Liu Z.-F.,Wuhan PU AI Hospital |
Wang J.-M.,Wuhan PU AI Hospital |
Wang L.,Wuhan PU AI Hospital |
Zeng X.-Y.,Wuhan PU AI Hospital |
And 3 more authors.
Journal of Clinical Rehabilitative Tissue Engineering Research | Year: 2010
BACKGROUND: An abroad study reported the distribution and expression of augmenter of liver regeneration (ALR) in the central nervous system. There are few literatures on how to prepare and evaluate ALR protein polyclonal antibody in recombinant rats, and how to construct prokaryotic expression vector. There are no reports concerning ALR in the central nervous system in China. OBJECTIVE: To express ALR fusion protein in E. coli BL21 and prepare and identify polyclonal antibody. METHODS: RNA was extracted from the hippocampus of Sprague Dawley rats. The prokaryotic expression plasmid pET28a-ALR was constructed and the positive recombinant plasmid was transformed into BL21. Protein ALR was expressed by inducing transformed BL21 with Isopropyl-β-D- thiogalactopyranoside (IPTG) and purified by Ni2+affinity chromatography column after immune the rabbit for 4 times, the serum of rabbits was extracted from heart as polyclonal antibody. The titer and specificity of the rabbit's antiserum was respectively measured by ELISA and Western blotting. The following parameters were measured: construction of prokaryotic expression plasmid pET28a-ALR; pET28a-ALR recombinant enzyme digestion evaluation; results of ELISA and Western-blotting. RESULTS AND CONCLUSION: Expecting bands were obtained by double enzyme digestion electrophoresis, respectively 5.3 kb and 0.4 kb. Nucleotide sequence analysis verified that prokaryotic expression vector pET28a-ALR was successfully constructed. The 19 ku fusion protein was successfully expressed. The titer of the antiserum measured by ELISA could achieve 1:2 000. This indicated that antibody and purified recombinant ALR had a good reaction, and high titer, could meet the experimental require. Western blotting analysis proved that the antibody could identify the prokaryotic expression product of ALR. Prokaryotic expression system expressed ALR fusion protein, prepared and purified polyclonal antibody of ALR protein, and could meet the experimental require of ALR immunoblotting.