GbWRKY1, a novel cotton (Gossypium barbadense) WRKY gene isolated from a bacteriophage full-length cDNA library, is induced by infection with Verticillium dahliae.

WRKY transcription factor proteins play important roles in diverse stress responses. In this study, we first cloned a novel WRKY from our constructed bacteriophage full-length cDNA library for cotton (Gossypium barbadense). The plants were stressed by exposure to a defoliating strain of Verticillium dahliae. The capacity of primary cDNA library was 1.28 × 106 PFU and the titer of the amplified cDNA library was >1010 PFU mL–1. The recombination rate of the library was 94% and average insert size was about 1.1 kb. This novel gene, named GbWRKY1 was 1971 bp long and encodes a protein of 489 amino acids. It contains two characteristic WRKY domains and two zinc finger motifs. The sub-cellular assay indicated that GbWRKY1–GFP fusion protein was localized in the nucleus. Furthermore, Northern blot analysis showed that expression pattern of GbWRKY1 was similar among tissue types (roots, stems and leaves), but differed between pathogen-infiltrated and Czapek medium-infiltrated (untreated control) plants. Quantitative real-time PCR showed that GbWRKY1 could also be induced by salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic acid (ACC). These findings clearly suggest that as a pathogen-inducible transcription factor GbWRKY1 plays an important role in plant defense responses.

Cloning and screening of cDNA of Psilgramma menephorn allergen / 药物分析学报

Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

Construction of cDNA library of human epithelial ovarian carcinoma tissue / 第三军医大学学报

Objective To construct a cDNA library of human epithelial ovarian carcinoma tissue for screening ovarian carcinoma specific-antigen.Methods The total RNA was separated from human epithelial ovarian carcinoma tissue.The mRNA from total RNA was isolated to synthesize the first and second strand cDNA.The ds-cDNA termini were blunted with pfu DNA polymerase.The blunted cDNAs were added EcoR Ⅰ adaptor and then digested by XhoⅠ.Small cDNA molecules(less than 400 bp) were removed through size fraction.After the cDNAs were ligated into ZAP expression vector,the ligated products were packaged in vitro and the bacteriophage particles infected the host strains XL1-Blue MRF′.Results The efficiency of the primary library was 5.5?10~(6)pfu/ml and the amplified library was 3.0?10~(11)pfu/ml with 96% clones positive.The average length of the inserted fragment was over 1 kb.Conclusion The quality of the constructed human epithelial ovarian carcinoma tissue cDNA library is excellent and helpful to screen ovarian carcinoma specific-antigen.

Construction of cDNA Library of Ovarian Carcinoma / 中国肿瘤生物治疗杂志

We isolated polyA~+ mRNA direcdy from tumor tissue of ovarian carcinoma using Oligotex~(TM) Direct mRNA Kit (QIAGEN) to synthesize the first and second strand cDNA. The ds-cDNA termini were blunted with pfu DNA poly-rnerase. The blunted cDNAs were added EcoR I adaptor, and then were digested by Xho I . Small cDNA molecules(

Construction of cDNA Expression Library from Lung Cancer Cells / 中国肿瘤生物治疗杂志

Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.

Construction of cDNA Expression Library of Unfed Female Haemaphysalis longicornis and Immuno-Screening / 中国寄生虫学与寄生虫病杂志

Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

Search colorectal cancer antigen genes by screening the colorectal tissue cDNA phage expression libraries with SEREX / 解放军医学杂志

Objective Identification of colorectal cancer specific antigens from cDNA phage expression library by recombinant expression cloning (SEREX). Methods The cDNA phage expression library derived from colorectal cancer tissue was constructed using the SMART (Switching Mechanism at 5'end of RNA Transcript) techniques. The cDNA phage expression library was screened with SEREX (serological identification of antigen by recombinant cDNA expression libraries), the positive clones encoding antigenic genes were obtained after immunoscreening, and the nucleotide sequences of cDNA inserts were determined and analyzed with BLST software in GenBank, and the antigenic gene function was analyzed by bioinformatics. Results The cDNA phage expression library derived from colorectal cancer tissues was successfully constructed. The primary library consisted of 2.39?10 6 recombinants, and the recombinant rate was more than 97.5%. The titer of the amplified cDNA phage expression library was 4.1?10 10pfu/ml, and the size of inserted cDNA varied from 0.5 to 4.0kb. Sixteen positive clones encoding antigenic genes were obtained after immunoscreening, and results showed that 16 reactive clones were derived from 12 different genes. Ten of 12 genes were highly homologous to the genes known in GenBank, such as IFITM1, CD24, Survivin, KLK6, et al. Two expressed sequence tags (ESTs) were not found in GenBank. The antigenic genes included structural gene, regulation gene and metabolizing gene. Conclusion The tumor-associated antigen genes and the two ESTs were obtained by SEREX were worthy of further study on their structures and functions.

Construction and primary characterization of cDNA expression library of psilgramma menephorn / 西安交通大学学报(医学版)

Objective To construct a cDNA expression library of psilgramma menephorn and provide the basis for screening the major allergen and producing recombination allergen of psilgramma menephorn. Methods Total RNA was extracted from the whole body of psilgramma menephorn with TRIZOL mRNA purified with Oligo (dT) Spin-Column. ds cDNA was synthesized through reverse transcription. EcoRI adapters were ligated to the blunt ends and the ends were phosphorylated. The XhoI digestion released EcoRI adapter. The fragments smaller than 400 bp were removed by GHROMA SPIN-400 column and the fragments longer than 400 bp were ligated to the Uni-ZAP XR vector. The lambda library was packaged in vitro and is plated on the E.coli cell line XL1-Blue MRF. The content and recombination rate of cDNA library were tested. The length of the inserted fragments was analyzed by PCR. Results The cDNA expression library contained 5?10 5 recombinants and the recombination rate was 67%. The average length of inserted cDNA fragments was about 1.49 kb. Conclusion The constructed cDNA expression library contains appropriate contents and size of cDNA fragments for screening target cDNA to clone.