Production of scFv-Fc fusion protein using genetically manipulated quails

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.     

Detection of the genetic modification in heat-treated products of Bt maize by polymerase chain reaction

 A method for the detection of the genetic modification in thermally treated products from insect-resistant maize expressing a synthetic gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis is described. The probability of detection of the transgene in thermally treated products was increased by choosing a short (211 bp) polymerase chain reaction (PCR) amplicon; the specificity was ensured by including a promoter region adjacent to the cryIA(b) gene in the target sequence. The influence of pH during thermal treatment of maize on the detectability of the transgene is demonstrated using polenta as a model for products made from this cereal. Received: 22 September 1997     

Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens

We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.     

普通烟草钙依赖蛋白激酶NtCDPK15的基因克隆及表达分析

钙依赖蛋白激酶(CDPK)对Ca²⁺信号转导通路下游元件的调控具有重要作用。本实验采用同源克隆技术从普通烟草中分离得到1 个CDPK 基因,命名为NtCDPK15(GenBank 登录号为JN662020),cDNA 全长为1963 bp,ORF 大小为1569 bp,编码522 个氨基酸。多序列比对结果表明,NtCDPK15 的编码产物具有典型的CDPK 保守序列,C 末端调控区有4个EF 手性结构。系统进化树分析显示,NtCDPK15 可能来源于绒毛状烟草,预测NtCDPK15 与NtCDPK1这对旁系同源基因在功能上可能非常保守。实时荧光定量PCR 分析结果表明,NtCDPK15 在根和叶中的表达量显著高于茎中的表达量,但经盐胁迫诱导后NtCDPK15 在茎中的表达量显著增加,同时,盐胁迫还使基因在叶中的表达量显著上调,ABA 胁迫可诱导基因在根中的表达,而干旱胁迫可使基因在根和叶中的表达量下调,这些结果说明NtCDPK15 在普通烟草中的表达受盐胁迫及ABA 胁迫诱导,但受干旱胁迫抑制。     

棘孢木霉木聚糖酶Ⅱ结构基因和上游调控区的克隆及分析

作者克隆及分析了棘孢木霉木聚糖酶Ⅱ结构基因和上游调控区,并获得了内源启动子.根据木霉属木聚糖酶Ⅱ结构基因及上游调控区的保守性,以棘孢木霉基因组DNA为模板,进行简并PCR扩增,产物纯化并克隆至T载体,经酶切鉴定后进行序列分析.扩增获得片段长1875 bp,由795 bp的木聚糖酶Ⅱ结构基因和1 080 bp的上游调控区...     

枯草芽孢杆菌P spovG启动子改造与表征

枯草芽孢杆菌是重要的工业生产菌株,广泛应用于外源蛋白质的表达。目前在枯草芽孢杆菌中常见组成型启动子中,P_spovG作为高强度组成型启动子在表达外源蛋白质时,具有表达量高、稳定性好和适用范围广等优势。作者首先对P_spovG上游序列截短,发现将启动子上游序列由原来的251个碱基缩短为26个,仍能保持启动子强度不降低,并确定了紧邻-35元件上游的3个碱基“AGC”为影响启动子强度的关键碱基。其次对启动子的上游A-T富含区以及spacer区域的G-C富含区进行随机突变,证明了上游激活序列中A-T碱基的排列对启动子强度有重要影响,并得到了强度提高的突变体。最后通过将不同表达时期启动子的关键调控序列分别插入突变体的上下游,得到了启动子强度为原始启动子135.1%的新型启动子SP_spovG-P_lytR。本研究为枯草芽孢杆菌表达系统开发新型强组成型启动子提供了重要的参考。     

香灰菌gpd启动子的克隆及功能验证

为了构建香灰菌的转化体系,研究香灰菌的基因功能,需要获得高活性的启动子元件。本实验通过PCR技术分别克隆得到gpd基因以及其基因上游约1000 bp序列,使用Neural Network Promoter Prediction、Signal Scan Search Request、PLANTCAREA database软件分析发现,gpd基因上游区域内不仅包含启动子所需的特征核心元件TATA-box、CAAT-box、G-box、GC-box、CT-rich等,还含有一个高分值的转录起始位点。为进一步验证该序列的启动子功能,将gpd启动子元件与增强型绿色荧光蛋白基因(egfp)和潮霉素基因(hph)连接构建成融合表达载体,通过根癌农杆菌介导将其转化香灰菌丝。潮霉素抗性筛选、绿色荧光蛋白检测结果显示,香灰菌gpd启动子成功驱动了潮霉素抗性基因和增强型绿色荧光蛋白基因的表达,根癌农杆菌介导转化成功,为香灰菌外源基因表达及基因功能的研究奠定了基础。     

