扩增抗体重链可变区的一个高效方法

从杂交瘤细胞或脾细胞中扩增抗体可变区，是构建单链抗体（Single-chain Fv fragment，ScFv）、克隆单克隆抗体和研究抗原与抗体相互作用的关键一步。而抗体可变区（Variable regions，Fv）高度变异，扩增相对困难，至今仍是一个难点，有待解决。我们在构建ScFv过程中，发现2株杂交瘤细胞用普通方法难于扩增出抗体重链可变区（Heavy—chain variable region，VH）抗体。于是提出了一种多序列比对和简并引物设计算法，并加以实现，设计出通用的鼠源性抗体VH引物；应用上述引物，采用反向多聚酶链反应（Reversepopymerase chain reaction，reversePCR）方法——3’RACE和5’RACE法，准确地扩增出2株杂交瘤细胞的VH基因。该算法很好地解决了引物特异性和最小化问题，具有简单、实现容易的特点，可用于基因家族中未知基因克隆和文库构建中的引物设计。与反向PCR方法结合，提高了难扩增的未知基因的成功率。     

Isolation of a gene encoding an integral membrane protein from the vicinity of a balanced translocation breakpoint associated with DiGeorge syndrome

Deletions within 22q11 have been associated with a wide varietyof birth defects embraced by the acronym CATCH22 and includingthe DiGeorge syndrome, Shprintzen syndrome (velocardiofacialsyndrome) and congenital heart disease. It is not known howmany genes contribute to this phenotype. Previous studies haveshown that a balanced translocation disrupts sequences withinthe shortest region of deletion overlap for DiGeorge syndrome.A P1 clone was isolated which spans this breakpoint and usedto isolate a cDNA encoding a transmembrane protein expressedin a wide variety of tissues. This gene (called IDD) is notdisrupted by the translocation, but maps within 10 kb of thebreakpoint. Mutation analysis of five affected cases with nopreviously identified chromosome 22 deletion was negative, buta potential protein polymorphism was discovered. No deletionsor rearrangements were detected in these patients followinganalysis with markers closely flanking the breakpoint, datawhich emphasize that large (i. e. over 1 Mb) interstitial deletionsare the rule in DiGeorge syndrome. The proximity of IDD to thebalanced translocation breakpoint and its position within theshortest region of deletion overlap indicate that this genemay have a role, along with other genes, in the CATCH22 haploinsufficiencysyndromes.     

Isolation of a gene expressed during early embryogenesis from the region of 22q11 commonly deleted in DiGeorge syndrome

DiGeorge syndrome (DGS) is one of several syndromes associatedwith deletions within the proximal long-arm of chromosome 22.The region of chromosome 22q11 responsible for the haplolnsufficiencysyndromes (the DiGeorge Critical Region or DGCR) has been mappedusing RFLPs, quantitative Southern blotting and FISH. Similardeletions are seen in the velo-cardio-facial syndrome (VCFS)and familial congenital heart defects. It Is not known whetherthe phenotypic spectrum is the result of the hemizygosity ofone gene or whether it is a consequence of contiguous genesbeing deleted. However, the majority of patients have a large(< =2Mb deletion). In this paper we report the isolationof a gene, lab name T10, encoding a serine/threonine rich proteinof unknown function which maps to the commonly deleted regionof chromosome 22q11. Studies in the mouse Indicate that it mapsto MMU16 and is expressed during early embryogenesis. Althoughnot mapping within the shortest region of overlap for DGS/VCFS,and therefore not the major gene Involved In DGS, the expressionpattern suggests that this gene may be involved in modifyingthe haplolnsufficient phenotype of hemizygous patients.     

Identification of a Novel Protein Antigen Encoded by a Mycobacterium tuberculosis-Specific RD1 Region Gene

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-14 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T vector system and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST-ORF-14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF-14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST-ORF-14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF-14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG-vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long-term contacts of TB patients also had antibodies reactive to the ORF-14 protein. These results suggest that the ORF-14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.     

