Construction of the full-length pseudorabies virus TNL strain genome as the bacterial artificial chromosome

• Main Authors: Pei-Yu Wu, 吳沛宇
• Other Authors: Tien-Jey Chang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/59281304081566925981

碩士 === 國立中興大學 === 獸醫學系 === 90 === Construction of full-length infectious clones of virus genome is useful for the study of viral pathogenesis and the application of vaccine development. We used the recently developed bacterial artificial chromosome (BAC) as a vector to carry 150 kb full-length genome of the pseudorabies TNL strain (a Taiwan local strain). A green fluorescent protein (eGFP) expression cassette flanked by two 34 base loxP sites was inserted into the segment of 11 k and 28 k genes for the homologous recombination. After 15 passages, green plaques were cloned and named TNL-GFP. By using the method of cre-loxP site-specific recombination, dark plaques, the TNL-loxP, were picked and proved to have the loxP site insertion of PRV genome by sequencing. We modified pCC1TM BAC vector from Epicentre to be inserted with another GFP cassette. This BAC vector containing a loxP site and the GFP expression cassette was integrated into PRV genome again by cre-loxP recombination thus resulted in green fluorescent plaques, named PRV-BAC. By plaque assay, we found the insertion of a GFP cassette between 11 k and 28 k genes would reduce the viral titer. Moreover, another large insertion as BAC vector could decrease the plaque size of PRV。By sequencing of the PCR fragment, we sugessted there might be rearrangements in TNL-BAC genome, thus the inserted GFP fragment could not be detected by PCR. Therefor the insertion of BAC vector was still identified by the dot-blot hybridization with linearised pCC1 as probe。After transforming E. coli with TNL-BAC infected cell DNA by electroporation, the plasmids from chloramphenicol resistant colonies were purified. However, these plasmids can only be detected to have the pCC1 sequence by dot-blot hybridization, but they didn’t show any detectable PRV-TNL sequence by PCR.