Construction of Tumor Specific Tubercle Bacillus Antigen Ag85A Gene Lentiviral Vector_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

WANGXiao-yong ¹ , LIAOBin ¹ , YUHai-qing ² , LIUGao-hua ¹ , LIDuo ³ , XIAOLin-lin ⁴ ,  TANGXiao-jun ¹

1. Department of Caridothoracic Surgery Affiliated Hospital of Luzhou Medical College, Luzhou 646000 Sichuan, P. R. China;
2. Laboratory of Molecular Biology, Affiliated Hospital of Luzhou Medical College, Luzhou 646000 Sichuan, P. R. China;
3. Department of Respiratory Medicine, Affiliated Hospital of Luzhou Medical College, Luzhou 646000 Sichuan, P. R. China;
4. Department of Pharmacy Affiliated Hospital of Luzhou Medical College, Luzhou 646000 Sichuan, P. R. China;

Corresponding author：

TANGXiao-jun, Email: tangxiaojun73@163.com

Keywords：

Tumor; Immuno-gene therapy; Telomerase catalytic subunit (TERT)promoter; Targeting expression; Tubercle bacillus antigen Ag85A

DOI：

10.7507/1007-4848.20150260

Video：

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Objective To construct tumor specific tubercle bacillus antigen Ag85A gene lentiviral vector driven by murine telomerase catalytic subunit promoter (PmTERT), paving the way for further research in tumor targeting immuno-gene therapy. Methods PmTERT was amplified by PCR method, with murine genomic DNA as template. Then, transcriptional activities of PmTERT in various murine and human cell strains were studied by luciferase assay. Ag85A expression lentiviral vectors driven by cytomegalo virus (CMV) promoter and PmTERT respectively (pLVX-Ag85ACMV and pLVX-Ag85A-PmTERT) were constructed with nucleic acid cloning approach. And above recombinants were verified with DNA sequencing and Western blot. Results Lucifease assay revealed that 331 bp PmTERT cloned in present research had transcriptional activity in murine Lewis lung cancer cells, human lung adenocarcinoma cells A549, and human esophageal cancer cells EC-109, while no transcriptional activity in murine fibroblasts NIH3T3 and human embryo fibroblasts MRC-5. Western blot revealed expression of Ag85A in pLVX-Ag85A-CMV transfected Lewis and NIH3T3 cells, pLVX-Ag85A-PmTERT transfected Lewis cells, no expression in pLVX-Ag85A-PmTERT transfected NIH3T3 cells. Conclusion PmTERT has tumor specific transcriptional activity. Ag85A gene can express selectively in tumor cells, driven by PmTERT.

Citation： WANGXiao-yong, LIAOBin, YUHai-qing, LIUGao-hua, LIDuo, XIAOLin-lin, TANGXiao-jun. Construction of Tumor Specific Tubercle Bacillus Antigen Ag85A Gene Lentiviral Vector. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2015, 22(11): 1050-1055. doi: 10.7507/1007-4848.20150260 Copy

