大鼠ZnT1基因重組腺病毒載體的構(gòu)建及鑒定
發(fā)布時間：2019-05-24 23:57
【摘要】：目的利用GatewayTM技術(shù)構(gòu)建含大鼠鋅轉(zhuǎn)運體1(ZnT1)基因重組腺病毒載體,鑒定外源基因在真核細胞中的良好表達。方法采用化學(xué)合成的方法,合成ZnT1/RES/EGFP目的基因片段,通過PCR方法在基因片段兩端加入attB重組位點,與含有attP重組位點的pDonr221供體載體BP反應(yīng)形成入門載體pDown-ZnT1。將含有attL重組位點的入門載體與含有attR位點目的載體Ad/CMV/V5-DEST通過LR反應(yīng)形成腺病毒表達載體pAd-ZnT1。酶切和測序鑒定后,由PacⅠ酶切線性化轉(zhuǎn)染HEK293A細胞包裝,提取病毒顆粒。采用終點稀釋法測定重組腺病毒滴度;Western blotting技術(shù)分析目的蛋白表達情況。結(jié)果目的基因按正確的方向重組入克隆載體中,重組腺病毒表達載體在HEK293A細胞中包裝成功,獲得成熟的腺病毒顆粒,病毒滴度為1.6×108 pfu/L,在HEK293A細胞中高表達。結(jié)論該實驗采用GatewayTM技術(shù)構(gòu)建了含有大鼠ZnT1基因的重組腺病毒載體,為下一步研究該基因在脊髓神經(jīng)細胞中的表達以及與BDNF/TrkB信號調(diào)節(jié)通路的關(guān)系奠定了基礎(chǔ)。
[Abstract]:Objective to construct recombinant adenoviral vector containing rat zinc transporter 1 (ZnT1) gene by GatewayTM and identify the good expression of foreign gene in eukaryotic cells. Methods the target gene fragment of ZnT1/RES/EGFP was synthesized by chemical synthesis. AttB recombination site was added to both ends of the gene fragment by PCR, and the entry vector pDown-ZnT1. was formed by reaction with pDonr221 donor vector BP containing attP recombination site. The entry vector containing attL recombination site and the target vector Ad/CMV/V5-DEST containing attR site were reacted with the target vector Ad/CMV/V5-DEST to form adenoviral expression vector pAd-ZnT1. by LR. After restriction endonuclease digestion and sequencing, the virus particles were extracted from HEK293A cells and packaged by HEK293A cells by restriction endonuclease digestion and sequencing. The expression of the target protein was analyzed by end-point dilution method. The recombinant adenoviral titer; Western blotting technique was used to analyze the expression of the target protein. Results the target gene was recombined into the clone vector in the right direction. The recombinant adenoviral expression vector was successfully packaged in HEK293A cells, and the mature adenoviral particles were obtained. the virus titer was 1.6 脳 108 pfu/L, and the expression of the recombinant adenoviral expression vector was high in HEK293A cells. Conclusion the recombinant adenoviral vector containing rat ZnT1 gene was constructed by GatewayTM technique, which laid a foundation for further study of the expression of the gene in spinal cord nerve cells and its relationship with BDNF/TrkB signal regulation pathway.
【作者單位】： 遼寧醫(yī)學(xué)院附屬第一醫(yī)院骨科;遼寧醫(yī)學(xué)院附屬第一醫(yī)院中心實驗室;
【基金】：國家自然科學(xué)基金資助項目(81171799)
【分類號】：R651.2
[Abstract]:Objective to construct recombinant adenoviral vector containing rat zinc transporter 1 (ZnT1) gene by GatewayTM and identify the good expression of foreign gene in eukaryotic cells. Methods the target gene fragment of ZnT1/RES/EGFP was synthesized by chemical synthesis. AttB recombination site was added to both ends of the gene fragment by PCR, and the entry vector pDown-ZnT1. was formed by reaction with pDonr221 donor vector BP containing attP recombination site. The entry vector containing attL recombination site and the target vector Ad/CMV/V5-DEST containing attR site were reacted with the target vector Ad/CMV/V5-DEST to form adenoviral expression vector pAd-ZnT1. by LR. After restriction endonuclease digestion and sequencing, the virus particles were extracted from HEK293A cells and packaged by HEK293A cells by restriction endonuclease digestion and sequencing. The expression of the target protein was analyzed by end-point dilution method. The recombinant adenoviral titer; Western blotting technique was used to analyze the expression of the target protein. Results the target gene was recombined into the clone vector in the right direction. The recombinant adenoviral expression vector was successfully packaged in HEK293A cells, and the mature adenoviral particles were obtained. the virus titer was 1.6 脳 108 pfu/L, and the expression of the recombinant adenoviral expression vector was high in HEK293A cells. Conclusion the recombinant adenoviral vector containing rat ZnT1 gene was constructed by GatewayTM technique, which laid a foundation for further study of the expression of the gene in spinal cord nerve cells and its relationship with BDNF/TrkB signal regulation pathway.
【作者單位】： 遼寧醫(yī)學(xué)院附屬第一醫(yī)院骨科;遼寧醫(yī)學(xué)院附屬第一醫(yī)院中心實驗室;
【基金】：國家自然科學(xué)基金資助項目(81171799)
【分類號】：R651.2
【二級參考文獻】
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1 徐小良,戴克戎,湯亭亭,郁朝鋒,徐e，
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