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通过高表达质粒在细菌中合成的单纯疱疹病毒胸苷激酶经原生质体融合转移至组织培养细胞。

Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.

作者信息

Waldman A S, Milman G

出版信息

Mol Cell Biol. 1984 Aug;4(8):1644-6. doi: 10.1128/mcb.4.8.1644-1646.1984.

DOI:10.1128/mcb.4.8.1644-1646.1984
PMID:6092926
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC368961/
Abstract

The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. We have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report we show that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography.

摘要

将一种蛋白质导入活的组织培养细胞可能会使得对该蛋白质的功能进行体内研究成为可能。我们之前描述过一种高效表达质粒pHETK2，它含有单纯疱疹病毒1型胸苷激酶（TK）基因，在温度诱导下，该基因会使TK的合成量超过细菌蛋白质总量的4%。在本报告中，我们表明，通过聚乙二醇介导与含有诱导水平TK的细菌制备的原生质体进行融合，具有酶活性的TK被转移到了小鼠Ltk-细胞中。通过放射自显影法测量[3H]胸苷掺入细胞核的情况来检测Ltk-细胞中TK的存在。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4572/368961/514a3af61e8d/molcellb00150-0225-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4572/368961/514a3af61e8d/molcellb00150-0225-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4572/368961/514a3af61e8d/molcellb00150-0225-a.jpg

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1
Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.通过高表达质粒在细菌中合成的单纯疱疹病毒胸苷激酶经原生质体融合转移至组织培养细胞。
Mol Cell Biol. 1984 Aug;4(8):1644-6. doi: 10.1128/mcb.4.8.1644-1646.1984.
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引用本文的文献

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J Virol. 1986 Aug;59(2):420-7. doi: 10.1128/JVI.59.2.420-427.1986.
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Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).哺乳动物细胞中的非法重组和同源重组：对聚（ADP-核糖基化）抑制剂的差异敏感性
Nucleic Acids Res. 1990 Oct 25;18(20):5981-8. doi: 10.1093/nar/18.20.5981.

本文引用的文献

1
High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.通过原生质体融合将克隆的单纯疱疹病毒1型序列高频转移至哺乳动物细胞。
Mol Cell Biol. 1981 Aug;1(8):743-52. doi: 10.1128/mcb.1.8.743-752.1981.
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Immunoglobulin gene expression in transformed lymphoid cells.转化淋巴细胞中的免疫球蛋白基因表达。
Proc Natl Acad Sci U S A. 1983 Feb;80(3):825-9. doi: 10.1073/pnas.80.3.825.
3
Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor.
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J Biol Chem. 1983 Oct 10;258(19):11571-5.
4
Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor.利用λPL启动子和CI857温度敏感型阻遏物的高效表达质粒产生的大肠杆菌鸟嘌呤-黄嘌呤磷酸核糖基转移酶的纯化及特性分析
J Biol Chem. 1983 Jun 25;258(12):7469-75.
5
A microsurgical methodology for human cells in vitro: evolution and applications.一种用于体外培养人类细胞的显微外科方法：演变与应用
Proc Natl Acad Sci U S A. 1970 Apr;65(4):911-8. doi: 10.1073/pnas.65.4.911.
6
Injection of foreign substances into single cells by cell fusion.通过细胞融合将外来物质注入单细胞。
Nature. 1974 May 31;249(456):449-50. doi: 10.1038/249449a0.
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Uptake of proteins by red blood cells.
Exp Cell Res. 1975 Jul;93(2):487-92. doi: 10.1016/0014-4827(75)90478-4.
8
Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker.脂质体与细胞的相互作用：捕获的荧光标记物的转移及细胞内释放
Science. 1977 Feb 4;195(4277):489-92. doi: 10.1126/science.835007.
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Ghost-mediated transfer of human hypoxanthine-guanine phosphoribosyl transferase into deficient Chinese hamster ovary cells by means of polyethylene glycol-induced fusion.
Exp Cell Res. 1978 Sep;115(2):423-8. doi: 10.1016/0014-4827(78)90299-9.
10
Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells.化学辅助将蛋白质显微注射到组织培养细胞的完整单层中。
Somatic Cell Genet. 1977 Nov;3(6):603-10. doi: 10.1007/BF01539068.