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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (5): 498-503    DOI: 10.3785/j.issn.1008-9292.2013.05.004
    
CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer
GONG Ting1, LI Hong-Li2, BA Yi2
1.Department of Oncology,Tianjin General Hospital,Tianjin 300052,China;2.Department of gastrointestinal Oncology,Tianjin Cancer Hospital,Tianjin 300060,China
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Abstract  

Objective: Toinvestigate the anti-tumor activity of CCL21-exCD40Leukaryotic expression vector.
Methods: CCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4 ,and then was cloned into expression vector pcDNA3.1+.pcDNA3.1+/CCL21 and pcDNA3.1+/exCD were constructed as negative control.Wsestern blot was used to identify the fusion protein.CHO cells was transfected with pcDNA3.1+/CCL21-exCD,pcDNA3.1+/CCL21 and pcDNA3.1+,respectively.The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.
Results: Gene sequencing and restrictive digestion proved the successful construction of pcDNA3.1+/CCL21-exCD40L,and its expression was conformed by western blot.The transfectant supernantes of pcDNA3.1+/CCL21-exCD40 group had a significant chmotactic function to DCs,of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field.After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse,the tumor mass reduced remarkablely,and all the mouse in fusion gene group survived after 4 weeks.
Conclusion: CCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time,which is more effective than all controlgroup.



Key wordsChemokine CCL21      CD40 ligand      Recombinant fusion proteins      Gene therapy      Colonic neoplasms      Disease models, Animal     
Received: 15 November 2012      Published: 25 September 2013
CLC:  R 730.51  
Cite this article:

GONG Ting, LI Hong-Li, BA Yi. CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer. Journal of ZheJiang University(Medical Science), 2013, 42(5): 498-503.

URL:

https://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.05.004     OR     https://www.zjujournals.com/med/Y2013/V42/I5/498


CCL21-CD40L融合蛋白的肿瘤免疫实验研究

目的:探讨CCL21-exCD40L融合基因的抗肿瘤作用。
方法:构建CCL21-exCD40L真核表达载体,Western blot法检测CCL21-exCD40L融合蛋白的表达,Transwell法检测CCL21-exCD40L融合基因趋化活性,体外转染CCL21-exCD40L融合基因,观察其在小鼠结肠癌肿瘤模型中的抗肿瘤作用。
结果:成功构建真核表达载体pcDNA3.1+/CCL21-exCD40L,Transwell法验证融合蛋白对树突细胞具有较强趋化功能,其每高倍镜视野穿膜细胞数是空载体对照组的14.95倍。将融合基因原位转染肿瘤局部,荷瘤小鼠全部存活,肿瘤体积缩小。
结论:CCL21-exCD40L融合基因能通过真核细胞正常表达,CCL21-exCD40L融合蛋白能够趋化树突细胞,在体内发挥抗肿瘤作用。


关键词: 趋化因子CCL21,  CD40配体,  重组融合蛋白质类,  基因疗法,  结肠肿瘤,  疾病模型, 动物 
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