Construction of subtracted cDNA library for differentially expressed genes associated with chronic optic nerve injury / 第二军医大学学报

Objective: To construct subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury in cats. Methods: Fifteen adult cats were randomly divided into 3 groups (n=5): control group, 4-w compression group and 8-w compression group. The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm. The total RNA was prepared from optic nerves of each group by TRIzol method. Double-stranded cDNA was produced by SMART PCR cDNA synthesis protocol. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the optic nerves after 4-w and 8-w compression. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library,followed by amplification of the libraries through E. coli transformation with calcium chloride and screening of blue and white clones. Three hundred positive bacterial clones were randomly picked in each library and identified by colony PCR. Results: Analysis of the white clones by PCR showed that 80% clones contained 200-800 bp inserts in each library. Conclusion: Four subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury have been successfully constructed by SSH and T/A cloning techniques,which lays a solid foundation for screening and cloning specific differentially expressed genes associated with chronic optic nerve injury.

Construction and identification of yeast cDNA expression library of human dermal papillae cells in primary culture / 中华皮肤科杂志

Objective To construct yeast cDNA expression library of human dermal papillae cells (DPCs) in primary culture.Methods Human dermal papilla cells (DPCs) were isolated by two-step digestion method and cultured in DMEM medium.Total RNA was extracted from primary DPCs that exhibited an aggregative behavior in culture,then,cDNA was synthesized and amplified by using CloneMinerTM cDNA Library Construction kit to construct primary cDNA library and yeast cDNA expression libary.Results The average titer and total clones were 7.0×106 colony forming units(cfu)/ml and 1.4×107 cfu respectively in the primary library,5.5×106 cfu/ml and 1.1×107 cfu respectively in the yeast expression library.The average insert size Was 1.2 kb and the recombination rate was above 95%.Conclusions The yeast cDNA expression library of DPCs in primary culture has been successfully constructed.which will lay a foundation for screening proteins interacting with HSPC016 gene in DPCs with yeast two-hybrid system.

Screening for rat vascular calcification related genes using suppression subtractive hybridization / 中华肾脏病杂志

Objective To determine the differentially expressed genes in the development of vascular medium calcification in rats using the suppression subtractive hybridization (SSH). Methods Twenty-four 6-week old SD rats of specific pathogen free grade were recruited and randomly allocated into calcified group (n=12) and control group (n=12). Rats were made for vascular calcification model in calcified group (vitamin D3 plus nicotine, VDN). All rats were sacrificed to measure concentration of calcium in the arterial tissue and examine the pathological lesion changes. RNA in rat aortic tunica tissue was extracted and reverse transcripted into cDNA. cDNA fragments which highly expressed calcification were isolated in calcified group using the SSH. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed to competent DH-5α by means of heating transfer. cDNA libraries of differentially expressed gene between calcified group and control group were successfully constructed. Recombinant vectors were analyzed by colony PCR. Positive genes were randomly selected for sequencing and analyzed by BLAST. Six genes, for example, were randomly selected for RT-PCR certification. Results (1) The pathological examination results demonstrated that in calcified group there were obvious calcium diposits and media squirm in tunica media of rat aortic wall, while in control group no calcium diposit was found. (2) There was statistical significance in calcium concentration in vascular tissue between calcified group[(15.34 ± 2.51)mg/g] and control group [(5.20 ± 0.75) mg/g] (P＜0.01). (3) Subtracted libraries in vascular calcification was successfully established. Ninety-two positive clones in positive library and 18 positive clones in reverse library were obtained after the colony PCR identification. The length of insertion fragments was concentrated between 150 bp and 400 bp. Calcification-related 43 up-regulated genes and 11 down-regulated genes were obtained through sequencing and BLAST analysis in positive clones. RT-PCR validation indicated that the expressions of 5 genes such as CytoP450 and Nell1 had greater increase in calcified group than those in control group, the average fold change was 1.71.Conclusions Model of vascular calcification induced by vitamin D3 plus nicotine is successfully constructed. Related gene expression spectrum is changed in the process of vascular calcification.Some ossification genes and genes associated with apoptosis, oxidation, inflammation and cytokines are up-regulated. At the same time, some genes which possibly inhibit vascular calcification are down -regulated.

