CAJ | 학술논문

背景:骨形态发生蛋白2是诱导成骨关键物质,参与调节从细胞增殖、决定种系分化方向到细胞死亡等一系列的生物过程. 目的:利用AdMax系统构建人骨形态发生蛋白2基因的重组腺病毒载体并鉴定. 方法:以人cDNA为模板PCR扩增人骨形态发生蛋白2基因,转化E.coli感受态细胞,经菌落PCR鉴定阳性转化子、阳性克隆测序无误后,扩增、抽提.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,PCR检测目的基因、Western Blot检测目的蛋白及终点稀释法检测病毒滴度. 结果与结论:PCR获得长度为1223 bp的人骨形态发生蛋白2目的基因片段,同源重组表达载体经阳性克隆PCR及测序鉴定,结果正确.293细胞内包装、扩增,经Western blot及PCR鉴定无误后,获得病毒滴度为5×1013 pfu/L的重组腺病毒.实验成功构建携带有人骨形态发生蛋白2基因的重组腺病毒载体.
배경:골형태발생단백2시유도성골관건물질,삼여조절종세포증식、결정충계분화방향도세포사망등일계렬적생물과정. 목적:이용AdMax계통구건인골형태발생단백2기인적중조선병독재체병감정. 방법:이인cDNA위모판PCR확증인골형태발생단백2기인,전화E.coli감수태세포,경균락PCR감정양성전화자、양성극륭측서무오후,확증、추제.장대유목적기인적선병독표체재체화휴대유선병독대부분기인적보조포장질립공전염293세포진행병독포장확증,PCR검측목적기인、Western Blot검측목적단백급종점희석법검측병독적도. 결과여결론:PCR획득장도위1223 bp적인골형태발생단백2목적기인편단,동원중조표체재체경양성극륭PCR급측서감정,결과정학.293세포내포장、확증,경Western blot급PCR감정무오후,획득병독적도위5×1013 pfu/L적중조선병독.실험성공구건휴대유인골형태발생단백2기인적중조선병독재체.
BACKGROUND:Bone morphogenetic protein 2 plays a key role in inducing osteogenesis. It involves in a series of bioprocess, including cel proliferation, determining the differentiation direction of germ line and cel 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 cel s 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 cel s for virus packaging and amplification. Final y, target genes were detected by PCR, and target protein was detected by Western blot method, as wel 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 cel s 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 successful y.