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目的:寻找更简便精确的方法来检测人凝血因子Ⅷ的抗原表位。方法:采用PCR技术从人凝血因子Ⅷ的克隆载体pENTR223.1/FⅧ上克隆出FⅧ重链(FⅧ-N)的cDNA。将cDNA插入原核载体pET21 a构建重组表达载体pET21 a-FⅧ-N,并转化大肠杆菌BL21(DE3),以IPTG诱导表达H is-FⅧ-N融合蛋白。以表达产物免疫BALB/c小鼠,制备小鼠抗FⅧ-N的抗血清。接着在重组表达载体pET21 a-FⅧ-N中引入内参F lagotag。对FⅧ-N进行定点突变,构建两个突变克隆N1和N2,对二者以及原FⅧ-N进行双色荧光免疫印迹分析。用双色红外激光成像系统进行扫描和定量分析,以绿荧光与红荧光的光密度比值表示突变克隆抗原性的大小。结果:双色荧光免疫印迹分析表明,突变克隆N1,N2的抗原性与原FⅧ-N相比均有显著下降(P<0.05)。FⅧ A2区的三个氨基酸残基Arg484,Arg489,Arg593对FⅧ-N抗原性起着重要的作用。结论:成功建立了人凝血因子Ⅷ重链抗原表位检测的双色荧光免疫印迹分析法。
OBJECTIVE: To find a more convenient and accurate method for detecting the epitope of human factor Ⅷ. Methods: The cDNA of FⅧ heavy chain (FⅧ-N) was cloned from the cloning vector pENTR223.1 / FⅧ of human coagulation factor Ⅷ by PCR. The cDNA was inserted into prokaryotic vector pET21 a to construct recombinant expression vector pET21 a-FⅧ-N and transformed into E. coli BL21 (DE3) to express H is-FⅧ-N fusion protein by IPTG. BALB / c mice were immunized with the expressed product to prepare mouse anti-FⅧ-N antiserum. Next, the reference F lagotag was introduced into the recombinant expression vector pET21 a-FⅧ-N. FⅧ-N was subjected to site-directed mutagenesis. Two mutant clones N1 and N2 were constructed and two-color fluorescent immunoblotting analysis was performed on the two and the original FⅧ-N. The bi-color infrared laser imaging system was used for scanning and quantitative analysis. The ratio of the optical density of green fluorescence to red fluorescence was used to indicate the antigenicity of the mutant clone. Results: Two-color fluorescent immunoblot analysis showed that the antigenicity of mutant clones N1 and N2 were significantly decreased compared with the original FⅧ-N (P <0.05). The three amino acid residues Arg484, Arg489, Arg593 of FⅧ A2 region play an important role in FⅧ-N antigenicity. Conclusion: Two-color fluorescence immunoblotting assay was successfully established for the detection of human coagulation factor Ⅷ heavy chain antigen epitopes.