Construct a subtractive library of Caski cell lines induced by exposing to the space environment by suppression subtractive hybridization / 中国医师杂志

Objectives Construct a subtractive library of Caski cell line induced by exposing to the space environment by suppression subtractive hybridization and pave the way to explain the molecular mechanisms of the changes at the gene level. Methods Super SMART cDNA synthesis and suppression subtractive hybridization (SSH) were performed to isolate differentially expressed cDNA fragments from strains subclonal 48A9 cell line. cDNA from the 48A9 cell line were used as " tester" , and the other from the control Caski cell line as "driver". Subtractive products were directly inserted into T/A cloning vector, and then transformed into host bacteria to set up a subtractive cDNA library of specially or highly expressed genes in strains subclonal 48A9 cell line. Results mRNA were directly extracted and purified with good quality. Double strand cDNA were reverse transcripted integratedly, and then cut by Rsa I into even length short segments. Liga-tion was identified as high effective. After two hybridizations, a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was successfully constructed by SSH. Conclusion SSH is an effective approach to isolate differentially expressed genes.

Screening of proteins binding to hepatitis C virus E1 protein from human pancreas cDNA library / 中华传染病杂志

Objective To screen proteins from human pancreas cDNA library,which interact with hepatitis C virus(HCV)E1 protein.Methods The human pancreas cDNA library was amplified,purified and evaluated,and then the purified library plasmids were transformed into yeast strain Y187.The reconstructed plasmid pGBKT7-E1 was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp.The transformed AH109 mated with Y187 that contained the library plasmids.The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-α-gal for selecting.The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5α.The plasmids in DH5α were extracted,sequenced and blasted.Result Sixteen proteins interacting with HCV E1 were found.Conclusion Some of the sixteen pancreatic proteins may be related with metabolisms of glucose and lipid.

Construction of a cDNA library from Agkistrodon acutus venom gland and identification of Agkihagin, a novel transcript for metalloproteinase / 中国药理学与毒理学杂志

AIM To construct a non-normalized cDNA library from Agkistrodon acutus venom gland as an imtial step to develop new and more effective venom by genetic engineering technique for screening and expressing target genes. METHODS The total RNA was extracted from fresh venom gland using Trizol. mRNA was reversely transcripted to cDNA using superscriptⅡ reverse transcriptase. Second-strand synthesis was performed using DNA polymeraseⅠ. After adding EcoRⅠ adaptor, phosphorylating the end and digesting with XhoⅠ, the cDNA was collected in five fractions (<0.25 kb, 0.25-0.5 kb, 0.5-1 kb, 1-2 kb and >2 kb) using the QIAquick Gel Extraction kit and ligated to pBluescriptⅡ vectors. The five libraries obtained were plated by infecting E.coli DH10B, constructing a cDNA library of Agkistrodon acutus venom gland. Sequencing clones at random, 8696 high quality 5′ end expressed sequenced tags (ESTs) were obtained and analyzed. The initial sequences were assembled into 2855 clusters. Among which, one of the clusters (Agkihagin) consisting of 74 ESTs was identified as a novel metalloprtoteinase based on RT-PCR and sequence analysis. RESULTSThe titers of library were 2.048×106. The novel metalloproteinase belonged to PⅢ type metalloproteinase. Its open reading frame was composed of 1827 nucleotides and coded a pre-zymogen of 608 amino acid with zinc-binding domain for metalloproteinase and Asp-Glu-Cys-Asp(DECD) domain for disintegrin. CONCLUSION The capacity of cDNA library of venom gland is above the general level of cDNA library. It would be a helpful platform to construct a catalog for transcripts in the venom gland of the Agkistrodon acutus. The sequence analysis indicates that the deduced amino acid sequence of the identified gene for metalloproteinase share the highest 87% identity with the metalloproteinase genes of other snakes in the GenBank. It lays a good foundation for the study of structure-function relationships of snake venom metalloproteinases.

Identification of proteins interacting with androgen receptor- associated coregulator 267-?(ARA267-?) with the yeast two-hybrid system / 北京大学学报(医学版)

