自发荧光猪瘟兔化弱毒重组病毒的构建

Five cDNA fragments covering the complete genome of HCLV were obtained by RT-PCR, sequenced and inserted into pGEM-T vector, separately. A clone of the full-length cDNA of the complete genome of HCLV was obtained by subcloning all of the five cDNA fragments into pPoly2. The genome is 12,310 nucleotides in length and contains a large open reading frame encoding 3,898 amino acids.The sequence has been submitted to GenBank and its accession number is AF531433. Phylogenetic analysis based on genomic sequences and their deduced amine acid sequences of known ninteen CSFV strains showed that there is no relatonship between homology and virulence. SK6 cell was transfected with the gemone RNA of HCLV obtained by by transcription of the full-length cDNA with T7 polymerase and passaged four days later. A transfering plasmid of fluorescence-emitted recombinant HCLV was obtained by inserting the EGFP gene into the Npro gene of the cDNA of HCLV gemone in frame. SK6 cell was transfected with the RNA of the transfering plasmid and fluorescence-emitted SK6 cell was observed in the second passage. T7 RNA polymerase (T7pol) gene was amplified by PCR from the gemone of E.coli strain BL21(DE3) and cloned into the pGEM-T vector. A T7pol prokaryotic expression plasmid pET28T7 was constructed by subcloning the T7pol gene into vector pET28b(+). A eukaryotic expression of T7pol, pIRT7, was constructed by subcloning the T7pol gene into pIRES1neo vector. A cell line expressing the T7pol was obtained by trasfecting the pIRT7 into SK6 cell.　The cDNA of porcine interleukin 6 (pIL-6) was amplified by RT-PCR from the total RNA extracted from the peripheral blood lymphocytes stimulated with Concavadin and cloned into the pGEM-T vector. Sequencing analysis showed that the encoding region of pIL-6 cDNA is 639pb including the stop coden. A prokaryotic expression plasmid of pIL-6, pETPIL6, was obtained by subcloning the encoding region of the pIL-6 mature peptide into pET-28a(+). The pIL-6 was expressed in pETPIL6-transformed BL21(DE3)LysS induced by IPTG with the yield accounting for 30.6～38.35% of the total bacterial protein.

. 将在可见下发荧光的绿色荧光蛋白GFP的突变体的mRNA插入到猪瘟兔化弱毒(HCLV)的Npro基因中，构建成自身带有检测标记的重组HCLV。通过观察该重组HCLV感染的细胞是否发荧光及发荧光细胞的多少即可对其效价进行测定，从而建立一种准确、简单、快速且经济的HCLV效价测定方法，以替代目前的家兔测定法。同时，该重组病毒由于其具有便利的检测条件，作为一种研究模型，将有助于研究猪瘟病毒在细胞中的增殖规律及在培养细胞上研究猪瘟病毒的生物学特征。并且，该课题的成果可对研究以HCLV为载体构建预防猪疫病的重组疫苗有借鉴作用。