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

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1 R PCR p215c3lyz R pqe-31 R IPTG Western blotting pqe- p215c3lyz COS- DH5α OD 600 Western blotting p215c3lyz Western blotting R R S A 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 China 2. Shanghai Jiaotong University Shanghai China 3. Shanghai Bright Holstein Co. LTD Shanghai 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 BE 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 Suppl 1 S24-S33. 9 Spradling A Drummodn Barrosa D Kai T. Stem cells find their Niche J. Nature RoseM T Aso H Yonekura S et al. In vitro differentiation of a cloned bovine mammary epithelial cell J. Journal of Dairy Research 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

2 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 ICR PCR R PCR 2 5'-CTGGATCCGAGC- FDA CATATTCAACGGGAAAC-3' 5'-TTGTCGACTTAG AAAAACTCATCG AGC ATC-3' 50 μl PCR 2 5 μl pmol /L dntps 10 pmol / L 100 ng p215c3lyz 1 U Pyrobest DNA Polymerase PCR 94 5 hlyz min s s 72 1 min min PCR 1% Wizard DNA Clean-Up System hlyz cdna p215c3lyz R 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 ml LB 100 μg /ml Amp 100 μg /ml μl 5 ml LB Wizard DNA Clean-up System 100 μg /ml Amp 100 μg /ml Promega DNA 4 37 OD ~ h Taq DNA TaKaRa DH5α M15 1 mmol /L IPTG 37 E. coli h pqe μl PBS 2 pcdnaklyz p215c3lyz 3 PCR SDS-PAGE 5 min

3 % 1. 6 SDS-PAGE 40 μl PBS ICR 50 μg / 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 IgG 800 nm 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 min 800 μl 1% pqe- IPTG FBS DMEM 3 h SDS-PAGE μl 10% FBS DMEM 24 h pqe ku 48 h 500 μl 1. 8 p215c3lyz pqe- COS μl / 50 μl /ml μl DH5α h DMEM OD 600 OD 600 Western blotting p215c3lyz μl μg / ml r /min pqe min 0. 5 ml 55 10% ph r /min 10 min 70% 15 min 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 IPTG pqe-- 2. IPTG pqe IPTG pqe-- 4. IPTG pqe-- 5. pqe31 6. M15 7. DH5α M. Marker 1 pqe- IPTG pqe- p215c3lyz

4 12 Animal Husbandry & Veterinary Medicine 2012 Vol. 44 No. 4 Western blotting pqe- 30 ku IPTG p215c3lyz pqe- p215c3lyz COS-1 24 h 48 h DH5α h OD 600 kan COS-1 pqe- p215c3lyz COS- OD IPTG pqe-- 3. IPTG pqe-- 4. pqe3 5. IPTG p215c3lyc 6. M15 7. DH5α M. Marker Western blotting 1 DH5α OD h 48 h pqe p215c3lyz p215c3lyz pqe- 2 1 mg p215c3lyz 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 IPTG pqe M. Marker 4 Western blotting

5 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 Ertl P F Thomsen L L. Technical issues in construction of nucleic Methods 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 J 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 Adv Drug Deliv Rev r. J

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Microsoft Word - 360145.doc 项目批准号 81273814 归口管理部门申请代码收件日期 H2716 360145 y 国家自然科学基金委员会资助项目计划书资助类别 : 面上项目亚类说明 : 非连续资助类项目附注说明 : 项目名称 : 益气养肺法拮抗 bfgf 对肺癌微环境介导化疗