Prokaryotic Expression, Purification Of Human Cl-l1 Neck-crd Fragment And The Polyclonal Antibody Production

Background and Objective: Human 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. CL-L1 is the only cytoplasmic protein, we understand very little about its structure, expression spectrum in body tissues and function. To investigate its structure, expression spectrum in body tissues and function, we need to obtain purified recombinant Neck-CRD fragment of human collectin liver 1 and prepare polyclonal antibodies.Methods: The target gene fragment was amplified from the constructed pcDNA3.1/myc-His-CL-Ll vector including full long CL-L1 gene, and was inserted into cloning vector pT7 blue. The target fragment was obtained from recombinant pT7 blue/NECK-CRDΔK digested by restriction endonucleases, and then oriently inserted into pRSET-C vector. Confirmed by enzymes and sequencing analysis, the recombinant vector pRSET-C/NECK-CRDΔK 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 and Western blot.Results: The pRSET-C/NECK-CRDΔK vector was successfully constructed. Induced by IPTG, the fusion protein of Mr 19 200 was expressed in E. coli BL21 andpurified with Ni-NTA column. Polyclonal antibodies were prepared from immunizedrabbits. The results of ELISA and Western blot indicated that the polyclonal antibodyhad high titer (1:20000) and high specificity.Conclusion: The purified Neck-CRD fragment of human CL-L1 and polyclonalantibodies have largely been acquired, which lays the foundation for further researchon human CL-L1.