中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (7): 1227-1230.doi: 10.3969/j.issn.1673-8225.2010.07.020

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

新型骨形态发生蛋白2反转录病毒载体的构建及活性检测

廖  青1，唐  颖2，赤仁杰3，陈晓春3，关景玉3，段有文3　   

  1. 1大庆油田总医院，黑龙江省大庆市  163001；2大庆龙南医院，黑龙江省大庆市  163301；3沈阳医学院附属奉天医院，辽宁省沈阳市 110024
  • 出版日期:2010-02-12 发布日期:2010-02-12
  • 作者简介:廖 青，女，1957年生，吉林省白城市人，汉族，1998年大庆职工医学院毕业，副主任医师，主要从事心血管疾病研究。 renjiechi@yahoo.com.cn

Construction and activity identification of recombinant retroviral vector expressing bone morphogenetic protein 2 gene

Liao Qing1, Tang Ying2, Chi Ren-jie3, Chen Xiao-chun3, Guan Jing-yu3, Duan You-wen3　   

  1. 1 Daqing Oilfield General Hospital, Daqing  163001, Helongjiang Province, China; 2 Longnan Hospital, Daqing  163301, Heilongjiang Province, China; 3 Fengtian Hospital of Shenyang Medical College, Shenyang 110024, Liaoning Province, China
  • Online:2010-02-12 Published:2010-02-12
  • About author:Liao Qing, Associate chief physician, Daqing Oilfield General Hospital, Daqing 163001, Helongjiang Province, China renjiechi@yahoo.com.cn

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421

被引次数

3

摘要：

背景：骨形态发生蛋白是一种具有潜在活性的蛋白质，当骨组织损伤时其迅速增加并活性的增强，与载体复合能修复动物骨缺损，但将其用做基因治疗的研究未见报道。
目的：构建表达重组人骨形态发生蛋白2基因的重组反转录病毒载体，探讨其在成骨细胞中的生物学作用。
方法：根据Genbank中人骨形态发生蛋白2基因序列设计并合成骨形态发生蛋白2特异性引物，高保真PCR扩增骨形态发生蛋白2基因，同源重组法将骨形态发生蛋白2 PCR片段连接与克隆载体pDNR-CMV，构成pDNR-CMV-BMP2，经酶切、PCR和测序鉴定后，将重组质粒pDNR-CMV-BMP2和反转录病毒空质粒pLP-LNCX以loxP位点进行同源重组，构成反转录病毒载体pLP-LNCX-BMP2，转染入包装细胞PT67进行病毒包装，并用NIH3T3细胞进行病毒滴度测定；将反转录病毒感染人成骨细胞，四甲基偶氮唑盐法检测细胞生长变化，转染48 h后Western blotting检测骨形态发生蛋白2蛋白表达。
结果与结论：pDNR-CMV-BMP2质粒SalI和EcoRI双酶切、PCR及测序结果均正确，重组质粒pLP-LNCX-BMP2经氯霉素及蔗糖筛选得到的阳性克隆骨形态发生蛋白2 PCR结果阳性，酶切产物与预期相一致；病毒载体pLP-LNCX-BMP2转染PT67后，G418筛选可得到稳定细胞克隆，其上清液中病毒滴度可达到5×108pfu；四甲基偶氮唑盐检测中反转录病毒组与正常对照组相比，72 h细胞抑制率无明显差别(P > 0.05)，转染48 h后Western blotting可见骨形态发生蛋白2蛋白高表达。结果说明，实验成功克隆了骨形态发生蛋白2基因并构建其反转录病毒表达载体。

关键词: 骨形态发生蛋白2基因, 反转录病毒, 成骨细胞, 四甲基偶氮唑盐, 蛋白表达

Abstract:

BACKGROUND: Bone morphogenetic protein (BMP) is a protein possesses potential activity, which can increase and enhance its activity when the bone issues are damaged, so it can be used to repair the bone defects when combined with carrier. However, there are few reports concerning it as gene therapy.
OBJECTIVE: To construct recombinant retroviral vector expressing human BMP2 gene and to discuss its biological function in osteoblasts.
METHODS: BMP2-specific primers were designed and synthesized according gene sequence of human BMP2 gene in Genbank，then BMP2 gene was amplified by Hifi PCR, which was recombined with cloning vector pDNR-CMV homologously into pDNR-CMV-BMP2 plasmid identified by BMP2 PCR and enzyme digestion of SalI and EcoRI as well as gene sequencing; recombinant plasmid pDNR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombined homologously in loxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67 and the supernatant was collected to assay viral titre. Human osteoblast was infected with retrovirus, then the growth of cells were observed by MTT, and the expression of BMP2 protein was detected by Western blotting at 48 hours transfection
RESULTS AND CONCLUSION: Digestion, BMP2 PCR and gene sequencing of pDNR-CMV-BMP2 were coincided with expected. After transfected with plasmid pLP-LNCX-BMP2, PT67 cells could be screened with G418 only to get stably integrated in BMP2, of whose supernanant viral titre amounted to 5×108 pfu/mL. MTT assay showed that there had no evident difference in cellular inhibition between the normal and retrovirus groups at 72 hours after transfection (P > 0.05); Western blotting showed that the BMP2 was strong expressed at 48 hours after transfection. It demonstrated that BMP2 gene was successful cloned and its retrovirus vector was constructed.  

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