HPV16L1。将mod.HPV16L1基因克隆到穿梭质粒PDC316上,与骨架质粒共转染293细胞,在细胞内包装重组腺病毒rAd-mod.HPV16L1。用免疫印迹法检测病毒感染的293T细胞中HPV16L1蛋白的表达。通过Optiprep密度梯度超速离心法纯化HPV16 L1病毒样颗粒(VLPs)。用磷钨酸负染,在电子显微镜下观察HPV16 L1蛋白自我装配形成的VLPs。结果显示,重组腺病毒载体可介导mod.HPV16 L1基因在哺乳动物细胞内的高效表达,L1蛋白可自我装配形成VLPs。     

人乳头瘤病毒16型E7过表达细胞的鉴定及其迁移效应的影响

鉴定人乳头瘤病毒16型早期蛋白7(HPV16E7)过表达细胞及其迁移效应的影响,为后续基于HPV16E7靶向分子作用机制的研究奠定工作基础.脂质体转染法将本室保存的pcDNA3.1-HPV16E7重组真核表达质粒分别转染人胚肾293T细胞(HPV16型DNA阴性)、宫颈癌SiHa细胞株(HPV16 DNA阳性),转染48 h后收集细胞,提取RNA,RT-PCR扩增相应的目的基因,Western Blotting和间接免疫荧光实验检测HPV16E7目的蛋白在细胞中的表达.转染24h的细胞进行细胞划痕和Transwell实验,检测过表达细胞迁移行为的变化.RT-PCR结果显示:分别从E7质粒转染的293T、SiHa细胞的cDNA中,均可扩增到250 bp的目的条带;Western Blotting分析结果显示:以HPV16E7单克隆抗体为检测抗体,转染细胞的裂解液中均能在相对分子质量(Mr)约为15 000处出现特异性目的条带;间接免疫荧光结果显示:转染细胞中均能检测到目的绿色荧光,且分布于胞浆及细胞核周围;细胞划痕和Transwell实验结果显示:转染E7细胞的迁移效应显著提高.本研究证实了 HPV16E7转染细胞后可成功表达,且过表达细胞明显促进了细胞迁移行为,为后续基于HPV16E7迁移相关分子机制及靶向干预等研究奠定了前期工作基础.     

表达HPV16E6和E7蛋白的非复制型重组痘苗病毒的构建

为研制HPV16的治疗性疫苗,首先将表达质粒pJSA1175与非复制型痘苗病毒NTVJTK+进行同源重组,构建了痘苗重组病毒NTVJLac.再将表达质粒pJSDME6E7R与NTVJLac进行同源重组,构建了表达HPV16 E6和E7蛋白的非复制型重组痘苗病毒NTVJmE6E7,并对获得的重组病毒进行了鉴定.Southern杂交显示,重组痘苗病毒NTVJmE6E7基因组中有E6和E7基因插入.该重组病毒在人源细胞中不复制.Western blot显示,重组病毒在人源TK-143细胞中能表达E6和E7蛋白.非复制型重组痘苗病毒NTVJmE6E7可作为HPV16相关肿瘤及其癌前病变免疫治疗的实验性疫苗株.     

HPV16 l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础.本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞.转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符.表达产物经Western blotting分析能与HPV16L1单克隆抗体特异结合.真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础.     

pEGFP-HPV16E7重组融合蛋白质粒的构建及其表达

应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达.酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达.构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达.由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对HPV16E7分子生物学特性、致瘤机理及APC提呈等的研究.为建立表达HPV16E7的实体瘤动物模型奠定了基础.     

不同地区HPV16 E7基因的克隆及序列差异分析

采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 ,与野生型有较大差异     

不同地区HPV16E7基因的克隆及序列差异分析

采用PCR扩增、pGEM-T载体克隆和核苷酸序列分析的方法对一 例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与 野生型(德国标准株)及已发表的HPV16湖北株(HPVHB)进行了比较.结果发现武汉地区HPV16 型E7基因仅第54位出现一个同义突变,而高发区HPV16型E7基因存在差异,第77位氨基酸由 精氨酸(Arg)变为半胱氨酸(Cys),第96位由谷氨酰氨酸(Gln)变为精氨酸(Arg),E7蛋白的二级 结构及亲、疏水性也相应改变,与野生型有较大差异.     