Objective:To search for proteins interacting with ARA267-? with the yeast two-hybrid system in order to further investigate the function of ARA267-?. Methods:We screened a pretransformed human brain cDNA library with the pGBKT7-PHD-SET recombinant plasmid as a bait which express four PHD(plant homeodomain) and one SET[Su(var)3-9, Enhancer-of-zeste, Trithorax] conserved domains in ARA267-?.The plasmids in positive yeast clones were selectively identified by restriction analysis and DNA sequencing. The interactions were retested by yeast two-hybrid assay. Results: There were about six hundreds positive yeast clones on SD/-Ade/-His/-Leu/-Trp/2.5 mmol/L 3-AT/ X-?-Gal high-stringency selection plates. The pACT2-cDNA plasmids in sixty-five yeast clones were isolated and thirty-five cDNA inserts were sequenced. Sixteen different genes,including DR6(death receptor-6), PIAS3 (protein inhibitor of activated STAT3)and RanBPM(Ran-binding protein in the microtubule-organizing center), were identified after BLAST in GenBank. The yeast two-hybrid retest showed that all but RanBPM were true interactors of ARA267-?-PHD-SET. Conclusion: The ARA267-?-PHD-SET can interact with several distinct proteins. This suggests that ARA267-? is a protein having multiple functions. RanBPM might be a transcriptional factor.

Identification of Differentially Expressed Genes in Anagen Dermal Papilla by Suppression Subtractive Hybridization / 中华皮肤科杂志

Objectives To construct a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) and clone differentially expressed genes related to DPCs in anagen. Methods Total RNA was isolated from DPC of anagen and telogen follicles. Then ds cDNAs were synthesized in turn using SMART cDNA synthesis technique. After cDNAs from anagen and telogen follicle DPCs were hybridized with each other twice and underwent two rounds of nested PCR, PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were verified by reverse Nothern blot and DNA sequencing, and the acquired sequences were analyzed for homology based on Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicle was set up successfully with high subtractive efficiency. Thirty-five genes were identified with 22 known functional genes and 13 unknown functional genes. Conclusions These results demonstrate the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression might be useful for elucidating the genetic events in hair follicle growth regulation.

Analysis and Screening of Differentially Expressed Genes of Dermal Papillae Cells with Aggregative Behavior / 中华皮肤科杂志

Objective To screen and analyze genes differentially expressed within dermal papillae cells(DPC)with aggregative behavior.Methods Total RNA was extracted from DPC with and without ag-gregative behavior,and double-stranded cDNA were synthesized by using SMART cDNA synthesis.The cD-NA fragments of differentially expressed genes in dermal papillae cells with aggregative behavior were isolat-ed by suppression subtractive hybridization,sequencing,and then subtracted library was set up.Positive clones were screened by PCR method and verified by cDNA dot blot and then analyzed through homologous retrieving.Results A subtractive cDNA library of DPC with aggregative behavior was successfully construct-ed.The results of screening and cloning of the library showed that DPC with aggregative behavior could ex-press genes related to homologous aggregation,regnlation of growth,differentiation and development,and sig-nal transduction proliferation and cycle control,which included known genes(capping protein,paladin,vas-cular endothelial growth factor),hematopoietic stem/progenitor cells(HSPC)related genes(HSPC011and HSPC016)and a new gene.Conclusions The construction of subtractive library of DPC lays solid founda-tion for screening and cloning new and specific genes related to aggregative behavior of DPC.Several genes may cooperatively involve in homologous aggregation,and regnlation of growth of DPC.Among these genes,capping protein and palladin may be closely related to aggregative behavior of DPC,and VEGF and HSPC re-lated clones may be responsible for the status of higher proliferation of DPC.

Construction and Analysis of cDNA Expression Library from the Leaves of Dendrobium candidum Wall. ex Lindl / 广州中医药大学学报

[Objective] cDNA expression libraries from the leaves of Dendrobium candidum Wall. ex lindl. were constructed to supply evidence for the clones of the full-length genes involved in the biosynthesis of bioactive compounds. [Methods] Total RNA was isolated and purified from tender leaves of 4-year-growing Dendrobium candidum by using Trizol single-step method. cDNA was synthesized by long distance polymerase chain reaction (LD-PCR) and then was connected to ?TripIEX2. After that, the recombinant bacteriophages were packaged and cDNA library of Dendrobium candidum was constructed. The titer of cDNA library was expressed as the number of phage formation unit (pfu) per milliliter and the inserted fragment was identified by PCR amphfication. [Results] cDNA expression libraries from the leaves of Dendrobium candidum were constructed successfully. The titer of the original library was 4.3?105 pfu/mL and 3.1?105 clones in total, with a recombinant rate of 97.6%. The amplified titer was 6.8?109pfu/mL. PCR amplification suggested that the inserted cDNA fragments ranged from 0.5 to 2.0 kb, mostly from 1.2 to 1.7 kb. [Conclusion] The constructed Dendrobium candidum cDNA library has a higher titer, a higher recombinant percentage and larger inserted fragments.

