人fgl2凝血酶原酶纤维蛋白原相关结构域cDNA的扩增、克隆和鉴定

Amplification,cloning and identification of the cDNA of FRED domain of human fgl2 prothrombinase

目的 :扩增人fgl2凝血酶原酶纤维蛋白原相关结构域 (FRED)cDNA ,构建原核表达质粒。方法 :从外周血单核细胞 (PBMNC)中提取细胞总RNA后 ,应用RT PCR方法扩增fgl2凝血酶原酶FRED段cDNA ,酶切后与pET2 2b(+)原核表达载体连接 ,然后经酶切和测序鉴定。结果 :从PBMNC中扩增了 72 7bp长的PCR产物 ,构建的重组原核表达质粒经酶切和测序鉴定与设计的相符。结论 :成功地从PBMNC中扩增了人fgl2凝血酶原酶FRED段cDNA ,构建了FRED pET2 2b(+)原核表达质粒 ,为进一步的研究奠定了基础。

Objective:To amplify the cDNA of fibrinogen related domain(FRED) of human fgl2 prothrombinase and to construct prokaryotic expression plasmid for FRED. Method:Total cell RNA was extracted from the peripheral blood mononuclear cells(PBMNC). The cDNA was amplified by RT-PCR. The digested cDNA and pET22b(+) were ligated by T4DNA ligase, Restriction endonucleases digestion and sequencing were used to identify recombinant plasmid. Result:A 727 bp cDNA was amplified from PBMNC,sequence analysis of cDNA had demonstrated that the recombinant vector FRED-pET22b was completely the same as we designed. Conclusion:The cDNA of FRED domain of human fgl2 prothrombinase was amplified successfully, a prokaryotic expression system for FRED of fgl2 prothrombinase was constructed,which laid foundation for further research.