Plasma levels of 16 alpha-hydroxypregnenolone, 16 alpha-hydroxyprogesterone and 16 alpha-hydroxydehydroepiandrosterone in the fetus and neonates.

Simultaneous determination of unconjugated 16 alpha-hydroxypregnenolone (16 alphaOH-Preg), 16 alpha-hydroxyprogesterone (16 alphaOH-Prog) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three 16 alpha-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16 alpha-OH-Preg in the umbilical arterial plasma increased from 11.2 +/- 3.1 at 24 weeks to 29.7 +/- 12.0 ng/ml at term, 16 alphaOH-Prog from 15.5 +/- 3.2 to 34.3 +/- 11.0 ng/ml and 16 alphaOH-DHEA from 5.1 +/- 1.2 to 5.9 +/- 1.0 ng/ml. In the anencephalic neonates, only 16 alphaOH-Preg showed an increase pattern under ACTH priming. 16 alpha-OH-Preg levels for normal full term neonates remain relatively constant at the first 24 hr and show a slight decrease at 3 days post partum. In small full term neonates, 16 alphaOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first 5 days post partum. 16 alphaOH-Prog and 16 alphaOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first 24 hr. Comparison with findings of the three 16 alpha-hydroxysteroids in fetal and neonatal plasma is discussed.     

Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16)

Membrane-bound matrix metalloproteinase 16 (MMP16/MT3-MMP) is considered a drug target due to its role(s) in disease processes such as cancer and inflammation. Biochemical characterization of MMP16 is critical for developing new generation MMP inhibitors (MMPi), which exhibit high efficacies and selectivities. Herein, a modified over-expression and purification protocol was used to prepare the catalytic domain of MMP16 (cdMMP16). The resulting recombinant enzyme exhibited steady-state kinetic constants of K _m = 10.6 ± 0.7 μM and k _cat = 1.14 ± 0.02 s^?1, when using FS-6 as substrate, and the enzyme bound 1.8 ± 0.1 eq of Zn(II). The enzymatic activity of cdMMP16 is salt concentration-dependent, and cdMMP16 exhibits autoproteolytic activity under certain conditions, which may be related to an in vivo regulatory mechanism of MMP16 and of other membrane-type MMPs (MT-MMPs). Co(II)-substituted analogs (Co₂- and ZnCo) of cdMMP16 were prepared and characterized using several spectroscopic techniques, such as UV–Vis, ¹H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening efforts.     

Overexpression of the key virulence proteases Bace16 and Bae16 in Bacillus nematocida B16 to improve its nematocidal activity

Proteases Bace16 and Bae16, an alkaline serine protease and a neutral protease, respectively, in the nematocidal bacterium Bacillus nematocida B16, have been identified as two key virulence factors and shown to have remarkable nematotoxic activities against the free-living nematode Panagrellus redivius and the plant parasite nematode Bursaphelenchus xylophilus. To facilitate the successful biological control application of this organism in the field, we genetically altered the strain B. nematocida B16 and optimized its growth condition to overexpress these two pathogenic proteases. The recombinant integration vectors of pAX01-Bace16 and pAX01-Bae16 for overexpressing the two proteases were constructed and successfully transformed into competent cells of the bacterium B. nematocida B16. The optimal induction condition for overexpressing Bace16 is 2% xylose at 37°C for 48 h. Our analyses showed that the proteolytic activity and nematocidal activity of the strain overexpressing Bace16 increased by about 62 and 80%, respectively, over the wild-type strain. However, our tested induction conditions could not significantly improve either the proteolytic activity or the nematocidal activity of the Bae16 overexpression mutant.     

On Spo16 and the Coefficient of Coincidence

spo16 mutants in yeast were reported to have reduced map lengths, a high frequency of nondisjunction in the first meiotic division, and essentially unchanged coefficients of coincidence. Were all crossing over in yeast subject to interference, such data would suggest that the “designation” of recombination events to become crossovers is separable from the “implementation” of that crossing over. In the presence of coexisting interference and noninterference phases of crossing over, however, lack of change in the coefficient of coincidence may show only that spo16 reduces crossing over in the two phases by a similar factor.