Substractive cDNA library construction of genes sensitive to mechanical stretch in human osteoblast like cells / 实用口腔医学杂志

Objective: To reconstruct a substractive complementary deoxyribornucleic acid (cDNA) library of genes sensitive to mechanical stretch in human osteoblast like cells.Methods: Mechanical stretch at 12 cycles per minute was applied to human osteoblast like cells Saos-2 and the deformation of the stretched cells was 12%. Twelve hours after loading, mRNAs were isolated from both stretched and unstretched cells. Substractive cDNA library of the genes sensitive to stretch was constructed with the technique based on polymerase chain reaction (PCR) and substractive hybridization. Primary sequencing of clones in the library was carried out. Results: A substractive cDNA library of genes sensitive to stretch was constructed with a capacity of about 200 clones. According to the results of sequencing, most genes in the library were related to the mechanical stimulation. One novel gene fragment was obtained. Conclusion: The method used in the experiment is effective in cloning genes sensitive to mechanical stretch.

Screening and sero-immunoscreening of ovarian epithelial cancer associative antigens / 中华妇产科杂志

Objective To explore epithelial ovarian cancer(EOC)antigens that are potentially useful for cancer early detection and therapy.Methods A high quality cDNA library derived from ascites tumor cells of EOC patients(3 cases of serous EOC,1 case of mucinous EOC,and 1 case of endometrial carcinoma of ovary)was constructed,and the method of combining serological analysis of recombinant cDNA expression libraries(SEREX)and suppression subtractive hybridization(SSH)was used for screening cDNA library.All of the positive clones were sequenced and bioinformatics analysis with BLAST software in GenBank was performed.Serological mini-arrays of recombinant tumor antigens(SMARTA)was used to investigate the prevalence of autoantibodies to these antigens in both 96 ovarian cancer patients and 96 cancer-free controls.Results Fifty-five positive clones encoding different antigenic genes of EOC recognized by IgG and(or)IgM were obtained.It showed that these 55 clones derived from 45 distinct genes and these genes could be grouped into 6 classes as following according to homology with known expressed sequence tag(EST):(1)known ovarian carcinoma related genes:BARD1,et al;(2)homologous genes with other tumors:TM4SF1,et al;(3)homologous genes with special tissues:ILF3,FXR1,et al;(4) homologous genes with special function:TIZ,C1 D,et al;(5)embryo originating genes:PKHD1,et al; (6)novel genes:OV-189,et al.SMARTA results showed that the positive ratio of five EOC antigens TM4SF1(28% vs 9%),CID(21% vs 6%),BARD1(23% vs 5%),FXR1(23% vs 8%),OV-189 (31% vs 13%)which reacting with their IgG autoantibodies,three antigens TIZ(26% vs 8%),FXR1 (28% vs 11%),and OV-189(18% vs 7%)which reacting with their IgM autoantibodies in patients was higher than in controls(P

Construction of cDNA library of hepatitis B virus with X protein C-terminally truncated 40 amino acids by suppression subtractive hybridization method / 第二军医大学学报

Objective:To construct subtractive cDNA library from human hepatocellular carcinoma cells transactivated by C-terminally truncated 40 amino acids using suppression subtractive hybridization(SSH) technique and to clone the associated genes.Methods: Huh-7 cells were separately transfected with pcDNA3(-) harboring the sequence of HBx protein C-terminally truncated 40 amino acids and pcDNA3(-) harboring the full length sequence of HBx protein vectors.The total RNAs were isolated from the transfected Huh-7 cells and were reversely transcripted into double strand cDNAs.After the cDNAs were digested with restriction enzyme RsaⅠ,they were divided into 2 groups and were ligated to the special adaptor 1 and adaptor 2R,respectively.The tester cDNAs were then hybridized with driver cDNAs twice and the products were amplified twice by nested PCR technique.The PCR products were connected with pUCm-T plasmid vectors to establish the subtractive library.Amplification of the library was carried out with E.coli strain JM109.The inserts of cDNAs were sequenced and analyzed in GenBank with Blast search.Results: The subtractive cDNA library was successfully constructed.The amplified library contained 154 positive clones,and colony PCR showed that these clones contained 200-800 bp inserts;some fragments coded proteins involved proto-oncogenes,cell signaling genes,cell growth factor genes,cell apoptosis genes,metabolism and protein synthesis genes.Conclusion: Subtractive cDNA library has been successfully constructed by SSH technique,which may help to clone novel genes transactivated by HBx C-terminally truncated 40 amino acids and to explore the molecular mechanism of hepatoma pathogenesis.

