Dmrt7基因重组腺病毒及腺相关病毒载体的构建及鉴定
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摘要
Doublesex和mab-3相关转录因子7(double sex and mab3- relatated transcrip- tion factor7,Dmrt7)在动物体内可表达Dmrt7蛋白。该蛋白是哺乳动物生殖系统的重要蛋白,主要含有DM结构域,其主要生物学功能是调控精子发生、控制性别的分化。应用Dmrt7蛋白在治疗雄性动物产弱精、不产精等方面具有丰富的使用价值和广阔的前景。然而,由于天然Dmrt7蛋白来源稀少,不能满足临床和科学研究的需要,因此利用转基因技术生产Dmrt7蛋白已经逐渐成为研究的热点。
本研究首先从小鼠睾丸组织中分离提取得到总RNA,利用RT-PCR法得到Dmrt7基因全长cDNA序列。对Dmrt7基因序列进行PCR扩增后,分别进行重组腺病毒载体和重组腺相关病毒载体的构建及鉴定。获得的实验结果如下:
(1)用一对预先设计好的引物,采用RT-PCR法,从小鼠睾丸组织总RNA中扩增出的产物,经1.0%的琼脂糖凝胶电泳,结果显示,在1100bp处有一条阳性目的条带。对所扩增的cDNA进行测序,结果显示与Genebank中所记载的序列一致,未发生碱基突变。
(2)细菌内同源重组法构建Dmrt7重组腺病毒载体。穿梭载体pShuttle -Dmrt7-IRES-GFP质粒经酶切鉴定正确后,用PacⅠ酶线性化,与pAdEasy-l共转染大肠杆菌BJ5183进行同源重组,获得pAd-Dmrt7重组腺病毒阳性克隆质粒,经PacⅠ酶切,通过1%琼脂糖凝胶电泳鉴定,得2.9kbp特异性小片段,获得正确的重组腺病毒载体Ad-Dmrt7。
(3)将重组Ad-Dnrt7转染293细胞,24h后可在倒置荧光显微镜下观察到绿色荧光,收集病毒上清,进行PCR鉴定及病毒滴度测定,鉴定结果显示,在1100bp处有一明显条带;采用半数荧光细胞法测定重组腺病毒滴度为0.95×10~8PFU/ml。
(4)细胞内同源重组法构建Dmrt7重组腺相关病毒载体。将测序正确的Dmrt7基因亚克隆至pAAV-MCS中,酶切鉴定获得的重组pAAV-Dmrt7质粒,纯化后与pRC质粒以及pHelper质粒共转染HEK 293细胞,包装获得重组Dmrt7腺相关病毒。转染24h后可在倒置荧光显微镜下观察到绿色荧光。重组Dmrt7腺相关病毒经酶切,PCR鉴定病毒上清,在1100bp处出现一明显条带。采用半数荧光细胞法测定重组腺相关病毒滴度为1.5×10~9PFU/ml。
Dmrt7 gene can express protein Dmrt7, which is important to mammalian reproductive system and contains DM domain. The main biological functions of Dmrt7 are the regulation of spermatogenesis and sex differentiation. There are rich values and broad prospects in the application of Dmrt7 protein in the treatment of low sperm production and no sperm production of male animals. While the natural Dmrt7 protein is scarce, and can not meet the needs of clinic and scientific research, the production of Dmrt7 protein using transgenic technology are becoming the main focus of current research.
In our study, total RNA was firstly separated and extracted from mouse testis and then the cDNA of Dmrt7 was obtained by RT-PCR. After being amplified by PCR, Dmrt7 was recombined with Adenovirus vector and adeno-associated virus vector.
The results of this study included as follows:
(1) The 1.0% agarose gel electrophoresis results of RT-PCR showed the 1100bp gene fragment was obtained from mouse testis total RNA and amplification with a pair of special primers designed at first. The sequence of Dmrt7 gene obtained was consistent with the reported, and had no mutation.
(2) Construction of Dmrt7 recombinant adenovirus vector using Homologous recombination in bacteria. After being digested and identified by restrictive endonuclease, pShuttle-Dmrt7 -IRES-GFP was linearized by PmeⅠand subsequently co-transfected into BJ 5183 with adenoviral backbone plasmid pAdEasy-1. The results of 1.0% agarose gel electrophoresis of recombinant Ad-Dmrt7 digested by restrictive endonuclease PacⅠshowed a special band of 2.9kb. It confirmed that the Ad-Dmrt7 was constructed successfully. The homologous recombination in bacteria was a convenient and efficient way to construction of recombinant adenoviral vectors.
(3) Recombinant Ad-Dmrt7 vector was transfected into HEK293 cell by liposome. After fluorescence was observed in 293 cells which were infected by 24h, the viral supernatant was collected and identified by PCR. The results indicated that there is a distinct band at 1100bp; the titer of recombinant Ad-Dmrt7 which was determined by half fluorescent cell method was 0.95×10~8PFU/ml.
