Synonymous Constraint Elements Show a Tendency to Encode Intrinsically Disordered Protein Segments

Synonymous constraint elements (SCEs) are protein-coding genomic regions with very low synonymous mutation rates believed to carry additional, overlapping functions. Thousands of such potentially multi-functional elements were recently discovered by analyzing the levels and patterns of evolutionary conservation in human coding exons. These elements provide a good opportunity to improve our understanding of how the redundant nature of the genetic code is exploited in the cell. Our premise is that the protein segments encoded by such elements might better comply with the increased functional demands if they are structurally less constrained (i.e. intrinsically disordered). To test this idea, we investigated the protein segments encoded by SCEs with computational tools to describe the underlying structural properties. In addition to SCEs, we examined the level of disorder, secondary structure, and sequence complexity of protein regions overlapping with experimentally validated splice regulatory sites. We show that multi-functional gene regions translate into protein segments that are significantly enriched in structural disorder and compositional bias, while they are depleted in secondary structure and domain annotations compared to reference segments of similar lengths. This tendency suggests that relaxed protein structural constraints provide an advantage when accommodating multiple overlapping functions in coding regions.     

CXXC5多克隆抗体的制备及表达研究

CXXC5基因是从人类胚胎心脏的cDNA文库中克隆出来的一个人类锌指基因,包含zf-CXXC5结构域,该基因编码322个氨基酸,在物种进化上高度保守.为了进一步研究该基因的功能,需要获得CXXC5蛋白并制备其抗体.通过PCR扩增方法扩增得到了CXXC5部分编码区序列,然后将其连接到PGEX4T-1上,转化到大肠杆菌BL...     

人源性抗体在HEK293T细胞中瞬时表达条件的优化

以HEK293T细胞为宿主对抗体基因的转染和表达条件进行优化。以GFP(green fluorescence protein)和人源性抗b FGF抗体1A2为报告基因,考察了3种转染试剂磷酸钙,PEI,FuGene HD的转染效率,确定FuGene HD的转染效率最高后,对DNA∶FuGene HD的比例,加入氯喹的浓度,转染后的孵育时间,最佳的低温培养温度以及加入组蛋白抑制剂的浓度进行了优化。结果显示,FuGene HD的转染效率最高,达到56.7%。当转染的DNA=2μg/4×106细胞时,DNA∶FuGene HD的最佳比例为1∶4(W/V);加入氯喹能够增强FuGene HD的转染效率,最佳浓度为100μmol/L,转染后的最佳孵育时间为6h;低温诱导能够提高293T细胞表达抗体的能力,最佳的温度为33℃;加入组蛋白抑制剂丁酸钠或丙戊酸能够明显提高抗体的表达量,丙戊酸的效果优于丁酸钠。通过对转染试剂,转染方法,培养温度,组蛋白抑制剂的加入等条件进行优化使人源性抗bFGF抗体的表达量从1.5mg/L提高到了15.1mg/L。     

Cell-Type-Specific Gene Transfer into Human Cells with Retroviral Vectors That Display Single-Chain Antibodies

The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from spleen necrosis virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 10⁵ CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.     

Effects of Maternal Antibodies on Protection and Development of Antibody Responses to Human Rotavirus in Gnotobiotic Pigs

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.     

抗HBsAg二硫键稳定性Fv抗体(dsFv)基因的构建与表达

采用PCR定点突变方法,成功地构建了抗HBsAg dsFv抗体的轻、重链突变基因,NdeI和EcoRI酶切后分别插入pET20b表达质粒,经测序证明在重链第44位氨基酸和轻链第100位氨基酸已突变形成半胱氨酸(Cys).VH和VL重组质粒分别转化到大肠肝菌BL21(DE3)中,IPTG诱导后,经SDS-PAGE电泳表明在12kD处有包含体蛋白表达,表达蛋白含量分别为28%和35%.VH和VL包含体蛋白经GuHCl变性后,等量混合在复性折叠液中结合,形成了一个约24kD并有一定活性的dsFv蛋白.抗HBsAg dsFv抗体表达及复性的成功,为今后基因工程抗体的研究及应用奠定了基础.     

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken V_L and V_H loci were knocked out in DT40 cells and replaced with human V_K and V_H genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human V_K and V_H regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens.     

Antiidiotypic Antibodies Interacting with cGMP-Dependent Channels of Frog Retinal Rod Outer Segments

Abstract

Antiidiotypic approach was used to obtain antibodies interacting with cGMP-binding site of the cGMP-activated channel of the photoreceptor cell. Monoclonal anti-BrcGMP antibodies having characteristics of binding of agonist and its analogs close to those for a natural receptor have been obtained. These antibodies were used to raise polyclonal antiidiotypic antibodies capable of interacting with a natural cGMP-receptor. Application of immunoglobulins, isolated from antiidiotypic serum, to inside-out fragments of the rod plasma membrane led to an irreversible increase of the conductance of cGMP-dependent channels.     

人类新基因Bzw2的克隆、抗体制备及表达分析

Bzw2( basic leucine zipper and W2 domains 2)基因是人类心脏发育候选基因,它由bzip结构域和W2结构域构成.为了研究该基因在心脏发育过程中的作用,利用生物信息学信息克隆了人类Bzw2基因全长,将所得的片段插入到原核表达载体pGEXT-4T-1载体中,利用BL21菌株表达该重组...     

