Prokaryotic Expression Of HnRNP A2/B1 Gene And Preparation Of Its Polyclonal Antibody

Objective: Heterogeneous nuclear ribonuclear protein A2/B1(hnRNP A2/B1)is a RNA binding protein, the most important member of hnRNPs family . HnRNPA2/B1 was involved in nucleocytoplasmic transport of mRNA and mRNA metabolism including regulation of mRNA translation,stability and mRNA localization .There is a intimate relation between hnRNP A2/B1 overexpression and disorder of growth regulation. At present, lots of research have manifestated hnRNP A2/B1 overexpression in lung cancer cell,even in early lung cancer and precancerous change, which reveal that hnRNP A2/B1 may be a tumor marker of early lung cancer.In our experiment ,we expressed and purified the recombinant human hnRNPA2/B1 in prokaryotic system and prepared the rabbit anti human hnRNPA2/B1 polyclonal antibody. The recombinant protein and the polyclony antibody can be used in further studies.Methods: (1) The gene hnRNPA2/B1 was got from the vector pGEX4T1-hnRNP A2/B1 by restriction endonuclease digestion with EcoR I and XhoⅠ, and was correctly link to eukaryotic expression vector pET28a(+) with the same restriction endonucleases digestion. And to identify by nucleotide sequencing and restriction endonuclease digestion. (2) The recombinant plasmid was transformed into Bacillus coli and to shake culture. Then it was inducted by IPTG , optimized cultural condition and analyzed existence and solubility of the fusion protein by SDS-PAGE. (3)The Bacillus coli with pET28a(+)-hnRNPA2/B1 was amplificated by shaking culture under optimized condition and collected by centrifugating. The bacterium was clearaged and the fusion protein was purified with affinity column. (4) After emulsifying purified fusion protein and Freund's adjuvant,the emulsion was immuned rabbit subcutaneously. We collected antiserum ,pured antibody the through ammonium sulfate precipitation and assessed the titer of polyclonal anti- hnRNPA2/B1 antibody by ELISA and the specificity by western-blot and immunohistochemistry.Results: (1) We got recombinant prokaryotic expression vector of pET28a(+)-hnRNP A2/B1,which was confirmed by nucleotide sequencing and restriction endonuclease digestion.(2) By induction of IPTG, the E.coli with pET28a(+)-hnRNPA2/B1 expressed the fusion protein ,which stranded at the 41KD by SDS-PAGE analysisthe comparing to E.coli uninduced and the control .The optimized condition for induction is 37℃,4h, IPTG 0.1mmol/L.(3) We purified soluble protein in supernatant of E.coli lysate with affinity column,and we found the protein is puried by SDS-PAGE analysis.(4) Rabbit anti- hnRNP A2/B1 polyclonal antibody was obtained by immuning rabbits with the purified protein. We confirmed that the titer of the polyclonal antibody was 1:8000 and the specificity of rabbit anti- hnRNP A2/B1 polyclonal antibody by western-blot and immunohistochemical staining.Conclusion: We got purified hnRNPA2/B1 and prepared high titer Rabbit anti-human hnRNP A2/B1 polyclonal antibody,which would provide experiment material for further research in the biological function of hnRNP A2/B1 and the relation between hnRNP A2/B1 and lung cancer.