Construction of Anti-CD3 Human/Murine Chimeric Antibody Genes for Expression in Mammalian Cells / 中国肿瘤生物治疗杂志

For construction of anti-CD3 human/murine chimeric antibody genes, a selective first-strand cDNA synthesis from mRNA or RNA of murine McAb HIT3a was performed using murine Ig constant region primers, and then cDNA of heavy and light chain variable domains of murine immunoglobulin were amplified by PCR using a set of degenerated oligonucleotide primers. Using these cDNA fragments as probes, the L and H chain V region exons encoding the murine McAb anti-CD3 were isolated from the gene library of HIT3a DNA and inserted into mammalian expression vectors containing the human ? and yl constant region exons for construction of human/murine chimeric antibody genes.

Cloning and Identifying the Differential Genes in Resistant Neisseria Gonorrhoeae / 中华皮肤科杂志

Objective To clone and study the antimicrobial-resistant gene in Neisseria gonorrhoeae.Methods The gene library,which contains the differential genes of antimicrobial resistant strains and stan-dard reference strains of Neisseria gonorrhoeae,was constructed using a technique known as suppression subtractive hybridization(SSH).Then the antimicrobial resistance associated genes were cloned and ana-lyzed.Results Subtractive gene library in antimicrobial-resistant Neisseria gonorrhoeae was successfully constructed,which contains2500positive clones.Sequence analysis was performed on5clones.The se-quences of these five clones were unknown previously.Conclusions The subtractive DNA library is succes-sively constructed which may provide an important clue for studying the mechanism of antimicrobial resis-tance in Neisseria gonorrhoeae.

A Convenient Method to Select DNA Fragments of Cephalosporium acremonium with Promoter Function / 微生物学通报

A promoter-trap vector pGBT14 for selecting promoters of fungus gene was constructed with E. coli-yeast shuttling plasmid pGBT9. Using this vector, a0. 5-2. 0kb chromosomal DNA library of Cepholosporium acremonium was constructed, and twenty four DNA fragments with promoter function in Saccharomyces oerevisiae Y153 were selected from this DNA library. And the promoter function of these DNA fragments was analyzed.

Construction of Suppression Subtracted cDNA Library of Deltamethrin-resistant Aedes albopictus / 中国寄生虫学与寄生虫病杂志

Objective To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Methods Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. Results The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150 bp to 750 bp. Conclusion The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

Screening of cDNA Clone for Putative RNA Polymerase Subunit of Cysticercus cellulosae / 中国寄生虫学与寄生虫病杂志

Objective To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library. Methods Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT)15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis. Results The amino acid sequence, encoded by the positive clone with a poly (A) 22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%. Conclusion The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

Construction and screening of cDNA library of alveolar macrophages / 解放军医学杂志

Objective To construct cDNA library of alveolar macrophages after lipopolysaccharide (LPS) and dexamethasone (Dex) treatment for exploring the protein molecule interactive with glucocorticoid receptor (GR) in condition of inflammation. Methods After the cultured AMs were treated with LPS (10mg/ml) and Dex (10 -5 mol/L) for 4h, total RNA was extracted from AMs, then the cDNA was synthesized from total RNA of AMs and amplified using primers SMARTⅢ TM and CDSⅢoligo (dT) as the base of recombination. The purified PCR products as well as the linearized plasmid pGADT7-Rec were co-transformed into the competent yeast AH109. They were recombined by yeast homologous recombinase in the yeast cells and became the active cyclic plasmid. The transformed yeasts grew in the SD/-Leu plates. All the growing clones were harvested and then constituted the cDNA library. Furthermore, the bait pGBKT7-rGR transformed the yeast AH109 of library was constructed, and the protein molecule interactive with GR was screened. Results cDNA library of AMs was constructed with high multiplication and good capacity. 1.07?106 recombinants were obtained from the cDNA library. The amplified PCR fragments were between 0.3-1.5kb in size. One true positive clone, obtained by screening the cDNA library, was confirmed to be BAG-1 by sequencing and BLAST. Conclusion The yeast two-hybrid cDNA library of AMs was successfully constructed by Clontech SMART method; GR can interact with BAG-1 in yeast cells.

Screening of binding protein of hepatitis C virus NS4A by phage display from human liver cDNA library / 解放军医学杂志

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.