Worldwide Genetic Variation at the 3'-UTR Region of the LDLR Gene: Possible Influence of Natural Selection

The low density lipoprotein receptor gene (LDLR) contains many Alu insertions, and is especially Alu‐rich at its 3′‐untranslated region (3′‐UTR). Previous studies suggested that the LDLR 3′‐UTR could regulate gene expression by the stabilization of its mRNA. Given the faster Alu evolutionary rate, and wondering about its consequences in a possibly regulatory locus, we have studied ～800 bp of 222 chromosomes from individuals of African, Asian, Caucasian and Amerind ancestry, to better understand the evolution of the worldwide genetic diversity at this locus. Twenty‐one polymorphic sites, distributed in 15 haplotypes, were found. High genetic diversity was observed, concentrated in one Alu insertion (Alu U), which also shows a fast evolutionary rate. Genetic diversity is similar in all populations except Amerinds, suggesting a bottleneck during the peopling of the American continent. Three haplotype clusters (A, B, C) are distinguished, cluster A being the most recently formed (～500,000 years ago). No clear geographic structure emerges from the haplotype network, the global F_st (0.079) being lower than the average for the human genome. When ancestral population growth is taken into account, neutrality statistics are higher than expected, possibly suggesting the action of balancing selection worldwide.     

Isolation of a Novel Strain of Mycobacterium iranicum from a Woman in the United States

A novel strain of Mycobacterium iranicum, a recently described nontuberculous Mycobacterium species, was isolated from the sputum of a woman. The source of infection was not determined; however, fomite transmission of inhaled aerosolized secretions from her husband''s sleep apnea equipment was historically possible.     

Isolation and Biochemical Characterisation of a Novel Collagen from Catostylus tagi

A preliminary biochemical approach to the study of collagen isolated from the medusa Catostylus tagi is reported and results are discussed in view of its use as a natural matrix for biomedical applications. Collagen from the jellyfish umbrella was isolated by pepsin digestion and purified by dialysis and salt precipitation. As expected, glycine represented almost one-third of the total amino acids. Aromatic amino-acid content was very low and imino acids were fewer than in collagens from fish and mammalian sources. Results from SDS-PAGE, ion-exchange chromatography and N-terminal amino-acid sequencing revealed an α1α2α3 heterotrimer, similar to vertebrate type V/XI. The molecular mass of two of the polypeptide chains was close to 85 kDa and 100 kDa for the third. However, the two chains presenting similar molecular mass, showed differences in charge and primary structure. Further characterisation showed a glycosylated protein with the carbohydrate moiety comprising almost 7% of the total mass, a denaturation temperature of 29.9°C and multiple isoelectric points. Incubation with glutamyl endopeptidase resulted in significant digestion, in agreement with the protein's high content of Asp and Glu.     

Isolation of cosmids and fetal brain cDNAs from the proximal long arm of human chromosome 22

The proximal portion of human chromosome 22q appears to carrygenes implicated in the pathogenesis of various developmentaldisorders, including the cat eye syndrome (CES) and the DiGeorgesyndrome (DGS). A cosmid library was prepared from a radiationhybrid selected for its content in chromosome 22 fragments.A large fraction of cosmids containing human DNA were foundto derive from the juxtacentromeric region of chromosome 22,as shown by fluorescence in situ hybridization (FISH) performedusing individual cosmids or cosmid pools as probes. Finer mappingwas obtained for individual cosmids by hybridization to a somaticcell hybrid mapping panel which splits the long arm of the chromosomeinto 14 bins numbered 1 to 14 from the centromere to the telomere.Of the 10 cosmids mapped, eight belonged to group 1, the othertwo to group 14, in agreement with FISH data. Rare endonucleasesites and fragments conserved between species were searchedin single cosmids, resulting in the selection of seven cosmidfragments which were used to screen a human fetal brain cDNAlibrary. Three cDNAs were identified, encoded from two chromosome22 genes which appeared to be novel, as determined from partialend sequence and comparison with the database entries. Finelocalization of the 30.9 cDNA indicated that the correspondinggene was located in a segment of proximal 22q overlapping withthe critical DGS region.     

Isolation of a novel gene from the DiGeorge syndrome critical region with homology to Drosophila gdl and to human LAMC1 genes

DiGeorge syndrome, and more widely the CATCH 22 syndrome, are associated with microdeletions in chromosomal region 22q11.2. A critical region of 500 kb has been delimited within which maps the breakpoint of a balanced translocation associated with mild CATCH 22 phenotypes. We report the isolation from this critical region of a novel gene, DGCR6, which maps 115 kb centromeric to the balanced translocation breakpoint. The DGCR6 gene product shares homology with the Drosophila melanogaster gonadal protein, which participates in gonadal and germ-line cells development, and with the human laminin. gamma-1 chain, which upon polymerization with alpha- and beta- chains forms the laminin molecule. Laminin binds to cells through interaction with a receptor and has functions in cell attachment, migration and tissue organization during development. DGCR6 could be a candidate for involvement in the DiGeorge syndrome pathology by playing a role in neural crest cell migration into the third and fourth pharyngeal pouches, the structures from which derive the organs affected in DiGeorge syndrome.      