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2.	Blankenstein T,Coulie PG,Gilboa E,et al.Viewpoint:The determinants of tumour immunogenicity.Nature Reviews Cancer,2012,12:307-313.
3.	Shaw DM,Connolly NB,Patel PM,et al.A phase Ⅱ study of a 5T4 oncofoetal antigen tumour-targeted superantigen(ABR-214936)therapy in patients with advanced renal cell carcinoma.Br J Cancer,2007,96(4):567-574.
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5.	舒晓燕,胡静.重组超抗原SEB-scFv融合蛋白的体内抑瘤作用.中华实验外科杂志,2009,26(12):1743-1743.
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9.	范文哲,杨建勇,陈伟,等.人端粒酶启动子调控双自杀基因治疗肝癌的实验观察.中华医学杂志,2011,91(43):3080-3085.
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13.	Li W,Deng G,Li M,et al.A recombinant adenovirus expressing CFP10,ESAT6,Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.Mol Immunol,2014,62(1):86-95.
14.	Zlotta AR,Drowart A,Van Vooren JP,et al.Superficial bladder tumors and increased reactivity against mycobacterial antigens before bacillus Calmette-Guerin therapy.J Urol,1998,159(6):1885-1891.
15.	Tarrant JP,Walsh MJ,Blanchard MC,et al.Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A.Int J Oncol,2004,25(6):1693-1699.
16.	Lee CF,Chang SY,Hsieh DS,et al.Immunotherapy for bladder cancer using recombinant bacillus Calmette-Guerin DNA vaccines and interleukin-12 DNA vaccine.J Urol,2004,171(3):1343-1347.
17.	Ma SH,Chen GG,Yi PJ,et al.Therapeutic effect of alpha-fetoprotein promoter-mediated tBid and Chemotherapeutic agents on orthotopic liver tumor in mice.Gene Ther,2010,17(7):905-912.
18.	孙运良,徐灿,苏长青,等.CEA启动子驱动下表达Hsp70基因的重组腺病毒及鉴定.中华胰腺病杂志,2011,11(4):251-254.
19.	Du YF,Shi Y,Xing YF,et al.Establishment of CXCR4-small interfering RNA retrovirus vector driven by human prostatespecific antigen promoter and its biological effects on prostate cancer in vitro and in vivo.J Cancer Res Clin Oncol,2008,134(11):1255-1264.
20.	Helder MN,Wisman GB,Van der Zee GJ.Teloerase and telomeres:from basic biology to cancer treatment.Cancer Invest,2002,20(1):82-101.
21.	Gu J,Fang B.Telomerase promoter-driven cancer gene therapy.Cancer Biol Ther,2003,2(4 Suppl 1):S64-S70.
22.	Fujiwara T,Urata Y,Tanaka N.Telomerase-specific oncolytic virotherapy for human cancer with the hTERT promoter.Curr Cancer Drug Targets,2007,7(2):191-201.
23.	唐小军,王艳萍,周清华,等.人端粒酶催化亚单位(hTERT)启动子的克隆及其在人肺癌细胞中转录活性的研究.中国肺癌杂志,2004,7(6):475-479.
24.	王艳萍,唐小军,周清华,等.人端粒酶催化亚单位启动子调控自杀基因HSV-TK靶向性表达治疗肺癌的体内外实验研究.四川大学学报(医学版),2008,39(5):701-705.
25.	Si SY,Song SJ,Zhang JZ,et al.Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.Oncol Rep,2011,26(2):377-382.
26.	Arendt ML,Nasir L,Morgan IM.The human and canine TERT promoter function equivalently in human and canine cell.Vet Comp Oncol,2010,8(4):310-316.

