稳定表达人乳头瘤病毒58型(HPV58)E6E7融合基因的人宫颈癌细胞株C33A的构建及验证

为构建稳定表达人乳头瘤病毒(HPV)58型E6E7融合基因的人宫颈癌C33A细胞系,将实验前期构建好并实现真核表达的重组慢病毒颗粒LV-HPV58E6E7转染入HPV(–)的人宫颈癌C33A细胞,经流式细胞仪分选出稳定转染的阳性克隆,利用四唑盐比色(MTT)法检测转染后细胞的生长情况以及流式细胞术检测细胞周期,并将稳定表达HPV58E6E7融合基因的C33A细胞LV-HPV58E6E7/C33A接种于裸鼠左腋下成瘤,用荧光定量PCR(q RT-PCR)、Western blot检测瘤组织中HPV58型E6E7融合基因的转录和表达。结果显示HPV58E6E7融合基因可促进C33A细胞的增殖;LV-HPV58E6E7/C33A细胞株能在裸鼠体内稳定转录及表达HPV58型E6E7融合基因。这表明我们成功建立了能稳定表达HPV58E6E7融合基因的人宫颈癌C33A细胞系LV-HPV58E6E7/C33A,为HPV58型治疗性疫苗的免疫效果检测提供了抗原细胞来源。     

人PSCA真核表达质粒的构建及稳定转染B16细胞系的建立

目的:构建真核表达质粒pcDNA3.1-PSCA,通过稳定转染建立高效表达人前列腺干细胞抗原(PSCA)的小鼠黑色素瘤B16细胞系。方法:利用PCR扩增出人PSCA全长基因的cDNA编码区序列,使用基因重组技术将其克隆到真核表达载体pcDNA3.1中,得到重组表达质粒pcDNA3.1-PSCA。双酶切及测序鉴定后,利用脂质体法将其转染入小鼠黑色素瘤B16细胞,G418加压筛选后得到阳性克隆。通过流式细胞术、免疫荧光及Western blot检测PSCA在细胞系中的表达情况。结果:经测序及双酶切鉴定,pcDNA3.1-PSCA重组质粒构建正确;稳定转染人PSCA的B16细胞系表达率接近100%。结论:建立的稳定转染人PSCA的B16细胞系能够高效表达PSCA基因,为抗前列腺癌疫苗的功能研究奠定了基础。     

HPV16 E6E7抗原表达模型的建立

目的 用基因重组技术构建pcDNA-E6E7真核表达载体.方法 经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCK法检测HPV16E6E7mRNA的生成,并将转染细胞接种小鼠皮下,观察成瘤情况.结果 酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入住点均正确,在转染的B16细胞中可见绿色荧光并检测到HPV16E6E7mRNA的生成,接种的转染细胞在小鼠皮下100%成瘤.结论 提示B16细胞转染E6E7后其致瘤性与转染空载体组和野生型B16细胞组无明显差异.     

人hCGβ真核表达载体的构建及稳定转染B16细胞系的建立

目的:构建人hCGβ真核表达载体,稳定转染入小鼠黑色素瘤B16细胞,建立稳定表达人hCGβ的小鼠黑色素瘤细胞系。方法:采用PCR方法扩增出人hCGβ全长基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×His标签,得到重组表达质粒pIRES-neo-hCGβ-(His)6。利用阳离子脂质体介导法将其稳定转染入小鼠黑色素瘤B16细胞,经G418加压筛选出阳性克隆。RT-PCR、Westernblot及免疫荧光检测人hCGβ在B16细胞中的表达。结果:经限制性内切酶鉴定及序列分析,pIRES-neo-hCGβ-(His)6重组体构建正确,最终建立的表达人hCGβ基因的B16细胞系阳性率高于90%。结论:成功构建了真核表达载体pIRES-neo-hCGβ-(His)6,建立的稳定转染人hCGβ小鼠黑色素瘤细胞系能够高效表达hCGβ基因。该稳定转染细胞系的建立为进一步研究人hCGβ抗肿瘤基因疫苗的功能提供了良好的实验基础,为hCGβ在肿瘤免疫治疗中的应用奠定了研究基础。     

