ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2013 5 29 5 475 ~ 481 T7 cdna 1 1 2 * 1 1 1 071002 2 Western University of Health Sciences California 91766 USA C cdna ORF PCR T7Select10-3b MCS cdna 3' 6 cdna cdna ORF 6 % 70 % cdna T7 Q78 Construction of Lung Cancer cdna T7 Phage Library Using Modified Polyhistidine Tagged Expression Vector DONG Xiao-Min 1 1 ZHONG Li 2 * FAN Jiang-Ping 1 ZHANG Xu-Fei 1 1 Laboratory of Biochip School of Life Sciences Hebei University Baoding 2 Western University of Health Sciences California 91766 USA 071002 China Abstract The commercially available cdna T7 phage library is constructed using C-terminal display mechanism which contains inadequate open reading frame ORF expressed protein epitopes To improve the selection of ORF proteins we modified the existing phage vector T7Select10-3b by inserting 6 His-tag genes into the multiple cloning sites MCS at the 3' terminus by PCR The obtained phage clone expressing His-tag was used for construction of a display library The cdna was extracted from the human lung cancer tissues and inserted into the His-tag modified vector The packaged phages with ORF inserts were enriched by Ni-chelating affinity chromatography and then screened by chemiluminescence immunoassay The result showed that the 6 His-tag allowed the enrichment of expressed peptides with an increased from 6 % to 70 % as compared to unselected the library Our His-taged phage library provided a convenient and practical method to improve the ORF selection Key words His-tag T7 phage library open reading frame ORF cdna 1 160 2 2013-02-04 2013-04-03 T7 cdna No 81272444 No 81071795 * Tel 0312-5079364 E-mail lee_z@ yahoo com Received February 4 2013 Accepted April 3 2013 Supported by National Natural Science Foundation of China No 81272444 No 81071795 3 4 * Corresponding author E-mail lee_z@ yahoo com Tel 0312-5079364
476 29 cdna 5 DNA PCR Xho I Sac I C cdna 6 T7 cdna 1 3 10% open T7 E coli BLT5615 reading frame ORF PCR 7 8 T7 1 2 2 6 His T7Select10-3b His ORF DNA cdna ORF Sal I Xho I 3' His T7 ORF 1 1 T7Select10-3b 1 1 1 10 μl 990 μl LB 1 100 100 μl 900 E coli BLT5615 T7Select μl LB 10 11 10-3b Novagen 12 RNeasy Mini 5% TBST Kit Oligotex Direct mrna Mini Kit QIAGEN TBST 10 4 HRP Orient Express Random Primer cdna Synthesis Kit EcoR I / Hind III End Modification Kit DNA Ligation Kit T7Select10-3b Cloning Kit Novagen Taq DNA β-agarase IPTG TaKaRa MiniBEST Agarose Gel DNA 1 3 1 RNA mrna Extraction Kit TaKaRa DNA QIAGEN RNeasy Mini HRP His Kit RNA RNA 6 His-Tagged Protein Purification Kit Oligotex Direct mrna Mini Kit mrna 10 μl RNase-free Water 1 3 2 cdna Orient 1 2 6 His T7Select10-3b Express Random Primer cdna Synthesis Kit 1 2 1 6 His 10 cdna T7Select10-3b DNA Directional EcoR Ⅰ / Hind Ⅲ Linkers P1 5'-CCCGAGCTC AAGCTTCATCATCATCATCATCATGTCGACGAGATT CTACAAGC-3' P2 5'- CCGCTCGAGTACCGAAGG Hind Ⅲ EcoR Ⅰ Mini Column Fractionation Kit cdna AGTCGTGAATCAGT-3' PCR PCR 94 5 min 94 1 min 66 1 min 72 1 min 32 72 10 min His ECL 1 3 T7 cdna RNA RNA 500 μg 1 3 3 DNA EcoRⅠ / HindⅢ 1% EcoRⅠ End Modification Kit HindⅢ
5 T7 cdna 477 cdna 3 1 16 2 1 4 ORF ORF 2 1 6 His P1 P2 T7Select10-3b DNA PCR 0 75 kb 6 10 mmol / L His DNA T7Select10-3b DNA Sac I Xho I 20 44 500 mmol / L kb 15 78 kb DNA Fig 1 PCR cdna Novagen T7Select PCR 6 ORF His T7Select10-3b Fig 1 Electrophoresis analysis of the modified T7 vector A M1 DL2000 DNA marker 1 Inserts of His-tag synthesized by PCR appeared at the size of 0 75 kb B M2 DNA ladder 2 Liner T7Select10-3b DNA that was digested by Sac I and Xho I appeared at 20 44 kb and 15 78 kb 2 2 5 6 μg mrna A 260 / A 280 6 His 2 02 1% mrna T7Select10-3b cdna T7Select10-3b Fig 3B 2 4 cdna HRP 1 6 10 6 pfu / ml PCR 1% Fig 2 85% 90% 2 3 RNA mrna 300 bp Fig 4 RNeasy Mini 2 5 cdna ORF RNA A 260 / A 280 1 92RNA 1% ORF 28S 18S rrna Fig 3A 6% ORF 70% Fig 5
478 29 Fig 2 Expression identification of His-tag by immunoblotting A The modified phage vector with Histag right and the negative control without insert left side were plated on BLT5615 bacterial plates at a similar density B Plaques in two plates were lifted onto nitrocellulose membranes respectively The expression of the His-tag in individual phage clones was analyzed by immunoblotting using HRP-conjugated anti His-tag monoclonal antibody and chemiluminescence detection Positive signals indicated that the phage expressed His-tag Fig 3 Electrophoresis analysis of total RNA and mrna A 1 Total RNA was isolated from lung cancer tissue and the 28S and 18S ribosomal RNA appeared clearly B 2 mrna was purified from total RNA and appeared as a smear M DL2000 DNA marker Fig 4 PCR analysis of cdna inserts in lung cancer T7 phage library The library was plated in limiting dilution on LB-Agar / agarose plates A total of 100 plaques were randomly picked followed by PCR amplification using T7SelectUp and T7SelectDown primers The sizes of PCR products were analyzed on agarose gel More than 85% of the clones had cdna inserts approximately 90% of inserts were longer than 300 bp M DL2000 DNA marker 1 ~ 11 PCR products of randomly selected clones
5 T7 cdna 479 3 Fig 5 Analysis of ORF library by immunoblotting The packaged phage cdna library was selected by Nichelating affinity chromatography to generate ORF enriched cdna library The library showed here before A and after B the enrichment was plated on BLT5615 bacterial plates respectively The expression of the His-tag was analyzed by immunoblotting and chemiluminescence detection as before Positive signals indicated that the clones expressed the His-tag with ORF cdna inserts Approximately 70% of phage clones in the ORF library had ORF inserts whereas only < 6% of the clones had ORF inserts in the non-his-tagged library 3 1 ORF T7 1985 Smith ORF 13 cdna ORF 90% T7Select10-3b 14 λ MSC 3' T7 cdna ORF 15 T7 C ORF 16 17 ORF T7Select10-3b DNA 36 kb DNA DNA PCR EDTA PCR PCR 450 bp DNA 750 bp PCR 1 200 bp 470 bp PCR 6 His PCR PCR 3 2 cdna ORF cdna ORF 2004 Faix 8 pg3orf cdna ORF ORF pucmg4-198 ORF 6% 87% 2010 Caberoy 6 T7DNA ORF T7 cdna 10B C cdna cdna ORF ORF 18 ORF cdna ORF ORF 19
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