(4) Construction of Dmrt7 recombinant AAV vector using Homologous recombination in HEK 293 cells. The Dmrt7 gene was subcloned into pAAV-MCS,then the obtained recombinant pAAV-Dmrt7 plasmid was digested and identified by restrictive endonuclease. After being purifed, the pAAV-Dmrt7 plasmid was co-transfected into HEK293 cells with pRC plasmid and pHelper plasmid and packaged into recombinant AAV-Dmrt7. The fluorescence can be observed after being transfected by 24h. The titer of recombinant AAV-Dmrt7 which was determined by half fluorescent cell method was 1.5×10~9PFU/ml.
本研究首先从小鼠睾丸组织中分离提取得到总RNA,利用RT-PCR法得到Dmrt7基因全长cDNA序列。对Dmrt7基因序列进行PCR扩增后,分别进行重组腺病毒载体和重组腺相关病毒载体的构建及鉴定。获得的实验结果如下:
(1)用一对预先设计好的引物,采用RT-PCR法,从小鼠睾丸组织总RNA中扩增出的产物,经1.0%的琼脂糖凝胶电泳,结果显示,在1100bp处有一条阳性目的条带。对所扩增的cDNA进行测序,结果显示与Genebank中所记载的序列一致,未发生碱基突变。
(2)细菌内同源重组法构建Dmrt7重组腺病毒载体。穿梭载体pShuttle -Dmrt7-IRES-GFP质粒经酶切鉴定正确后,用PacⅠ酶线性化,与pAdEasy-l共转染大肠杆菌BJ5183进行同源重组,获得pAd-Dmrt7重组腺病毒阳性克隆质粒,经PacⅠ酶切,通过1%琼脂糖凝胶电泳鉴定,得2.9kbp特异性小片段,获得正确的重组腺病毒载体Ad-Dmrt7。
(3)将重组Ad-Dnrt7转染293细胞,24h后可在倒置荧光显微镜下观察到绿色荧光,收集病毒上清,进行PCR鉴定及病毒滴度测定,鉴定结果显示,在1100bp处有一明显条带;采用半数荧光细胞法测定重组腺病毒滴度为0.95×10~8PFU/ml。
(4)细胞内同源重组法构建Dmrt7重组腺相关病毒载体。将测序正确的Dmrt7基因亚克隆至pAAV-MCS中,酶切鉴定获得的重组pAAV-Dmrt7质粒,纯化后与pRC质粒以及pHelper质粒共转染HEK 293细胞,包装获得重组Dmrt7腺相关病毒。转染24h后可在倒置荧光显微镜下观察到绿色荧光。重组Dmrt7腺相关病毒经酶切,PCR鉴定病毒上清,在1100bp处出现一明显条带。采用半数荧光细胞法测定重组腺相关病毒滴度为1.5×10~9PFU/ml。
Dmrt7 gene can express protein Dmrt7, which is important to mammalian reproductive system and contains DM domain. The main biological functions of Dmrt7 are the regulation of spermatogenesis and sex differentiation. There are rich values and broad prospects in the application of Dmrt7 protein in the treatment of low sperm production and no sperm production of male animals. While the natural Dmrt7 protein is scarce, and can not meet the needs of clinic and scientific research, the production of Dmrt7 protein using transgenic technology are becoming the main focus of current research.
In our study, total RNA was firstly separated and extracted from mouse testis and then the cDNA of Dmrt7 was obtained by RT-PCR. After being amplified by PCR, Dmrt7 was recombined with Adenovirus vector and adeno-associated virus vector.
The results of this study included as follows:
(1) The 1.0% agarose gel electrophoresis results of RT-PCR showed the 1100bp gene fragment was obtained from mouse testis total RNA and amplification with a pair of special primers designed at first. The sequence of Dmrt7 gene obtained was consistent with the reported, and had no mutation.
(2) Construction of Dmrt7 recombinant adenovirus vector using Homologous recombination in bacteria. After being digested and identified by restrictive endonuclease, pShuttle-Dmrt7 -IRES-GFP was linearized by PmeⅠand subsequently co-transfected into BJ 5183 with adenoviral backbone plasmid pAdEasy-1. The results of 1.0% agarose gel electrophoresis of recombinant Ad-Dmrt7 digested by restrictive endonuclease PacⅠshowed a special band of 2.9kb. It confirmed that the Ad-Dmrt7 was constructed successfully. The homologous recombination in bacteria was a convenient and efficient way to construction of recombinant adenoviral vectors.
(3) Recombinant Ad-Dmrt7 vector was transfected into HEK293 cell by liposome. After fluorescence was observed in 293 cells which were infected by 24h, the viral supernatant was collected and identified by PCR. The results indicated that there is a distinct band at 1100bp; the titer of recombinant Ad-Dmrt7 which was determined by half fluorescent cell method was 0.95×10~8PFU/ml.
(4) Construction of Dmrt7 recombinant AAV vector using Homologous recombination in HEK 293 cells. The Dmrt7 gene was subcloned into pAAV-MCS,then the obtained recombinant pAAV-Dmrt7 plasmid was digested and identified by restrictive endonuclease. After being purifed, the pAAV-Dmrt7 plasmid was co-transfected into HEK293 cells with pRC plasmid and pHelper plasmid and packaged into recombinant AAV-Dmrt7. The fluorescence can be observed after being transfected by 24h. The titer of recombinant AAV-Dmrt7 which was determined by half fluorescent cell method was 1.5×10~9PFU/ml.
引文
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