人乳头瘤病毒58型治疗性复合DNA疫苗载体的构建

目的:构建和表达人乳头瘤病毒(Human papilloma virus,HPV)58型相关宫颈癌治疗用PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体。方法:将HPV58mE6E7融合基因片段插入真核表达载体pCI-Fc-GPI中,构建重组真核表达质粒pCI-sig-HPV58mE6E7-Fc-GPI。再将得到的sig-HPV58mE6E7-Fc-GPI片段插入新型疫苗载体PVAX1-IRES-hIL12中,构建PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体,流式细胞术、免疫荧光及ELISA分别检测该疫苗载体的表达。结果:经限制性内切酶鉴定及测序分析证实PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体构建成功。流式细胞术、免疫荧光及ELISA检测表明该疫苗载体中的融合抗原sig-HPV58mE6E7-Fc-GPI及分子佐剂人白介素12(hIL12)能够同时实现真核表达。结论:PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体可作为治疗HPV58相关肿瘤及其癌前病变的候选疫苗载体。     

稳定表达hTERT/Luc的小鼠黑色毒瘤肺转移模型的建立

目的 建立稳定共表达荧光素酶基因和人端粒酶逆转录酶(hTERT)的小鼠黑色素瘤B16细胞系,并通过尾静脉注射的方式建立小鼠肿瘤肺转移模型.方法 利用DNA重组技术将hTERT基因和荧光素酶基因Luc定向插入到真核表达载体,构建真核表达质粒pIRES-neo-hTERT和pIRES-hyg3-Luc,利用阳离子脂质体LipofectamineTM 2000共转染小鼠黑色素瘤B16细胞,经G418及潮霉素B加压筛选出稳定转染的细胞株.应用Western blot法及免疫荧光法检测hTERT和Luc基因在B16细胞中的表达;将稳定共表达hTERT和Luc的B16-hTERT/Luc细胞株通过尾静脉注射的方式接种雄性C57BL/6小鼠建立肿瘤肺转移模型,并通过活体成像技术检测小鼠肺部肿瘤的生长.结果 建立了稳定共表达hTERT和Luc的小鼠黑色素瘤B16单克隆细胞株B16-hTERT/Luc,经检测hTERT基因和荧光素酶基因Luc在单克隆细胞株中的表达分别为84％和98％.通过尾静脉注射的方式成功建立了小鼠肿瘤肺转移模型,应用活体成像技术能方便地检测到B16-hTERT/Luc肿瘤在小鼠体内的生长情况.结论 成功建立了可用于活体成像技术检测的稳定表达hTERT的小鼠黑色素瘤肺转移模型.     

登革2型病毒NGC株E基因在NIH3T3细胞中的表达及初步鉴定

目的　构建登革 2型病毒E基因的真核表达载体 ,实现登革病毒E蛋白的真核表达。方法　采用逆转录 多聚酶链反应 (RT PCR)扩增登革 2型病毒 (NGC株 )包膜糖蛋白E基因全长片段 ,克隆入真核表达载体pcDNA3的Pcmv启动子下游 ,构建重组真核表达质粒pcDNA3 E ,用脂质体转染法转染NIH3T3细胞 ,表达产物以免疫荧光、SDS PAGE和蛋白质印迹进行分析检测。结果　成功构建了重组真核表达质粒pcDNA3 E ,通过脂质体转染法导入NIH3T3细胞 ,免疫荧光、SDS PAGE和蛋白质印迹分析检测表明 ,E基因在NIH3T3细胞实现了真核表达 ,产物相对分子质量 (Mr)为 6 0× 10 3。结论登革病毒E基因真核表达载体的构建及E基因的真核表达为研究登革病毒E蛋白的结构与功能、研制登革病毒诊断试剂及核酸疫苗奠定了基础     

