摘要
应用ｐＢｌｕｅｓｃｒｉｐｔｌｌｋｓ质粒构建了含人肿瘤坏死因子－α ｃＤＮＡ的过渡性载体ｐＢＴ１和ｐＢＴ２。用限制性内切酶ＢａｍＨＩ与ＥｃｏＲＶ消化ｐＢＴ１ＤＮＡ，分离出了ＴＮＦ－α ｃＤＮＡ基因片段，并在其平末端加入了ＢａｍＨＩ接头。进而将带有ＢａｍＨＩ粘性末端的ＴＮＦ－αｃＤＮＡ克隆到了逆转录病毒载体ｐＺＩＰＮｅｏＳＶ（Ｘ）１５′－端长末端重复序列（ＬＴＲ）下游的ＢａｍＨＩ切点上，构建了重组质粒ｐＺＩＰＴＮＦ。该重组质粒经琼脂糖凝胶电泳，限制性内切酶消化和核酸杂交鉴定分析，证明ｐＺＩＰＮｅｏＳＶ（Ｘ）１中插入了ＴＮＦ－α ｃＤＮＡ，并且方向正确。本研究为应用重组逆转录病毒介导ＴＮＦ－α基因转移与治疗奠定了基础。
The secondary vectors pBT1 and pBT 2 carrying human tumor
necrosisfactor α cDNA were constructed with pBluescript 11ks plasmid. The TNFα cNDA
frag-ments were isolated through digestion of pBT1 with restriction enzymes Bam HI and
EcoRV , and the Bam HI linkers were added to the blunt ends of the cDNA fragments. Fur-ther
experiments constructed the recombinant plasmid pZIPTNF by inserting TNFα cD-NA with Bam
HI cohesive termini into the Bam HI site followed 5′-long terminal repeat(LTR) of retroviral
vector pZIPNeoSV(X)1.Using agarose gel electrophoresis,restric-tion enzyme analysis and
nucleic acid hybridization for pZIPTNF, it is evident thatpZIPTNF contained the inserted TNFα
cDNA and the translating direction of the cDNAin pZIPTNF was correct. This study established a
fundament for using recombinantretrovirus-mediated TNFα gene transfer and therapy.
出处
《中国兽医学报》
CAS
CSCD
1994年第2期135-138,共4页
Chinese Journal of Veterinary Science
关键词
人
肿瘤坏死因子
逆转录病毒
载体
human tumor necrosis factor α
retroviral vector
expressing plasmid Construction
