中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (50): 9376-9381.doi: 10.3969/j.issn.2095-4344.2012.50.012
• 组织构建实验造模 experimental modeling in tissue construction • 上一篇 下一篇
邱俊钦,尹承慧,曾昭勋,陈宗雄
出版日期:
发布日期:
Qiu Jun-qin, Yin Cheng-hui, Zeng Zhao-xun, Chen Zong-xiong
Online:
Published:
Abstract:
BACKGROUND: Bone morphogenetic protein 2 plays a key role in inducing osteogenesis. It involves in a series of bioprocess, including cell proliferation, determining the differentiation direction of germ line and cell death. OBJECTIVE: To construct and identify the recombinant adenovirus vectors encoding human bone morphogenetic protein 2 gene by using AdMax system. METHODS: First, human bone morphogenetic protein 2 gene sequencing was amplified by PCR from human cDNA template and then cloned. Second, the recombinant shuttle plasmid was constructed and transformed into Escherichia coli competent cells DH5α. After the positive colonies were identified by colonies PCR and sequencing, the expression vectors were amplified and extracted. Next, the adenovirus expression vectors with target gene and the helper packaging plasmid carrying a majority of adenovirus genes were co-transfeced into 293 cells for virus packaging and amplification. Finally, target genes were detected by PCR, and target protein was detected by Western blot method, as well as infectious titer of the recombinant adenovirus was detected by end point dilution method. RESULTS AND CONCLUSION: Gene fragment of a length of 1 223 bp human bone morphogenetic protein 2 was obtained by PCR. The expression vectors constructed by homologous recombination techniques were identified by PCR cloning and sequencing; the results were correct. After virus packaging and amplification in 293 cells were identified by Western blot and PCR methods, the virus titer of recombinant adenovirus was 5×1013 pfu/L. These results suggest that the recombinant adenovirus vectors carrying human bone morphogenetic protein 2 gene have been constructed successfully.
邱俊钦,尹承慧,曾昭勋,陈宗雄. 人骨形态发生蛋白2基因重组腺病毒的构建与鉴定[J]. 中国组织工程研究, 2012, 16(50): 9376-9381.
Qiu Jun-qin, Yin Cheng-hui, Zeng Zhao-xun, Chen Zong-xiong. Construction and identification of recombinant adenovirus vectors encoding human bone morphogenetic protein 2 [J]. Chinese Journal of Tissue Engineering Research, 2012, 16(50): 9376-9381.
0 / / 推荐
导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks
链接本文: https://www.cjter.com/CN/10.3969/j.issn.2095-4344.2012.50.012
https://www.cjter.com/CN/Y2012/V16/I50/9376
于体外构建并鉴定表达人骨形态发生蛋白2基因的重组腺病毒,将带有目的基因的腺病毒表达载体和携带有腺病毒大部分基因的辅助包装质粒共转染293细胞进行病毒包装扩增,成功构建了携带有人骨形态发生蛋白2基因的重组腺病毒载体。
病毒载体包括逆转录病毒、腺病毒、疱疹病毒等,分为整合型和非整合型。整合型病毒载体目前常用有逆转录病毒、慢病毒、单纯疱疹病毒等,因其可能携带基因随机整合到宿主细胞,有诱发肿瘤的可能性,以及免疫原性的问题现已少用。非整合型重组腺病毒载体是目前应用最广泛的一种病毒载体。腺病毒的基因组为线形双链DNA,长度约为36kb,分为编码区和非编码区。目前应用的腺病毒载体主要是在人类腺病毒C亚群的Ad2和Ad5的基础上构建的。