狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系建立

中国生物工程杂志

2009, Vol. 29

Issue (04): 46-50

研究报告

狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系建立

于恒智^1,2,张守峰²,扈荣良²,张乐萃¹

1. 青岛农业大学动物科技学院
2.中国人民解放军军事医学科学院军事兽医研究所

Establishment of Transformed Mammalian Cell Lines Stably Expressing the Rabies Virus Glycoprotein

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摘要： 试验首先根据GenBank上所发表的狂犬病病毒CVS-24株糖蛋白基因序列，设计并合成一对特异性引物，通过反转录聚合酶链式反应（RT-PCR），获得糖蛋白全长cDNA，连接在pMD18-T载体上，测序证明克隆的正确性后，将其插入真核表达载体pIRES1neo，构建了糖蛋白单一表达载体pICG，表达质粒通过脂质体转染BHK-21细胞，在G418抗性压力下出现细胞克隆，通过PCR检测，确定启动子与糖蛋白基因在细胞基因组中共同整合。借助Western blot检测，证明所表达的糖蛋白与狂犬病抗血清有特异的反应性。采用间接ELISA法，筛选出3株高效表达糖蛋白的细胞株，分别命名为ICG1、ICG2、ICG3。

关键词： 转染;稳定表达;狂犬病病毒;糖蛋白;转染;稳定表达;狂犬病病毒;糖蛋白

Abstract: The glycoprotein of rabies virus strain CVS-24 was amplified by reverse transcription-polymerase chain reaction and cloned it and its fusion gene with green fluorescent protein into eukaryotic expression vector pIRES1neo. The resultant plasmid pICG was transfected into BHK-21 cell line by liposome transfection. After screening with G418, G418-resistant BHK-21 colonies were obtained and amplified. With Western blot analysis, there appeared a reaction band between the specific antibody and the expressed protein, which indicated that the protein has been expressed. By screening with indirect ELISA, three cell clones of expression vector with high level expression were established and used for further identification, named respectively ICG1, ICG2, ICG3.

Key words: transfection;stable expression;rabies virus;glycoprotein;transfection;stable expression;rabies virus;glycoprotein

收稿日期: 2008-08-27 出版日期: 2009-04-27

基金资助: 国家自然科学基金资助项目（30571379）

通讯作者: 张乐萃

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引用本文:

于恒智,张守峰,扈荣良,张乐萃. 狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系建立[J]. 中国生物工程杂志, 2009, 29(04): 46-50.

XU Heng-Zhi- Zhang-Shou-Feng- Hu-Rong-Liang- Zhang-Le-Cui. Establishment of Transformed Mammalian Cell Lines Stably Expressing the Rabies Virus Glycoprotein. China Biotechnology, 2009, 29(04): 46-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I04/46

［1］ WHO. Expert Consultation on Rabies (First Report). Geneva, 2004，13: 48~58 ［2］ Marinez X, Brandt C, Saddallah F, et al. DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life. Proc Natl Acad Sci USA, 1997,94:8726~8731 ［3］ Dai X, Hajos J P. Isolation of a Spodoptera exigua baculovirus recombinant with a 10. 6 kb Pgenome deletion that retains biological activity. J Gen Virol, 2000, 81(10):2545~2554 ［4］ J. 萨姆布鲁克, D.W.拉塞尔.分子克隆实验指南.第3版.北京:科学出版社，2002. 880~887，888~897 J. Sambrook, D. W. Russell. Molecular Cloning: A Laboratory Manual. 3rd ed, Beijing:Science Press,2002. 880~887，888~897 ［5］王化磊，王承宇，杨松涛，等.狂犬病病毒重组免疫毒素的表达及其生物活性鉴定.中国生物工程杂志，2008,28(5):1~5. Wang H L,Wang C Y, Yang S T, et al. China Biotechnology, 2008,28(5):1~5 ［6］殷震，刘景华．动物病毒学．第2版.北京：科学出版社，1997.329~331 Yin Z,Liu J H.Animal Virology.2nded.Beijing:Science Press,1997.329~331 ［7］ Klinmam D,Yi A K,Beaucage S L,et al.CpG motifs present in bacterial DNA rapidly induce lymphocytes to secreteIL-6,IL-12 and IFN-γ.Proc Nati Acad Sci USA,1996,93:2879~2883 ［8］ Petrounkina A M,Harrison R A,Petzddt R,et al.Cyclical changes in sperm volume during in vitro incubation under capaci2 tating conditions:a novel boar semen characteristic.J Reprod Fertil,2000,118(2):1283～1293 ［9］扈荣良，涂长春．狂犬病病毒糖蛋白转基因小鼠的建立．中国兽医学报，1995，15（3）209~213 Hu R L,Tu C C.Chinese Journal of Veterinary,1995,15(3):209~213

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