The Subcellular Localization Of Recombinant Humam Collectin Liver 1 In Cho Cells

Background and ObjectiveHuman collectin liver 1 (CL-L1) is a new member of collectin family, and first cloned by Ohtani. CL-L1 is deduced to have typical structural characteristics of collectins, consists of four regions:N-terminal cystine-rich region, collagen-like region, neck region and carbohydrate-recognition domain (CRD). Most of collectins are secreted proteins, playing an important role in immunological defence through aggregation, complement activation, opsonization, activation of phagocytosis, and inhibition of microbial growth, and so on. We understand very little about the structure, expression spectrum in body tissues and function of CL-L1. To investigate its subcellular localization, we need to prepare polyclonal antibodies of recombinant Neck-CRD fragment of human CL-L1 and construct eukaryotic expression system pEGFP-CLL1 (including full length cDNA sequence of human CL-L1).MethodsThe target gene fragment was obtained from recombinant pT7 blue/NECK-CRD△K digested by restriction endonucleases, and then oriently inserted into pRSET-C vector. Confirmed by enzymes and sequencing analysis, the recombinant vector pRSET-C/NECK-CRD△K was expressed in E. coli BL21, the expressed fusion protein was purified with Ni-NTA column. Rabbits were immunized with the purified protein and antiserum was obtained. The titers and specificity of antibodies were measured by ELISA. The constructed eukaryotic expression vector pEGFP-CLL1 was transiently transfected into Chinese hamster ovary cells (CHO) by polyethylenimine(PEI). Immunofluorescent assay and Western blot was employed to detect the localization of EGFP-CLL1 fusion protein in CHO cells.ResultsThe pRSET-C/NECK-CRDAK vector was successfully constructed. Induced by IPTG, the fusion protein of Mr 19 200 was expressed in E. coli BL21 and purified with Ni-NTA column. Polyclonal antibodies were prepared from immunized rabbits. The results of ELISA indicated that the polyclonal antibody had high titer (1:20000) and high specificity. Influorescence microscope observation and Western blot analysis showed that EGFP-CLL1 fusion protein was located in cytoplasm, not in supernatant, endoplasmic reticulum(ER), Golgi body and nuclear.ConclusionThe purified Neck-CRD fragment of human CL-L1 and high specific polyclonal antibodies have largely been acquired. Recombinant human CL-L1 may be only one cytoplasmic protein in all identified collectin members.