2012 44 4 9 2 2 3 1. 225009 2. 200240 3. 200072 R PCR p215c3lyz R pqe-31 R IPTG Western blotting pqe- p215c3lyz COS- DH5α OD 600 Western blotting p215c3lyz Western blotting R R S852. 4 A 0529-5130 2012 04-0009-05 Detection of kanamycin resistant gene expression in vector-transfected mammalian cells and milk samples of vector-injected cow WENG Yu-gen 1 JI Yu-hui 1 SUN Huai-chang 1 ZHANG Xin-yu 1 ZHU Jian-guo 2 YI Ming-mei 2 YUAN Yao-ming 3 1. College of Veterinary Medicine Yangzhou University Yangzhou 225009 China 2. Shanghai Jiaotong University Shanghai 200240 China 3. Shanghai Bright Holstein Co. LTD Shanghai 200072 China Abstract To investigate whether the kanamycin resistant R gene in mammary-specific expression vector p215c3lyz for treating bovine mastitis could express active protein in mammalian cells under the control of its own promoter R gene was amplified by PCR using mammary-specific expression vector p215c3lyz as the template and subcloned into prokaryotic expression vector pqe-31. After transformation and cultivation on -containing agar plates -resistant E. coli colonies were obtained and an expected recombinant protein was expressed after IPTG induction. After separation on SDS-PAGE the protein band was excised and specific antiserum was obtained by immunization of mice for 6 times. The specificity of the antiserum was confirmed using Western blotting. The prokaryotic expression vector pqe- and eukaryotic expression vector p215c3lyz both of which contained R gene were transfected into COS-1 cells and the supernatants were collected after cultivation in antibiotic-free medium DH5α E. coli were inoculated into the supernatants in the absence or presence of and the OD 600 values were detected after 24h cultivation. The results showed that growth of the indicator bacterium was inhibited signifi- 2011-09-23 2012-01-03 2005 10-2 BE2004611 1975- 櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔櫔 8 Tarnawski A S. Cellular and molecular mechanisms of gastrointestinal ulcer healing J. Dig Dis Sci 2005 50 Suppl 1 S24-S33. 9 Spradling A Drummodn Barrosa D Kai T. Stem cells find their Niche J. Nature 2001 414 6859 98-104. 11 RoseM T Aso H Yonekura S et al. In vitro differentiation of a cloned bovine mammary epithelial cell J. Journal of Dairy Research 2002 69 345-355. 10 Fleming J M Desury G Polanco T A et al. Insulin growth factor-1 and epidermal growth factor receptors recruit distinct upstream signaling molecules to enhance AKT activation in mammary epithelial cells J. Endocrinology 2006 147 12 6027-6035.
10 Animal Husbandry & Veterinary Medicine 2012 Vol. 44 No. 4 cantly in the presence of indicating that the resistant protein was not expressed in the supernatants of the vector-transfected cells. Western blotting of the cell lysates confirmed that resistant protein was not expressed in the vector-transfected cells. In addition no resistant protein was detected in concentrated milk samples from cows after intramammary injection with vector p215c3lyz. These experimental data demonstrate that the promoter of R p215c3lyz vector for treating bovine mastitis. gene is inactive in mammalian cells providing additional safety evidence using the Key words E. coli kanamycin resistant gene recombinant vector mammalian cell expression cow 1990 Wolff DNA TaKaRa OXOID 1 1. 2 ICR - 1. 