抗HBsAg二硫键稳定性Fv抗体（dsFv）基因的构建与表达

采用ＰＣＲ定点突变方法,成功地构建了抗ＨＢｓＡｇｄｓＦｖ抗体的轻、重链突变基因,ＮｄｅＩ和ＥｃｏＲＩ酶切后分别插入ｐＥＴ２０ｂ表达质粒,经测序证明在重链第４４位氨基酸和轻链第１００位氨基酸已突变形成半胱氨酸（Ｃｙｓ）。ＶＨ和ＶＬ重组质粒分别转化到大肠肝菌ＢＬ２１（ＤＥ３）中,ＩＰＴＧ诱导后,经ＳＤＳＰＡＧＥ电泳表明在１２ｋＤ处有包含体蛋白表达,表达蛋白含量分别为２８％和３５％。ＶＨ和ＶＬ包含体蛋白经ＧｕＨＣｌ变性后,等量混合在复性折叠液中结合,形成了一个约２４ｋＤ并有一定活性的ｄｓＦｖ蛋白。抗ＨＢｓＡｇｄｓＦｖ抗体表达及复性的成功,为今后基因工程抗体的研究及应用奠定了基础。     

HIV gp41基因分段低温诱导表达

人类免疫缺陷病毒(Human immunodeficiency virus,HIV)GP41跨膜蛋白由于具有特殊的穿膜拓扑结构,对宿主菌细胞膜产生毒性作用而使其难以在E.coli中有效表达[1].本室前期工作发现GP41蛋白中有三个区域对表达菌细胞具有毒性作用,其分别为:位于N端2/3区域(N3片段:nt7373-8006)的融合肽(aa512-527)和跨膜区(Aa 684-705),它们含有丰富的疏水性氨基酸;以及位于C端1/3区域(C片段:nt8007-8339)的慢病毒裂解肽LLP1(aa 826-854)和LLP2(aa 768-788),可形成2个两亲性α螺旋,从而对宿主菌产生较强的细胞膜毒性作用使细菌大量死亡,最终致使GP41蛋白难以获得有效表达[2].为此我们尝试在低温条件(16℃)下对HIV-gp41N端2/3和C端1/3区域在大肠杆菌BL21(DE)3中进行诱导表达,并对低温条件下GP41蛋白表达对细菌细胞膜的毒性作用特征进行初步探讨.     

从噬菌体抗体库中淘选血纤维蛋白特异的单链抗体

为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 .     

HIV gp41基因分段低温诱导表达

Clone N3 and C from Human immunodeficiency virus(HIV) gp41 gene were expressed using the pET expression system. When induced by IPTG at 37℃, both two clones did not express in E.coli BL21(DE)3. Howerver, when induced at 16℃, the two clones were both overexpressed, and the amount of the product was about 20% of the total bacteria protein. In Western blotting test, the protein product could react with HIV-positive serum. After IPTG induction, E. coli cells had much higher death rate at 37℃ than at 16℃; [^3H]uridine release assay also showed that after IPTG induction, E. coli had a higher release at 37℃. The results suggested that overexpression of the two proteins was due to their decreased toxicity at lower temperature.     

Gene Conversion between Unlinked Sequences in the Germline of Mice

Gene conversion between homologous sequences on non-homologous chromosomes (ectopic gene conversion) is remarkably frequent in fungi. It is thought to be a consequence of genome-wide homology scanning required to form synapses between homologous chromosomes. This activity provides a mechanism for concerted evolution of dispersed genes. Technical obstacles associated with mammalian systems have hitherto precluded investigations into ectopic gene conversion in the mammals. Here, we describe a binary transgenic mouse system to detect ectopic gene conversion in mice. Conversion events are visualized by histochemical staining of spermatids, and corroborated by polymerase chain reaction amplification of transgenes in spermatozoa. The results show that conversion between unlinked, hemizygous lacZ transgenes is frequent in the male germline, ranging from 0.1 to 0.7% of spermatids. Genomic location may affect the susceptibility to recombination, since the frequency varied between lines. The results suggest that homologous genes can undergo concerted evolution despite being genomically dispersed. However, mechanisms may exist to modulate this activity, enabling the divergence of duplicated genes.     

A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato

Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon Ca2+-ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots.     

UAA and UAG may Encode Amino Acid in Cathepsin B Gene of Euplotes octocarinatus

The ciliate Euplotes deviates from the universal genetic code by translating UGA as cysteine and using UAA and UAG as the termination codon. Here, we cloned and sequenced the Cathepsin B gene of Euplotes octocarinatus (Eo‐CTSB) which containing several in‐frame stop codons throughout the coding sequence. We provide evidences, based on 3′‐RACE method and Western blot, that the Eo‐CTSB gene is actively expressed. Comparison of the derived amino acid sequence with the homologs in other eukaryotes revealed that UAA and UAG may code for glutamine in Eo‐CTSB. These findings imply an evolutionary complexity of stop codon reassignment in eukaryotes.