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目的制备人载脂蛋白A5(ApoA5)重组蛋白及其多克隆抗体,利用该抗体检测ApoA5在体内的组织分布情况,为进一步研究该分子的功能和血清水平等提供条件。方法2004年3月至6月,中南大学湘雅二医院心内科通过聚合酶链反应(PCR)和基因重组技术,构建了含ApoA5编码基因的表达载体,表达了His-ApoA5融合蛋白,免疫新西兰大白兔制备了抗ApoA5多抗;经酶联免疫吸附分析技术(ELISA)、Western-blot等方法进行鉴定,并初步用于临床组织标本的检测,观察ApoA5在体内的表达。结果所表达的His-ApoA5融合蛋白相对分子质量约为40ku),以其为抗原制备的ApoA5特异性抗血清具有良好的免疫反应性、较高的效价和特异性。Western印迹表明,ApoA5存在于人血清和肝组织中(无论正常血脂和高血脂者),其他组织中(心、血管、小肠、脾、肺)未见到阳性结果。结论ApoA5存在人血清中,ApoA5在肝内表达对于血脂在肝内代谢可能具有重要的意义。
OBJECTIVE: To prepare ApoA5 recombinant protein and its polyclonal antibody and to detect the distribution of ApoA5 in vivo using this antibody, providing the conditions for further study on the function and serum level of ApoA5. Methods From March 2004 to June 2004, the expression vector containing ApoA5 gene was constructed by polymerase chain reaction (PCR) and gene recombination in the Second Xiangya Hospital of Central South University. The fusion protein of His-ApoA5 was expressed and immunized in New Zealand Rabbits were prepared anti-ApoA5 polyclonal antibody by ELISA, Western-blot and other methods for identification and preliminary clinical tissue samples were detected to observe the expression of ApoA5 in vivo. As a result, the expressed His-ApoA5 fusion protein has a relative molecular mass of about 40 ku). The ApoA5-specific antiserum prepared by using the antigen as the antigen has good immunoreactivity, high potency and specificity. Western blotting showed that ApoA5 was present in both human serum and liver tissue (both normal lipids and hyperlipidemia) and no positive results were found in other tissues (heart, blood vessels, small intestine, spleen, lung). Conclusion The existence of ApoA5 in human serum and the expression of ApoA5 in the liver may play an important role in the metabolism of lipids in the liver.