3 PCR R PCR 2 5'-CTGGATCCGAGC- FDA CATATTCAACGGGAAAC-3' 5'-TTGTCGACTTAG AAAAACTCATCG AGC ATC-3' 50 μl PCR 2 5 μl 10 10 pmol /L dntps 10 pmol / L 100 ng p215c3lyz 1 U Pyrobest DNA Polymerase PCR 94 5 hlyz min 94 45 s 50 45 s 72 1 min 30 3 72 10 min PCR 1% Wizard DNA Clean-Up System hlyz cdna p215c3lyz R 3 1. 4 R Sal I 4 Bam HI PCR pqe-3 R T4 DNA M15 E. coli p215c3lyz PCR 100 μg /ml 100 μg / R ml Amp LB 37 LB Bam R HI Sal I p215c3lyz 1. 5 10 ml LB 100 μg /ml Amp 100 μg /ml 37 1. 50 μl 5 ml LB Wizard DNA Clean-up System 100 μg /ml Amp 100 μg /ml Promega DNA 4 37 OD 600 0. 4 ~ 0. 6 3 h Taq DNA TaKaRa DH5α M15 1 mmol /L IPTG 37 E. coli 32 30 28 4 h pqe-31 500 μl PBS 2 pcdnaklyz p215c3lyz 3 PCR SDS-PAGE 5 min
2012 44 4 11 10% 1. 6 SDS-PAGE 40 μl PBS ICR 50 μg / 1 6 1. 6 pqe- 10 d p215c3lyz pqe- SDS- 2 PAGE 350 ma /20 min Western blotting Odyssey Infrared Imaging System LI-COR Biosciences Tween20 PBS 37 /2 h 1 1 000 1 10 000 IgG 800 nm 2. 2 1. 7 PCR pqe-31 5 p215c3lyz Bam HI /Sal I M15 E. coli pqe- COS- 35 mm Amp 37 10% FBS DMEM PCR R 37 /5% CO 2 18 h 80% 2 μl 2 μg 100 μl DMEM 2 μl 4 μg PEI 100 μl DMEM 10 min 2. 3 15 min 800 μl 1% pqe- IPTG FBS DMEM 3 h SDS-PAGE 1 000 μl 10% FBS DMEM 24 h pqe-31 30 ku 48 h 500 μl 1. 8 p215c3lyz pqe- COS-1 96 200 μl / 50 μl /ml 2 3 5 μl DH5α 37 24 h DMEM OD 600 OD 600 Western blotting 1. 6 1. 9 p215c3lyz 5 500 μl μg / 1 2 4 8 2. 4 5 1 ml 12 000 r /min pqe- 6 10 min 0. 5 ml 55 10% ph 4. 6 12 000 r /min 10 min 70% 15 min 4 12 000 r /min 20 min Western blotting 2. 1 PCR R 5' 3' R PVDF 0. 1% p215c3lyz 30 PCR 800 bp Bam HI /Sal I 3. 4 kb 0. 8 kb 30 1 1. IPTG pqe-- 2. IPTG pqe- - 3. IPTG pqe-- 4. IPTG pqe-- 5. pqe31 6. M15 7. DH5α M. Marker 1 pqe- IPTG pqe- p215c3lyz
12 Animal Husbandry & Veterinary Medicine 2012 Vol. 44 No. 4 Western blotting pqe- 30 ku IPTG p215c3lyz 2 2. 5 pqe- p215c3lyz COS-1 24 h 48 h DH5α 37 24 h OD 600 kan COS-1 pqe- p215c3lyz COS- OD 600 1 2 1. 2. IPTG pqe-- 3. IPTG pqe-- 4. pqe3 5. IPTG p215c3lyc 6. M15 7. DH5α M. Marker Western blotting 1 DH5α OD 600 24 h 48 h 0. 652 1. 002 0. 603 1. 090 pqe31-0. 681 1. 167 0. 610 1. 102 p215c3lyz 0. 621 1. 178 0. 632 1. 009 2 2. 6 p215c3lyz pqe- 2 1 mg p215c3lyz 2 4 8 1 ml Western blotting 2 IPTG pqe- pqe- Western blotting 3 p215c3lyz pqe- 4 d 4 1. IPTG pqe- 2. IPTG p215c3lyz 3. p215c3lyz COS- 48 h 4. p215c3lyz COS- 24 h 5. pqe- COS- 48 h 6. pqe- COS-1 24 h M. Marker 3 Western blot 1. 2. IPTG pqe- 3. 1409 4. 2092 5. 2103 6. 204 7. 98168 M. Marker 4 Western blotting
2012 44 4 13 3 DNA Western blotting pqe- 6 p215c3lyz R COS-1 FDA p215c3lyz β 1 ml 20 2 R μl rhlyz 3 II aph II npt II apha2 90% R R 7 R R COS- 7 R R R PCR R pqe-31 R Western blotting R R acid vaccines J. pqe- p215c3lyz COS-1 24 h 48 h bovine mastitis J 4 DH5α R 5. R J diseases of farmed animals J R 24 h 48 h OD 600 R pqe- p215c3lyz p215c3lyz 1 Rolland A. Gene medicines The end of the beginning J. Adv Drug Deliv Rev 2005 57 5 669-673. 2 Ertl P F Thomsen L L. Technical issues in construction of nucleic Methods 2003 31 199-206. 3 Sun H C Xue F M Qian K et al. Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating. Zhejiang Uni Sci 2006 7 4 324-330.. J. 2003 23 6 9-12.. 2007 27 2 76-79. 6 van Drunen Littel-van den Hurk S GerdtsV Loehr B I et al. Recent advances in the use of DNA vaccines for the treatment of 1 13-28.. Adv Drug Deliv Rev 2000 43 7. r. J 2001 36 2 18-19.