普通烟草碳酸酐酶家族基因的生物信息学分析

为挖掘普通烟草碳酸酐酶基因的信息并探讨其功能,本研究利用生物信息学方法,在烟草基因组数据库中对普通烟草碳酸酐酶家族成员进行了检索,并对其理化性质、遗传进化、基因结构、蛋白保守基序、顺式作用元件和组织表达模式进行了分析。结果显示,在普通烟草中至少含有9个α和6个β亚家族成员。在进化关系上,不同亚家族成员之间,序列同源性较低;与水稻相比,拟南芥和普通烟草两个亚家族间的亲缘关系较近。亚细胞定位分析结果显示,烟草α亚家族成员在细胞壁、细胞膜、线粒体、叶绿体、细胞质等细胞器中均有分布,而β亚家族成员均存在于叶绿体中。基因结构和保守基序分析说明,各亚家族成员的基因结构和蛋白保守基序呈现较高的一致性。启动子顺式作用元件分析显示,烟草碳酸酐酶基因的启动子中,均含有多个参与光反应、激素响应、非生物胁迫和机械损伤等相关的顺式作用元件。基因组织表达模式分析表明,烟草的碳酸酐酶基因不同成员在不同组织表达水平存在差异,并呈现出时空特异性。本研究结果为烟草碳酸酐酶基因的功能研究奠定了基础。     

源于 GAP 启动子的毕赤酵母组成型表达系统的研究进展

巴斯德毕赤酵母表达系统是目前应用非常广泛的外源基因真核表达系统。与源于醇氧化酶启动子(pAOX1)的诱导型表达系统相比, 源于三磷酸甘油醛脱氢酶启动子(pGAP)的毕赤酵母组成型表达系统在表达外源蛋白时不需使用甲醇, 表达时间短, 并且在高密度发酵时采用连续发酵模式, 因此成为近年研究的热点。本文分别从pGAP表达载体的构建、碳源、基因拷贝数、信号序列、高密度发酵等方面对该表达系统进行了综述, 以期为其在蛋白生产中的深入研究和应用提供参考。     

普通烟草NtNDPK4基因的克隆与表达分析

为揭示核苷二磷酸激酶在烟草中的功能和作用机制,从烟草K326克隆获得了1个核苷二磷酸激酶基因并命名为NtNDPK4,CDS长度为678 bp,编码225个氨基酸。生物信息学分析表明,NtNDPK4蛋白分子量大小24.95 kDa,pI约8.34,亚细胞定位预测在叶绿体上,进化分析显示该蛋白属于NDPKⅡ型,顺式作用元件分析预测该基因启动子含有激素和光照响应的作用元件。组织表达特异性分析结果显示,NtNDPK4在烟草各组织中均有表达。在MeJA、ABA、GR-24、IAA、GA和6-BA等激素处理下,NtNDPK4基因的表达显著上调,尤其是GA处理后NtNDPK4基因表达上调了近50倍。黑暗生长的烟草幼苗恢复光照24 h后,NtNDPK4基因表达明显上调。这表明烟草NtNDPK4基因可能参与激素和光照响应。     

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.     

Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter

The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H(+)-ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.     

Cloning and functional analysis of the Aspergillus oryzae conidiation regulator gene brlA by its disruption and misscheduled expression

We cloned the brlA gene from Aspergillus oryzae genomic DNA using the A. nidulans brlA gene as a probe. A 3.1-kb EcoRI-BalI genomic DNA fragment was cloned and sequenced. The deduced amino acid sequence revealed 70% identity with A. nidulans BRLA and contained two C2H2 zinc finger motifs in its carboxyl terminus, and the promoter sequence contained a 43-bp highly conserved region, indicating that the cloned gene was an A. oryzae homologue of A. nidulans brlA. Disruption of the brlA gene by homologous recombination resulted in the loss of ability to form conidiophores. These results suggest that the brlA gene of A. oryzae plays a fundamental role in controlling conidiophore development. When the brlA gene was expressed under the control of the amyB promoter in A. oryzae transformants, highly differentiated and compact colonies were observed on a solid medium. The misscheduled expression of the brlA gene in submerged culture, in which conidiation does not normally occur, caused the development of complex conidiophore structures with vesicles, phialides and conidia.