1. Loisel-Meyer S,Foey R,Medin JA.Immuno-gene therapy approaches for cancer:from in vitro studies to clinical trials.Front Biosci,2008,13:3202-3214.
2. Blankenstein T,Coulie PG,Gilboa E,et al.Viewpoint:The determinants of tumour immunogenicity.Nature Reviews Cancer,2012,12:307-313.
3. Shaw DM,Connolly NB,Patel PM,et al.A phase Ⅱ study of a 5T4 oncofoetal antigen tumour-targeted superantigen(ABR-214936)therapy in patients with advanced renal cell carcinoma.Br J Cancer,2007,96(4):567-574.
4. Terman DS,Bohach G,Vandenesch F,et al.Staphylococcal superantigens of the enterotoxin gene cluster(egc)for treatment of stage Ⅲb non-small cell lung cancer with pleural effusion.Clin Chest Med,2006,27(2):321-334.
5. 舒晓燕,胡静.重组超抗原SEB-scFv融合蛋白的体内抑瘤作用.中华实验外科杂志,2009,26(12):1743-1743.
6. Liu XY.Targeting Gene-virotherapy of cancer and its prosperity.Cell Res,2006,16(11):879-886.
7. Liu L,Wu W,Zhu G,et al.Therapeutic efficacy of an hTERT promoter-driven oncolytic adnovirus that expresses apoptin in gastric carcinoma.Int J Mol Med,2012,30(4):747-754.
8. Kim YC,Kim BG,Lee JH.Thymosin β10 expression driven by the human TERT promoter induces ovarian cancer-specific apoptosis through ROS production.PLoS One,2012,7(5):e35399.
9. 范文哲,杨建勇,陈伟,等.人端粒酶启动子调控双自杀基因治疗肝癌的实验观察.中华医学杂志,2011,91(43):3080-3085.
10. Belisle JT,Vissa VD,Sievert T,et al.Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.Science,1997,276(5317):1420-1422.
11. 李岩,王大南,杨芳莉,等.Ag85A-CD226 DNA疫苗的制备及对小鼠免疫功能的影响.中华微生物学和免疫学杂志,2014,(3):235-240.
12. Meyer J,Harris SA,Satti I,et al.Comparing the safety and immunogenicity of a candidate TB vaccine MVA85A administered by intramuscular and intradermal delivery.Vaccine,2013,31(7):1026-1033.
13. Li W,Deng G,Li M,et al.A recombinant adenovirus expressing CFP10,ESAT6,Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.Mol Immunol,2014,62(1):86-95.
14. Zlotta AR,Drowart A,Van Vooren JP,et al.Superficial bladder tumors and increased reactivity against mycobacterial antigens before bacillus Calmette-Guerin therapy.J Urol,1998,159(6):1885-1891.
15. Tarrant JP,Walsh MJ,Blanchard MC,et al.Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A.Int J Oncol,2004,25(6):1693-1699.
16. Lee CF,Chang SY,Hsieh DS,et al.Immunotherapy for bladder cancer using recombinant bacillus Calmette-Guerin DNA vaccines and interleukin-12 DNA vaccine.J Urol,2004,171(3):1343-1347.
17. Ma SH,Chen GG,Yi PJ,et al.Therapeutic effect of alpha-fetoprotein promoter-mediated tBid and Chemotherapeutic agents on orthotopic liver tumor in mice.Gene Ther,2010,17(7):905-912.
18. 孙运良,徐灿,苏长青,等.CEA启动子驱动下表达Hsp70基因的重组腺病毒及鉴定.中华胰腺病杂志,2011,11(4):251-254.
19. Du YF,Shi Y,Xing YF,et al.Establishment of CXCR4-small interfering RNA retrovirus vector driven by human prostatespecific antigen promoter and its biological effects on prostate cancer in vitro and in vivo.J Cancer Res Clin Oncol,2008,134(11):1255-1264.
20. Helder MN,Wisman GB,Van der Zee GJ.Teloerase and telomeres:from basic biology to cancer treatment.Cancer Invest,2002,20(1):82-101.
21. Gu J,Fang B.Telomerase promoter-driven cancer gene therapy.Cancer Biol Ther,2003,2(4 Suppl 1):S64-S70.
22. Fujiwara T,Urata Y,Tanaka N.Telomerase-specific oncolytic virotherapy for human cancer with the hTERT promoter.Curr Cancer Drug Targets,2007,7(2):191-201.
23. 唐小军,王艳萍,周清华,等.人端粒酶催化亚单位(hTERT)启动子的克隆及其在人肺癌细胞中转录活性的研究.中国肺癌杂志,2004,7(6):475-479.
24. 王艳萍,唐小军,周清华,等.人端粒酶催化亚单位启动子调控自杀基因HSV-TK靶向性表达治疗肺癌的体内外实验研究.四川大学学报(医学版),2008,39(5):701-705.
25. Si SY,Song SJ,Zhang JZ,et al.Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.Oncol Rep,2011,26(2):377-382.
26. Arendt ML,Nasir L,Morgan IM.The human and canine TERT promoter function equivalently in human and canine cell.Vet Comp Oncol,2010,8(4):310-316.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Construction of Tumor Specific Tubercle Bacillus Antigen Ag85A Gene Lentiviral Vector

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