具全期启动子盒的杆状病毒高效表达载体的组建与应用

通过ＰＣＲ扩增，得到苜蓿丫纹夜蛾核型多角体病毒（ＡｕｔｏｇｒａｐｈａｃａｌｉｆｏｒｎｉｃａＮｕｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ，ＡｃＮＰＶ）具早晚期启动子元件的ｐ３５基因启动子，将其插入到杆状病毒转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３多克隆位点上游，使之与ｐＳＸＩＶＶＩ＋Ｘ３质粒中的人工合成后期启动子（ＰＳｙｎ）、多角体ＸＩＶ启动子（ＰＸＩＶ）串联构成早期、晚期、极晚期能持续启动外源基因表达的转移载体质粒ｐＳＸ３５．将ｐＳＸ３５用于组建含ＨＢｓＡｇ基因并形成多角体的重组ＴｎＮＰＶ，ＨＢ－ｓＡｇ基因的表达量显著提高，表达时间亦明显提前，从而实现了外源基因在杆状病毒表达系统的全期、高效表达．ｍＲＮＡ引物延伸试验结果显示，Ｐｐ３５在重组病毒中可产生２套转录本，分别于病毒感染的早期和晚期起始ＨＢｓＡｇ基因的表达．     

AcNPV增强子hr5增强HBsAg基因表达的研究

用形成包涵体（ＯＯＣ＋）并能利用人工合成启动序列和多角体ＸＩＶ启动子表达外源基因的转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３将多角体基因、乙型肝炎病毒表面抗原（ＨＢＳＡｇ）基因和苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的增强子ｈｒ５部分序列同时插入无包涵体的粉纹夜蛾核型多角体病毒ＴｎＮＰＶ－ＳＶＩ－Ｇ基因组中，得到两株高效表达ＨＢｓＡｇ基因又形成包涵体的重组病毒ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋．对重组病毒的酶切鉴定、ＤＮＡ斑点杂交和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证实，外源基因及其相应的启动子和增强子序列已正确插入病毒基因组中．插入顺序中，ｈｒ５增强子是插入ＨＢｓＡｇ基因下游，多角体基因与ＨＢｓＡｇ基因方向相反．１２５Ⅰ－固相放射免疫检测和Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，ＨＢｓＡｇ基因在昆虫离体细胞中得到高效表达并保留了抗原活性．ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋表达的ＨＢｓＡ吕蛋白与没有插入增强子序列的重组病毒ＴｎＮＰＶ—ＨＢｓ８５－ＯＣＣ＋的比较，分别提高了４０％和４６％．     

大肠杆菌β－半乳糖苷酶基因在小菜蛾细胞中的表达

用携带大肠杆菌β－半乳糖苷酶（β－ｇａｌ）基因的杆状病毒转移载体．ｐＡｃ３６０－β－ｇａｌ与苜蓿银纹夜蛾核型多角体病毒（ＡｃＮＰＶ）ＤＮＡ经钙磷沉淀法共转染小菜蛾细胞系（ＢＣＩＲＬ－ＰＸ２－ＨＮＵ２,Ｐｘ）,利用Ｘ－ｇａｌ空斑检测分析,β－ｇａｌ基因成功地插入ＡｃＮＰＶ基因组中,得到重组病毒Ａｃ－β－ｇａｌ,重组病毒在Ｐｘ细胞中表达出受ＡｃＮＰＶ多角体蛋白基因启动子控制的具有生物活性的外源基因表达产物──β－ｇａｌ．     

应用核型多角体病毒载体在家蚕和家蚕培养细胞中高…

将人尿激原ｃＤＮＡ分别插入家蚕核型多角体病毒转移载体ｐＢＫ２８３和ｐＢＦ４中，构建成两个重组质粒。所构建的这两个重组质粒与野生型家蚕核型多角体病毒ＤＮＡ共转染家蚕培养细胞，经病毒斑实验筛选出含ｐｒｏ－ＵＫ ｃＤＮＡ的稳定重组病毒株ＢｍＮＰＶ－ｐｋ１和ＢｍＮＰＶ－ｐｋ２。将此两株蛋白平板溶圈测活法和Ｗｅｓｔｅｒｎ印迹法分析细胞培养上清及细胞和家蚕的体液及组织，证实均有ｐｒｏ－ＵＫ表达。家蚕培养细胞的     

电击法转移含人凝血因子IX基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒颗粒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者合ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸｃＤＮＡ，后者仅含ｈＣＭＶ启动于控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动于驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１０８０细胞中的ＦＩＸ表达量分别为２２０、２１２ｎｇ／（１０６细胞·２４ｈ）通过与ｐＣＭＶＩＸ共转染靶细胞，可以增加人ＩＸ因子表达１．５～３．５倍，显示了用质粒载体转染靶细胞在血友病Ｂ基因治疗中是一条潜在可行的途径．本项研究用自制的电击仪转移ＰＡ３１７和ＨＴ１０８０等细胞，最高的转移效率达１０－３，并探讨了细胞种类，ＤＮＡ量，ＤＮＡ结构，电压，脉冲时间与转移效率及表达量的关系．     

