Infectivity Resume Of The Full-length Clone Of C Strain And The Construction Of Its Marker Vaccine

Classical swine fever is the most harmful disease in swine breeding. The C strain of CSFV is one of the best attenuated vaccine in the world. To discuss the tropism of spleen derived virus in SK-6 cell, SK-6 cell was selected as the tropism cell and the antigen express and the sequence of CSFV was detected by direct immuno-fluorescent staining method, ELISA and RT-PCR. The result indicated that the spleen derived C strain of CSFV can growth at a low level in SK-6 cell. The ELISA and RT-PCR method is suitable for the detection of cultured CSFV, while the direct immuno-fluorescent staining method is not suitable. The study can served as a useful guide for the detection of attenuated CSFV. And it also constructed a solid foundation for its culture in SK-6 and its reverse genetics study. By sequencing, several leathal mutation sites were fount in the full-length cDNA clone we constructed before. To resume its infectivity, it was reconstructed. Using RT-PCR, nested PCR and half nested PCR, three fragments were obtained from the total RNA of experimentally infected rabbit spleen. After correspondingly cloned in pMD18-T vector, they were sequenced. The corresponding sequence in 5'half cDNA and 3'half cDNA were substituted by recombinant. The two half cDNA were then ligated into full-length cDNA. After sequencing, it was verified that the 3 lethal mutation sites were corrected. Then it was transfected into SK-6 by lipofectin. The antigens and special sequence of CSFV was detected. The result indicated that the full-length cDNA have infectivity. The study successfully constructed the reverse genetic system of CSFV. The infective cDNA clone was used as a skeleton, the GP5gene of PRRSV was used as a marker gene and was inserted into the genome of CSFV right down the initiation codon ATG in Npro. After transfected the SK-6, a special sequence containing GP5 gene the antigens of CSFV were detected. The results indicated that the GP5gene was stably inserted in the CSFV genome even after 5or 10 passages and the antigen of CSFV can express in SK-6. Due to the field virus can be differentiated from the recombinant by detecting the special sequence containing GP5, and the marker is the major antigen gene of PRRSV, the recombinant maybe used as a marker vaccine to control CSF and PRRS. It is hopefully a live marker vaccine of CSFV C strain.