摘要
以互为模板的ＰＣＲ合成技术及克隆技术体外重组人血管生长因子基因片段（ｈＡｎｇｆ）并获得ｐＢｌｕｅｓｃｒｉｐｔ－ｈＡｎｇｆ重组体。方法：设计目的基因ｈＡｎｇｆ的上、下游引物。二引物以１８ ｈｐ区段互补，二引物的５’端分别具有加入的或利用简并密码设计的酶切位点，以ＰＣＲ合成目的基因前体，经ＥｃｏＲＩ作用，获目的基因。以ｐＢｌｕｅｓｅｒｉｐｔ为克隆载体，目的基因插入该载体ＥｃｏＲＩ及ＥｃｏＲＶ之间。克隆后经Ｋｐｎｌ、Ｓｃａｌ及ＥｃｏＲＩ、ＥｃｏＲＶ作用，筛选并电泳定征。结果３以ＰＣＲ合成目的基因，并获ｐＢｌｕｅｓｃｒｉｐｔ－ｈＡｎｇｆ重组质粒。经克隆并定征，该重组体确含１３４ ｈｐ（含上游加入的酶切位点及起始密码子）的目的基因序列。结论：成功获得了 ｐＢｌｕｅｓｃｒｉｐｔ－ｈＡｎｇｆ重组体，以作为进一步研究的基础。
Enough human angiogenin gene fragments were obtained with PCR and cloning for research. A couple of primers, the upstream and downstream primer of human angiogenin gene fragment(h-Angf) (# 1 ～ # 123 bp) were designed. The length of upstream primer was 76 nt and the other was 78 nt. The EcoRI cleavage site was introduced to the 5' end of upstream primer and the initator codon was provided at this region. According to code degeneracy, the 5' end of downstream primer just was the EcoRV cleavage site. There was a complementary region of 18 bp between the 3' ends of both primers,so the primers were used as template each other. The synthesis of h-Angf occurred with PCR, and then the PCR prod-ucts were indentified by electrophoresis. The PCR products were inserted into pBluescript vectors and cloned. The clones were treated with KpnI, ScaI, EcoRI and EcoRV. Because of the different lengths of nuclwtide sepuences between containing the insert and not, the positive clones were screened and identified with electrophoresis. The electrophoretogram showed that the h-Angf wanted was obtained by means properly adopted. As the part of foundation for further investigation, enough human angiogenin gene fragments were synthesized by gene recombination in vitro.
出处
《天津医药》
CAS
1999年第4期223-225,共3页
Tianjin Medical Journal
