Computational analysis and prediction for exons of PAC579 genomic sequence

To isolate the novel genes related to human hepatocellular carcinoma (HCC), we se-quenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC. Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0-60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.     

Computational analysis and prediction for exons of PAC579 genomic sequence

To isolate the novel genes related to human hepatocellular carcinoma (HCC), we sequenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC. Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0–60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.     

扎伊尔埃博拉病毒糖蛋白基因的克隆和表达

埃博拉病毒属丝状病毒科,能引发动物和人出血热症状,人感染后病死率高达90%以上,目前还没有有效预防和治疗的药物和疫苗。近年来,这种烈性传染病病毒传入我国的可能性不断加大,给我国公共卫生应急体系带来新的挑战。本研究针对埃博拉病毒的最主要结构蛋白——糖蛋白(GP),构建了重组原核表达载体pET28a(+)-GP1(33~313aa)、pET28a(+)-GP1(190~313aa)、pET28a(+)-GP2(502~632aa)、pET28a(+)-sGP,以及重组真核表达载体pcDNA3.1(+)-edited GP、pcDNA3.1(+)-GP1、pcDNA3.1(+)-GP。结果表明,GP1(33~313aa)、GP1(190~313aa)和sGP能在大肠埃希菌BL21(DE3)中以包涵体的形式表达,GP、GP1和GP2能在HEK293T细胞中表达,但均不能在BHK21细胞中表达。本研究为进一步探索埃博拉病毒GP的结构和功能及GP抗体制备奠定了基础。     

PAC579基因组序列外显子计算机分析与预测

为了寻找肝癌相关基因,选择位于人原发性肝细胞肝癌染色体17p13.3杂合性缺失最小共同缺失区内含有D17S926位点的PAC579克隆进行了基因组测序,通过实验分离和克隆了位于PAC579上的4个全长新基因,并将它们在该基因组上进行了外显子定位.使用5种计算机预测外显子的方法和4种剪接位点识别的方法对PAC579前60 kb序列(含上述4个基因)进行扫描,将得到的外显子预测结果和剪切位点结果与实验结果进行了对照与评析,又对未获得基因的区域加以预测.     

基于WWW与UNIX的核酸序列分析实用软件的开发

讨论了计算机序列分析的工作,介绍了基于WWW和UNIX的核酸序列分析实用软件,其特点是快速,易用,提高了工作效率。     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini-F replicon、一个卡那霉素抗性基因和一个lacZα基因8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内,构建了既能在E.coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid-HZ8).另外将HaSNPV的多角体蛋白基因和P10启动子序列取代pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列,构建插入HaSNPV多角体蛋白基因和 P10启动子序列的HapFastBacPhP10供体质粒.利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上,随后将含有eGFP基因的重组HaBacmid DNA转染至HZAm1细胞内.转染5d后,细胞核内能形成典型的多角体,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光.结果证明我们构建的HaBac to Bcac 表达系统能有效的表达外源基因.     

以减毒沙门氏菌为SARS-CoV N DNA口服疫苗载体的初步研究

目的:以减毒沙门氏菌为载体运送SARS-CoV N DNA疫苗至小鼠体内,研究其诱导的免疫应答情况,评价减毒鼠伤寒沙门氏菌作为口服疫苗的免疫效果。方法：将含SARS-CoV N基因的pcDNA-N质粒导入减毒鼠伤寒沙门氏菌CS022中,采用口服和滴鼻相结合的方法免疫BALB/c小鼠,以ELISA检测不同时间免疫小鼠血清中抗体及其亚型;以MTT法测定特异性淋巴细胞增殖反应;ELISPOT检测细胞因子;流式检测T细胞亚型。结果：pcDNA-N DNA疫苗口服免疫后2周就可以诱生特异性IgG抗体,且以IgG2a占优势;诱导了较高水平的淋巴细胞特异性增殖反应和IFN-γ,主要以Th1免疫为主。结论：减毒沙门氏菌可以有效运送pcDNA-N重组质粒并诱导产生特异体液和细胞免疫应答,为减毒细菌作为DNA疫苗运送载体的研究提供了参考依据,也为SARS疫苗研究开辟了新方法。     

中华民族基因组多态性数据库的构建

为配合总体的实验研究构建了中华民族基因组多态性(Genomic Polymorphism of Chinese Ethnic Groups,简称GPCEG)数据库,现已初步建成包括民族名称、基本情况介绍、体态特征、基因多态性数据、永生细胞株系、参考文献、国际相关数据库连接等内容的数据库,并完成了其可视化浏览及查询系统的建立,为建成具有中国特色的国家自有数据库奠定了基础,也可为从事相关研究的科学工作者提供信息服务。