The full-length of ORF5 gene was amplified from the genomic RNA of Henan variant PRRSV strain HN07-01 by RT-PCR.The amplified fragment was cloned into pMD19-T vector and then sequenced.Compared with the ORF5 gene sequences of BJ-4,classical American genotype VR-2332,HB-1 and JXA-1,the homology of HN07-01 ORF5 gene was 88%,89%,96% and 99%,respectively.The ORF5 gene was subcloned into the prokaryotic expression vector pGEX-6p-1,transformed into E.coli BL21 and induced with IPTG.The results of SDS-PAGE and Western-Blot confirmed that the recombinant protein of 41.5kD was successfully expressed in E.coli BL21.

An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken β-actin/rabbit β-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3′-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.

To prepare hyperimmunized rabbit serum against classical swine fever virus(CSFV) C strain,the Zelanian rabbit were immunized with the attenuated CSFV vaccine.The serum titer of immunized rabbit was determined by indirect ELISA and the IgG was purified by saturated ammonium sulfate precipitation.Then,the purity of IgG was checked by SDS-PAGE electrophoresis and the safety was confirmed by microorganism test and inoculating piglets.The results showed that the protein concentration of the purified IgG was 6 mg/mL,and the ELISA titer was 1∶6400.Taken together,we have produced serum IgG against CSFV,which shows high titer,high safety,low cost,and can be used to treat or prevent swine fever.It is an ideal biological agent for the detection of CSFV and the control of CSF.

综述了国内外IBDV细胞受体的研究情况,概括介绍了近年来的研究进展,以期对IBDV细胞受体的结构和功能研究提供参考.