Identification Of The Interaction Site Of Truncated Rabbit Ferritin Heavy Chain And RHDV

Rabbit Hemorrhagic Disease(RHD), commonly known as "Rabbit Plague", was first described in China’s jiangsu area. RHDV is a member of calicivirus in the Lagovirus, and the virus can cause acute, high contact and septic infectious diseases of rabbits, which can usually bring devastating damage to rabbit industry. The liver is the main organ of RHDV multiplication. So, the research of the protein which interact with RHDV is one of the important areas. Through SMART technology, our lab has previously screened and identified the relevant candidate protein interacted with VP60. Rabbit ferritin heavy chain is one of the candidate protein interacted with VP60, and the results of GST pull down, ELIS A, Co-IP and HA showed that the FTH had interaction with RHDV. Our study shortened ferritin heavy chain and expressed it in vitro. Thus, we identified the interaction site between FTH and RHDV, laying a founding for the development of antiviral drugs.To precisely positioning the protein-protein interaction sites between FTH and RHDV, total RNA of rabbit liver was extracted and FTH gene was amplified by means of RT-PCR. FTH gene was segmentally cloned into pGEX-6P-1vector, corresponding protein was expressed in BL21(DE3)pLysS with IPTG induction, and we obtained five truncated recombinant proteins of FTH named rF1, rF2, rF3, rF4and rF5. The results of ELISA assay showed that OD450nm value of recombinant proteins rF4was1.531, and recombinant proteins rF1, rF2, rF3and rF5’s OD450mn value were1.084,1.443,1.104and1.152, respectively. This showed that the recombinant protein rF4could interact with RHDV, and there was no interaction between recombinant proteins rF1, rF2, rF3, rF5and RHDV. As a result of hemagglutination inhibition,3.1ug rF4can neutralize the hemagglutination activity of8HAU RHDV, but recombinant proteins rF1, rF2, rF3and rF5cannot neutralize the hemagglutination activity of8HAU RHDV. The results of haemagglutination test showed that the rF4had interaction with RHDV. And the accurate position was between80-123amino acids.By the yeast two-hybrid technology, we wanted to examine whether there were interactions between differentially expressed proteins of infected or non-infected rabbit liver tissue, truncated FTH and RHDV. Total RNA of liver tissue and viral RNA of HYD strain were extracted by Trizol method and reverse transcription to cDNA, viral structural protein VP60, minor structural protein VP10and7nonstructural proteins p16, P23,2C-like protein, P29, VPg,3C-like protease, RdRp and49differentially expressed proteins were amplified by PCR, and then constructed to pGBKT7and pGADT7vector respectively. Recombinant plasmid pGBKT7-X and pGADT7-Y were tested to be no autoactivation and toxicity and then co-transformation into AH109to identify the interactions. As a result, positive interactions between VP60and fumarylacetoacetase like protein, VP10and laminin receptor precursor, member2were identified.In this study, FTH were truncately expressed. We found the interaction site of Rabbit Ferritin heavy chain and RHDV is F4(80-123amino acids) by ELISA assay and haemagglutination test. This research provides a theoretical basis to develop short peptide drugs. Yeast two-hybrid technology identified the interaction between VP60and fumarylacetoacetate enzyme, and between VP10and laminin receptor precursor. Both of which would be further studied at the level of protein and layed a foundation to dissect the rabbit viral hemorrhagic disease virus infection mechanism.