The third component of complement C3 and its fragments have a central role in a variety of host defense mechanisms. The identification of functionally relevant C3 domains is important because of the marked functional versatility of the C3 molecule. Several human C3 cDNA clones from a human liver cDNA library were isolated and characterized. A bacterial expression vector system was used to express cDNA clones that were identified by an immunological screening procedure. The C3 cDNA clones produced in E. coli the hybrid proteins consisting of cro-beta-galactosidase and polypeptide segments of human C3, as revealed by Western blotting with antisera to human C3. The C3 moiety of the hybrid proteins had a m.w. of up to 46.000. Polyclonal antibodies against the C3 segments expressed by one of the C3 cDNA clones (ReC3-1) have been raised in mice and rabbit, and in addition, a monoclonal antibody was produced. The antisera and the monoclonal antibody reacted in Western blotting analysis selectively with the alpha-chain, but not the beta-chain of human C3. Restriction mapping of the different cDNA clones was performed, and revealed that the different clones were partially overlapping. The ReC3-1 cDNA clone included a 0.7 kb noncoding region at the 3' terminal end of the C3 cDNA. One of the restriction sites (Hind III) identified in the ReC3-1 cDNA clone was not present in the recently published sequence of human C3 cDNA. This difference in nucleotide sequence provides direct evidence for C3 polymorphism at the DNA level. The combination of immunologic procedures with recombinant DNA methodology should facilitate additional analysis of the structure-function relationship of the C3 molecule.

译文

补体C3的第三部分及其片段在多种宿主防御机制中起着核心作用。由于C3分子具有明显的功能多样性,因此对功能相关的C3域的鉴定非常重要。从人肝脏cDNA文库中分离出几个人C3 cDNA克隆并进行了表征。使用细菌表达载体系统表达通过免疫筛选程序鉴定的cDNA克隆。 C3 cDNA克隆在大肠杆菌中产生了杂种蛋白质,该杂种蛋白质由cro-β-半乳糖苷酶和人C3的多肽片段组成,这是通过对人C3的抗血清进行Western印迹证实的。杂合蛋白的C3部分具有m.w。高达46.000。已经在小鼠和兔子中产生了针对由C3 cDNA克隆之一(ReC3-1)表达的C3节段的多克隆抗体,并且还产生了单克隆抗体。抗血清和单克隆抗体在Western印迹分析中与人C3的α链(而非β链)选择性反应。进行了不同cDNA克隆的限制性作图,并揭示了不同克隆部分重叠。 ReC3-1 cDNA克隆在C3 cDNA的3'末端包含一个0.7 kb的非编码区。 ReC3-1 cDNA克隆中鉴定出的一个限制性酶切位点(Hind III)在最近发布的人C3 cDNA序列中不存在。核苷酸序列的这种差异为DNA水平的C3多态性提供了直接证据。免疫程序与重组DNA方法的结合应有助于对C3分子的结构-功能关系进行额外的分析。

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