Molecular Cloning Of Infectious Full-length CDNA Of Attenuated Lapinized CSFV Chinese-strain (Derived From Spleen)

Successfully cloned and constructed infectious full-length cDNA of attenuated lapinized CSFV Chinese-strain (derived from spleen) could make us get pure RNA virus genome of CSFV C-strain, and further study and utilize mutation, deletion, insertion and substitution of CSFV gene on DNA molecular level. It has made the strong basis for further studying mechanism of replication, virulence and determinant, attenuation, pathogenesis, functions of genetic products, specific diagnosis, cell and host tropism, development of DNA vaccine and marker vaccine of CSFV, and provided an excellent tool for molecular virology. Main research contents include:Based on published nucleotide sequences of CSFV and by the help of computer analysis software, high conservative regions and single restriction enzyme sites of genome were selected. Utilizing RT-PCR and nested-PCR techniques, 7 overlapping cDNA fragments covering the full genome of CSFV C-strain were successfully amplified. After 7 fragments were cloned into pMD-18T or pGEM-T Easy vectors and transformed into E.coli JM109 respectively, genomic cDNA library were constructed. Genomic nucleotide sequence was spliced after 7 fragments were sequenced. Sequence comparison with other strains of CSFV revealed that the overall nucleotide sequence homologies of self-sequenced C-strain are 95.3% with Shimen, 94.9% with Berscia, 95.1% with Cap, 96.0% with Eystrup, 95.6% with F114, 95.2% with Glentorf, 98.9% with Riems, 93.6% with Russia C-strain and 85.4% with Alfort, the overall deduced amino acid sequence homologies of self-sequenced C-strain are 96.6% with Shimen, 96.7% with Berscia, 96.2% with Cap, 97.1% with Eystrup, 97.0% with F114, 96.1% with Glentorf, 98.9% with Riems, 95.5% with Russia C-strain and 92.4% with Alfort.T7 RNA polymerase promoter and helper restriction enzyme site were introduced into 7 overlapping cDNA fragments severally using gene modification techniques. Recombinant plasmid pMD-18T/F34 of connected fragment F3-F4 had been obtained after PCR amplified fragments F3 and F4 were both digested by restriction enzyme Ace III and further connected and cloned into pMD18-T vector by T4 DNA ligase. Recovery fragments from recombinant plasmid pMD-18T/Fl double-digested by Not I & Pvu I, pGEM-T Easy/F2 by Pvu I & Nsi I, pMD-18T/F34 by Nsi I & Sal I, and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct 5'half-length cDNA pGEM-5zf(+)/Fl-4 whose length is 6 517bp. Recovery fragments from recombinant plasmid pMD-18T/F5 double-digested by Not I & Fsp I, pMD18T-18T/F6 by Fsp I & Csp45 I, pMD-18T/F7 by Csp45 I & Sal I, and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct 3'half-length cDNA pGEM-5zf(+)/F5-7 whose lengthis 5 940bp. Finally recovery fragments from pGEM-5zf(+)/F1-4 double-digested by Not I & Spl I, pGEM-5zf(+)/F5-7 double-digested by Spl I & Sal I and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct full-length cDNA pGEM-5zf(+)/F1-7. artificially using T-A cloning techniques and initiated one-step ligation of multi-fragments method were the main factors in the process of full-length cDNA construction.Constructed full-length cDNA pGEM-5zf(+)/Fl-7 was linearized by Srf I so as to obtain the exact 3'terminal of genome. Linearized full-length cDNA was used as template then genomic RNA of CSFV was in vitro transcriped by T7 RNA polymerase. Genomic RNA was transfected into plating cells SK-6 by liposome transfection reagent and transfected cells were passaged 10 times continuously. Cells were collected after being passaged 5 times and used to identify infection by RT-PCR, direct fluorescent antibody, sandwich ELISA. Results indicated that integrated particles of CSFV could be obtained from constructed full-length cDNA. Successful construction of infectious full-length cDNA of CSFV C-strain has provided an excellent tool for further study CSFV C-strain on level of molecular biology.