Conserved and Non-Conserved Regions in the Sendai Virus Genome: Evolution of a Gene Possessing Overlapping Reading Frames

We have sequenced the entire genome of a virulent field isolate of Sendai virus, the Hamamatsu strain, and compared the sequence with that of a distant related strain, the Z strain. Calculation of synonymous and non-synonymous (amino acid changing) nucleotide substitutions revealed regions where changes were permissive and non-permissive, and the experimentally determined functional region were found to be conserved, showing that important regions for function were conserved during evolution. In the cistron-overlapping regions in the P gene, one reading frame was conserved, whereas the other overlapping frame was flexible. The priority of one frame could be a strategy for evolution of an overlapping gene of RNA viruses. We found that the carboxyl two thirds of the C protein was conserved over the amino-terminal one third, possessing priority to the overlapping P polypeptide. This suggests that the carboxyl two thirds of the C protein have a functional importance. We also found a highly variable region between the L coding frame and the 5 trailer sequence. The relevance of these findings to actual viral replication should be clarified in the future.     

Evidence for a Conserved Polydnavirus Gene Family: Ichnovirus Homologs of the CsIV Repeat Element Genes

In Campoletis sonorensis Ichnovirus (CsIV), the repeat element genes constitute a gene family of 28 members. In the present work, we document the presence of members of this gene family in two additional ichnoviruses, Hyposoter didymator Ichnovirus (HdIV) and Tranosema rostrale Ichnovirus (TrIV). Two repeat element genes, representing at least one functional gene, were identified in TrIV, whereas HdIV was found to contain at least three such genes. In both HdIV and TrIV, the known repeat element genes are encoded on single genome segments, with hybridization studies suggesting the presence of other, related but as yet uncharacterized genes. The HdIV and TrIV repeat element genes are all transcribed in infected caterpillars, although differences exist among genes in levels and in tissue specificity of expression. A heuristic tree was generated indicating that the repeat element genes are more similar within a species of wasp than between species, with TrIV genes being more closely related to the CsIV than to the HdIV genes. These results suggest that the most significant duplication, divergence, and expansion of the repeat element genes occurred after speciation. The finding that repeat element genes form an interspecific family within the genus Ichnovirus supports the view that the proteins they encode play an important role in ichnovirus biology.     

In Vivo Efficacy of a Chimeric Peptide Derived from the Conserved Region of the M Protein against Group C and G Streptococci

The J8 peptide from the conserved region of the M protein protects against group A streptococcus infections. In this study, we demonstrate that vaccination with a J8-containing formulation induces IgG that recognizes and binds group C and G streptococci. Moreover, this formulation has the potential to provide protection against infections caused by these organisms.     

Molecular Cloning of the Gene for a Conserved Major Immunoreactive 28-Kilodalton Protein of Ehrlichia canis: a Potential Serodiagnostic Antigen

A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.     

Ficoll分离和MACs分选对T细胞BCL11b表达水平的影响

目的 了解Ficoll分离和MACs分选过程对T细胞中BCL11b基因表达水平的影响情况。方法 利用Ficoll分离3例正常人外周血单个核细胞（PBMC）。进一步经MACs负性分选CD3^＋T细胞。分别收集培养0、6和20h的PBMC和CD3^＋T细胞，利用实时定量PCR方法检测各组样本中BCL11b基因的表达水平。结果 经Fieoll分离的PBMC和MACs分选后CD3^＋T细胞在3个时间点BCL11b的表达水平无显著性差异（P〉0．05）。根据CD3＋T细胞％标化后，各组BCL11b表达水平也均无显著性差异。结论Fieoll分离和MACs负性分选过程不影响T细胞中BCL11b表达水平。提示其不会影响T细胞的活化。     

Characterization of a Novel Methyl-Accepting Chemotaxis Gene, dmcB, from the Oral Spirochete Treponema denticola

Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named dmcB. Northern blot analysis suggested that dmcB was part of an operon structure containing prtB. Insertional inactivation of dmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of the dmcB gene also attenuated the methylation of the DmcA protein. These results suggest that the dmcB gene codes for an MCP in T. denticola which may interact with other MCPs in these organisms.