Cloning of parvovirus H-1 NS partial gene by PCR

HUANG Qing-Shan; HUANG Jian; LUO Jie-Yu; WANG Shun-De; LIU Wei-Ping

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August 1995

HUANG Qing-Shan, HUANG Jian, LUO Jie-Yu, WANG Shun-De and LIU Wei-Ping. Cloning of parvovirus H-1 NS partial gene by PCR[J]. Virologica Sinica, 1995, 10(4).

Citation: HUANG Qing-Shan, HUANG Jian, LUO Jie-Yu, WANG Shun-De, LIU Wei-Ping. Cloning of parvovirus H-1 NS partial gene by PCR .VIROLOGICA SINICA, 1995, 10(4) : 323.

用PCR技术克隆细小病毒H-1部分非结构蛋白基因

1.
复旦大学生理学与生物物理学系复旦大学遗传工程国家重点实验室

摘要

应用ＰＣＲ技术定向克隆了细小病毒Ｈ－１的非结构蛋白（ＮＳ）部分基因片段。自行设计并合成了ＰＣＲ引物△Ｐ３和△Ｐ４，在两个引物中分别引入两个突变碱基，使扩增后的ＤＮＡ片段的两端含有限制性核酸内切酶ＨｉｎｄⅢ或ＢａｍＨＩ的酶切位点，经双酿切法把该ＤＮＡ片段重组到ｐＵＣ１１８质粒中。对插入片段的ＤＮＡ序列测定和分析结果证实该片段为Ｈ－１ＮＳ－１基因序列。以此重组质粒为探针，采用分子杂交的方法，分别测定了Ｈ－１及ＭＶＭＤＮＡ在细胞内的复制水平。这一基因的克隆为制备Ｈ－１的质量监测、Ｈ－１及ＭＶＭＮＳ－１蛋白抑瘤作用机理及其在肿瘤细胞及正常组织中的转录表达等研究奠定了基础。
- 自主性细小病毒H1 聚合酶链反应（PCR）基因克隆抑瘤作用

Cloning of parvovirus H-1 NS partial gene by PCR

Abstract

Two modified primers,△P3 and △P4,were designed and,for each of them,two mutatedbases were included.A fter PCR amplification, the DNA fragment contains a restriction endonu-clease recognition site,BamHⅠ or Hind Ⅲ at its ends,Cloning of the parvovirus H-1 NS-1 partialgene was conducted by double digestion uf the aniplified DNA fragement with endonucleases,andits presence in the inserted fragement was confirmed by DNA sequence analysis.The techniquedescribed here provides the basis for the quality control of H-1 propagation,and for the studies ofoncosuppressive action of H-1 NS-1 protein and of transcription and expression of NS-1 gene incancer cells and normal tissues.
- Autonomous parvoviruses H1 and MVM Polymerase chain reaction Gene cloning Oncosuppression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning of parvovirus H-1 NS partial gene by PCR

Abstract: Two modified primers,△P3 and △P4,were designed and,for each of them,two mutatedbases were included.A fter PCR amplification, the DNA fragment contains a restriction endonu-clease recognition site,BamHⅠ or Hind Ⅲ at its ends,Cloning of the parvovirus H-1 NS-1 partialgene was conducted by double digestion uf the aniplified DNA fragement with endonucleases,andits presence in the inserted fragement was confirmed by DNA sequence analysis.The techniquedescribed here provides the basis for the quality control of H-1 propagation,and for the studies ofoncosuppressive action of H-1 NS-1 protein and of transcription and expression of NS-1 gene incancer cells and normal tissues.