Prokaryotic expression and polyclonal antibody preparation of IFN-β and IFN-γ of Tupaia belangeri yaoshanensis

CAO Ying-ying;LI Bao-ying;WANG Ting;LIANG Liang;YUN Chen-xia;TANG Hai-bo;LENG Jing;Guangxi University of Chinese Medicine/Guangxi Key Laboratory of Translational Medicine for Treating Highincidence Infectious Diseases with Integrative Medicine,Guangxi Key Laboratory for Complementary and Alternative Medicine Experimental Animal Models;

Tupaia belangeri yaoshanensis;;interferon(IFN);;IFN-β;;IFN-γ;;prokaryotic expression;;polyclonal antibody

【Objective】To construct prokaryotic expression vector of IFN-β and IFN-γ genes in Tupaia belangeri yaoshanensis and prepare their polyclonal antibody,so as to lay the foundation for further research on animal models for viral infections of tree shrew.【Method】Peripheral lymphocytes of T. belangeri yaoshanensis were taken as material and total RNA was extracted from them. IFN-β and IFN-γ genes,amplified by reverse transcription-polymerase chain reaction(RT-PCR),were subcloned into recombinant eukaryotic expression plasmids of pcDNA3.1(+)to censtruct eukaryotic expression vector,and transiently transfected into renal cells of Cricetidae(BHK-21). At the same time,the genes were also were subcloned into prokaryotic vector p ET-28a(+) to construct prokaryotic expression vector and transformed into Escherichia coli BL21(DE3)competent cell,and fusion protein of IFN-β and IFN-γ was induced by IPTG. Mice were immunized with purified recombinant proteins to obtain polyclonal antibodies of IFN-β and IFN-γ,and their reactivity were detected by Western blotting and indirect immunofluorescence assay(IFA).【Result】Fragments of IFN-β and IFN-γgenes in T. belangeri yaoshanensis obtained through amplification were 564 and 501 bp. They were subcloned to plasmids of pcDNA3.1(+),so eukaryotic expression vectors pcDNA3.1-IFN-β and pcDNA3.1-IFN-γ were constructed;and they were subcloned to prokaryotic vector of pET-28a(+),so prokaryotic expression vectors pET-28a-IFN-β and pET-28a-IFN-γwere constructed. After prokaryotic expression vectors p ET-28a-IFN-β and pET-28a-IFN-γ transformed into BL21(DE3) competent cell,fusion proteins of IFN-β and IFN-γ were obtained induced by 0.5 mmol/L IPTG for 6 h at 30 ℃. The fusion proteins were expressed in a form of inclusion body. After washing with a washing solution containing 2 mol/L urea,fusion proteins of relatively high purity could be obtained with less loss. Mice were immunized with purified and emulsified fusion proteins of IFN-β and IFN-γ to obtain polyclonal antibodies of IFN-β and IFN-γ. Western blotting and IFA results showed that these two polyclonal antibodies were of good reactivity.【Conclusion】With prokaryotic expression system induced in E. coli,IFN-β and IFN-γ in T. belangeri yaoshanensis was obtained;renatured fusion proteins are of good reactivity and immunogenicity. Polyclonal antibodies prepared through mice immunization were of good reactivity and high titer,and it can be taken as test tools for studying viral infection model of T. belangeri yaoshanensis.