修饰后中国山东地方株HPV16 E6E7基因的转化活性检测及免疫原性研究

目的 利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型（HPV16）E6E7基因，研制HPV16 DNA疫苗。方法 定点突变E6的终止密码，并保证E7读码框架不定；定点突变E7蛋白的Rb结合区中对其转化活性维持起关键作用的第24位氨基酸。突变修饰后的基因命名为fmE6E7。PCR扩增fmE6E7，重组人pLNCX载体，脂质体法转染3T3细胞，免疫荧光组织化学及Western blot检测转染细胞蛋白的表达。经软琼脂集落培养法和BALB／c裸鼠皮下接种法检测fmE6E7的转化活性。然后PCR扩增fmE6E7，构建pVR1012-fmE6E7真核表达质粒，于C57BL／6小鼠肌肉内直接进行裸DNA免疫，^51Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性，间接ELISA法检测免疫鼠血清中E7特异性抗体。结果 测序证实获得了预期的突变结果。pLNCX-fmE6E7转染细胞体外软琼脂培养3周未见集落形成；裸鼠皮下接种2月后未见移植瘤形成（0／3）。免疫鼠获得了较好的E7特异性的抗体E和抗原特异性的CTL。结论 修饰后E6E7基因可融合表达，转化活性消除的同时还可诱发特异的细胞免疫和体液免疫，表明中国山东地方株的E6E7基因可作为HPV16治疗性DNA疫苗的靶基因。     

HPV16型E7重组蛋白的表达及其多克隆抗体的制备

目的制备人乳头瘤病毒(HPV)16型E7原核表达蛋白及其多克隆抗体。方法用PCR方法从宫颈癌组织中扩增HPV16 E7基因,克隆至pET21a(+)载体并构建pET21a(+)/HPV16 E7重组质粒,测序鉴定;将重组质粒转化至大肠杆菌BL21(DE3),经异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导后表达重组蛋白,用Ni-NTA亲和层析法纯化并经SDS-PAGE及Western blot分析鉴定;纯化的HPV16 E7重组蛋白免疫日本大耳白兔制备多克隆抗体,用ELISA检测多克隆抗体的效价,并用Western blot法及免疫荧光技术分析多克隆抗体的特异性。结果 HPV16 E7重组蛋白可通过原核表达系统进行表达和纯化;通过兔免疫后可制备特异性的多克隆IgG抗体,效价达1∶30 000;经Western blot法和免疫荧光染色结果证实兔多克隆抗体可特异性识别HPV16 E7蛋白。结论成功进行了HPV16 E7原核表达并制备了HPV16 E7兔多克隆抗体。     

丙肝病毒NS5B-EGFP融合蛋白真核表达载体的构建及稳定转染HepG2细胞系的建立

目的　构建可表达丙型肝炎病毒 (HCV)NS5B EGFP融合蛋白的真核表达载体 ;获得重组质粒稳定转染的HepG2 细胞系。方法　利用PCR技术从HCV基因组中扩增出ns5b基因片段 ,XhoⅠ KpnⅠ双酶切后连接到经同样酶切的pEGFP N3真核表达载体 ,转化TG1菌株感受态细胞 ,获得阳性重组质粒pEGFPN3 ns5b。将阳性克隆用脂质体法转染HepG2 细胞 ,经持续G4 18压力选择和有限稀释法克隆化获得稳定转染的细胞系。结果　成功构建了真核表达载体pEGFPN3 ns5b ;建立了其重组质粒稳定转染的HepG2 细胞系。结论　重组质粒稳定转染的HepG2 细胞系可表达NS5B EGFP融合蛋白 ;该HepG2 细胞系可以应用于以ns5b基因为靶位的抗HCV感染研究     

E.E.G. preprocessing by an on-line amplitude-and-frequency analyser

A four-channel e.e.g. preprocessing device is described, based on the signal's wave durations and amplitude measurement and their classification into four frequency and three amplitude bands. The analysis results are ordinarily written back on the e.e.g. machine for visual interpretation, in an appropriate polarity-and-amplitude coded form, occupying two channels per each processed channel. Outputs are provided for oscilloscope monitoring, further computer processing, etc. The possibilities of this device are discussed and examples of humans' and cats' e.e.g. analysis are given. About 10 devices of this type have been built and used for the last two years in several laboratories dealing with e.e.g. monitoring methods for experimental purposes.     

The stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.     

A novel interaction between the human papillomavirus type 16 E2 and E1^E4 proteins leads to stabilization of E2

The E4 (also called E1^E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1^E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1^E4. All this raises the possibility that E1^E4 acts to influence E2 activity by varying the amount of available E2 in the cell.     

戊型肝炎病毒结构区基因在大肠杆菌中的表达

目的获得可用于研制戊型肝炎疫苗的基因工程抗原。方法用非融合蛋白表达载体ｐＥＴ１１ａ,表达戊型肝炎病毒（ＨＥＶ）第二读码框区（ＯＲＦ２）２２４６６０ａａ段蛋白。结果得到分子量为５００００的表达产物,经Ｗｅｓｔｅｒｎｂｌｏｔ证实,表达产物具有ＨＥＶ特异抗原性。结论该基因工程抗原可能具有制备戊型肝炎疫苗的前景。     

Apolipoprotein E5 and E7 in apparently healthy Japanese males: Frequencies and relation to plasma lipid levels

Summary In order to determine the frequencies of apolipoproteins (apo) E5 and E7 and their relation to plasma lipid levels, apo E phenotypes were determined in 608 healthy Japanese male adults by two-dimensional gel electrophoresis. Apo E5 and E7 were observed in 2.8% of the subjects, in addition to the three common apo E isoforms, E2, E3, and E4. Apo E5 was divided into two subtypes based on the migration rate on SDS/PAGE, E5f is the type with faster migration and E5s slower migration. The gene frequencies were: the 3 allele, 0.841; the 4 allele, 0.095; the 2 allele, 0.049; the 7 allele, 0.009; the 5 allele encoding apo E5f (the 5f allele), 0.004; and the 5 allele encoding apo E5s (the 5s allele), 0.001. The five individuals with apo E5f and the eleven with apo E7 were heterozygotes and normocholesterolemic. Also plasma apo B and apo E levels were not increased in any subjects with apo E5f or apo E7. The data suggests that apo E5f and E7 are not rare in the Japanese population but that neither apo E5f nor E7 are associated with hypercholesterolemia in most of the heterozygotes.     

Non-apolipoprotein E and apolipoprotein E genetics of sporadic Alzheimer's disease

The genetic epidemiology of sporadic Alzheimer's disease (SAD) remains a very active area of research, making it one of the most prolifically published areas in medicine and biology. Numerous putative candidate genes have been proposed. However, with the exception of apolipoprotein E (APOE), the only confirmed genetic risk factor for SAD, all the other data appear to be not consistent. Nevertheless, the genetic risk for SAD attributable to the APOE gene in the general population is 20–70%, providing a strong evidence for the existence of additional genetic risk factors. The first part of the present article was dedicated to non-APOE genetics of SAD, reviewing chromosomes-by-chromosomes the available data concerning the major candidate genes. The second part of this article focused on some recently discovered aspects of the APOE polymorphism and their implications for SAD. An attempt to identify the future directions for non-APOE genetic research in SAD was also discussed.     

人乳头瘤病毒16型修饰后E6E7基因免疫原性增强

利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7)，研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后，插人真核表达质粒获得pVRl012-fmE6E7，瞬时转染Cos-7细胞，免疫荧光法检测证实其表达后，在C57BL／6小鼠后腿肌肉进行裸DNA免疫，5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte，CTL)，间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应，与单独野生型E7基因免疫相比，E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。