斜纹夜蛾NPV多角体基因的克隆和部分测序

本文对ＳＩＮＰＶ基因组作了酶解分析，测得其基因组大小为１４５ｋｂ，并用双酶法确定了ＳＩＮＰＶ基因组的ＨｉｎｄⅢ和ＰｓｔⅠ物理图谱。以含ＡｃＮＰＶ多角体基因的质粒ｐＡＣ－Ⅰ的ＳａｌＩ－Ｃ片段为探针，对ＳＩＮＰＶＤＮＡ酶切片段ｓｏｕｔｈｅｒｎ转印杂交结果，初步判断多角体基因定位于ＰｓｔＩ－Ｂ／Ｃ／Ｄ片段、ＢｇｌⅡ－Ｃ／Ｄ片段、ＢａｍＨＩ－Ｂ／Ｃ片段和ＥｃｏＲＩ－Ａ／Ｂ片段上，且ＳＩＮＰＶ与ＡｃＮＰＶ多角体蛋白基因有６４％的同源性，而以大肠仟菌质粒ｐＵＣ１９为载体对ＳＩＮＰＶ的多角体基因试克隆，得到带有ＢｇｌⅡ－ＰｓｔⅠ双酶切片段的２个克隆子。对这两个杂交阳性克隆子之一的核苷酸序列测定，表明插入片段与ＢｍＮＰＶ多角体基因上游序列亦有一定同源性。     

人粒细胞集落刺激因子在昆虫细胞中的表达

利用苜蓿尺核型多角 体病带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标志的非融合蛋白基因转移载体ｐＢＢ成功地构了重组杆状病毒ＡｃＮＰＶ－Ｇ－ＣＳＦ．在感染重组病毒的草地夜蛾细胞中ｈＧ－ＣＳＦ得到了高效表达。表达产物由ＷｅｓｔｅｒｎＢｌｏｔ检出，其分子量约为１９ＫＤａ。     

小菜蛾颗粒体病毒DNA限制性酶切分析及基因组DNA片段克隆

小菜蛾颗粒体病毒ＤＮＡ用ＢａｍＨＩ和ＸｈｏＩ酶切，经０．７５％琼脂糖凝胶电泳，分别得到１２条带和８条带，平均分子量为１１３．０ｋｂ。用Ｓｕｐｅｒｃｏｓ１ Ｃｏｓｍｉｄ作载体，将Ｓａｕ３ＡＩ部分消化的ＰｘＧＶ－ＤＮＡ随机片段插入该载体的ＢａｍＨＩ酶切位点，转化于ＸＬ１－Ｂｌｕｅ受体菌，经Ａｍｐ平板筛选转子，挑出２５６个Ａｍｐ＾＋菌落，以＾３２Ｐ－ｄＣＴＰ标记的ＰｘＧＶ－ＤＡＮ为探针，斑点杂交筛选得到     

家蚕核型多角体病毒gp64基因的定位及克隆

以苜蓿丫纹夜蛾核型多角体病毒ＡｃＭＮＰＶ的膜表面糖蛋白基因ｇｐ６４作为探针，对经不同内切酶酶切的家蚕核型多角体病毒基因组ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析，发现ＢａｍＨⅠ酶切产生的４．２ｋｂ和７．６ｋｂ片段呈阳性杂交，经ＤＮＡ片段回收，将４．２ｋｂ片段进行了克隆     

电击法转移含人凝血因子Ⅸ基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者含ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸ ｃＤＮＡ，后者仅含ｈＣＭＶ启动子控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动子驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Surface phenotypes in T-cell leukaemia are determined by oncogenic retroviruses

Spontaneous thymic leukaemia in experimental mice is the result of a complex series of genetically controlled events. An important step in this process involves the production by thymocytes of recombinant polytropic retroviruses (MCF viruses). These leukaemogenic agents arise by recombination of genes from the env regions of endogenous precursor viruses. Sequences in these regions encode the envelope glycoprotein gp70 (ref. 6). Thus far, each cloned isolate of recombinant virus from AKR and HRS/J mice has been found to possess unique oligonucleotide sequences in its env region, as well as clone-specific peptides in its gp70 (refs 7,8). Therefore, the polytropic viruses of these leukaemia-susceptible mice are extremely diverse. These findings suggest that random recombination of env genes gives rise to leukaemogenic polytropic viruses. McGrath and Weissman have proposed that thymocytes with cell surface receptors for the gp70 of a particular leukaemogenic virus are the target cells for malignant transformation by that specific virus. In view of the diversity of polytropic viral gp70, their hypothesis would predict extensive phenotypic diversity among spontaneous thymic leukaemias. In contrast, leukaemias induced by a particular leukaemogenic recombinant virus would always have the same phenotype. Here we verify these predictions experimentally.     

mRNA transcripts related to full-length endogenous retroviral DNA in human cells

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.     

