Preparation Of Virus-Like Particles Of RHDV And Construction Of The Expression CDNA Library Of RK13 Cells

Rabbit hemorrhagic disease virus (RHDV; genus Lagovirus family Caliciviridae) is the causative agent of a highly contagious disease of wild and domestic rabbits. Caliciviruses are a diverse virus family with a wide range of host and tissue tropisms. The first step of viral infection is the binding of the virus to cellular receptor. Caliciviruses bind to host cellular receptors and then enter into host by endocytosis. A few progresses have been made about the cellular receptors of calicivirus in some kinds of this family, but a blank to RHDV. To screen the cellular receptor of RHDV, we expressed the capsid protein of RHDV with the baculovirus expression system and constructed the full-length cDNA library of RK13 with SMART technique.1. Preparation virus-like particles of RHDV JX/97 strainThe vp60 fragment containing SalⅠand HindⅢsites were separately amplified by PCR with pBLRHDV as a template. Then the vp60 gene were cloned into pFastBacl. The recombinant plasmids obtained were transformed into DH10Bac and the positive clone could be screened in the LB culture plate including Kanamycin, gentamincin, tetracycline, X-gal and IPTG. Then the recombinant Bacmid DNA was extracted and transfected into insect cell Sf9. The supernatant was collected six days later. And twice repetitive infection was carried out with the supernatant collected. VP60 protein expression were identified by indirect immunofluorescence and Western Blot assay. Furthermore, the results of transmission electron microscope observation show that expressed proteins were capable of self-assembling to form virus-like particles.2. Construction of full-length cDNA library of RK13 cellsThe total RNAs and mRNAs were isolated and purified from RK13 cells. Then the first strand cDNAs were synthesized by reverse transcription (RT) with RT primer (CDSⅢ) and the double-strand cDNAs were amplified by Long-distance PCR. The large fragments(>500 bp) of ds cDNAs have been separated by cDNA Size Fractionation system after the total ds cDNAs digested by SfiⅠ. Then the large fragments obtained were cloned into pEXP-Lib vector. The ligation product was transformed into E.coli DH5a by electrotransformation. The result showed that the cDNA library contained 2.55×105 clones with average inserts about 1.0 kb.The VLPs prepared with the insect cells-baculovirus expression system and the full-length cDNA library of RK13 constructed by SMART technique laid the foundation of identifying cellular receptor of RHDV.