Sequencing of p35 gene fromHeliothis armigera polyhedrosis virus and its homologous comparison

Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino-acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification. Biography: WANG Ye-fu(1962-), male, Ph D.     

以精子载体法制备转基因小鼠的初步研究

将外源融合基因BaLA-HI与DOSPER脂质体等比较混合,加入获能精子悬液中,37度,5％CO2共培养0．5h,以这种处理精子作为转基因载体乾鼠的体外受精及胚胎移植,在获得的40只移植后代后,经PCR特异片段扩增和Southern杂交,共检测出2只呈相性的转基因小鼠,证明人胰 基因已在小鼠染色体上实现了整合,基因整合率为5％。     

DMY is a Y-specific DM-domain gene required for male development in the medaka fish

Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. Here, we used recombinant breakpoint analysis to restrict the sex-determining region in medaka fish (Oryzias latipes) to a 530-kilobase (kb) stretch of the Y chromosome. Deletion analysis of the Y chromosome of a congenic XY female further shortened the region to 250 kb. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only the DM-related PG17 was Y specific; we thus named it DMY. Two naturally occurring mutations establish DMY's critical role in male development. The first heritable mutant--a single insertion in exon 3 and the subsequent truncation of DMY--resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. During normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for testicular development and is a prime candidate for the medaka sex-determining gene.     

A Drosophila Minute gene encodes a ribosomal protein

Minute genes have long constituted a special problem in Drosophila genetics. For at least 50-60 different genes scattered throughout the genome, dominant mutations and/or deficiencies have been recognized which result in a common phenotype consisting of short thin bristles, slow development, reduced viability, rough eyes, small body size and etched tergites. Schultz proposed that the Minute loci encode similar but separate functions involved in growth and division common to all cells. Atwood and Ritossa suggested that Minute loci encode components of the protein synthetic machinery, specifically the transfer RNA genes; this now seems unlikely on grounds of both mapping and mutability studies. More recently, we and others suggested that the Minute loci are ribosomal protein genes. We report here that transformation with a cloned 3.3-kilobase (kb) region containing the gene encoding the large subunit ribosomal protein 49 (rp49) suppresses the dominant phenotypes of Minute (3)99D, a previously undescribed Minute associated with a chromosomal deficiency of the 99D interval. This activity is specific to the 99D Minute as it does not suppress other Minute loci elsewhere in the genome. This result provides direct evidence that the Minute locus at the 99D interval encodes the ribosomal protein 49.     

人RP2基因在果蝇胚胎细胞中的表达

将质粒pJLA503中的RP2编码cDNA用PCR的方法扩增,并与pIND-3myc质粒上的人c-myc编码序列连接到果蝇转基因载体pUAST上,构建了UAS－RP2－3myc和UAS－RP2(del6)-3myc融合质粒。用脂质体转染的方法,将质粒导入果蝇培养细胞系Schneider line2(S2),并用PCR加以证实,然后通过Western blot检测证明了RP2和RP2 del6的表达。证明了人的RP2基因在果蝇细胞中表达的可能性,说明果蝇系统是可以用来分析人的RP2基因功能的。     

Unexpectedly similar rates of nucleotide substitution found in male and female hominids

In 1947, it was suggested that, in humans, the mutation rate is dramatically higher in the male germ line than in the female germ line. This hypothesis has been supported by the observation that, among primates, Y-linked genes evolved more rapidly than homologous X-linked genes. Based on these evolutionary studies, the ratio (alpha(m)) of male to female mutation rates in primates was estimated to be about 5. However, selection could have skewed sequence evolution in introns and exons. In addition, some of the X-Y gene pairs studied lie within chromosomal regions with substantially divergent nucleotide sequences. Here we directly compare human X and Y sequences within a large region with no known genes. Here the two chromosomes are 99% identical, and X-Y divergence began only three or four million years ago, during hominid evolution. In apes, homologous sequences exist only on the X chromosome. We sequenced and compared 38.6 kb of this region from human X, human Y, chimpanzee X and gorilla X chromosomes. We calculated alpha(m) to be 1.7 (95% confidence interval 1.15-2.87), significantly lower than previous estimates in primates. We infer that, in humans and their immediate ancestors, male and female mutation rates were far more similar than previously supposed.