逆转录病毒载体介导表达伪狂犬病病毒PK基因MDBK细胞系的建立

Construction of MDBK Cells Expressing Pseudorabies Virus PK Gene by Retroviral Expression Vector

Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant plasmid pLXSNPK was co transfected with packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (300 mg/L) for 2 weeks. Viral supernatants gaining stable cell clones were collected, the total RNA was extracted and the PK gene was amplified by RT PCR. It was shown that the PK gene had been inserted into the retroviral genome. The positive viral supernatant was collected to infect the interesting target cell MDBK. The infected MDBK cells were selected by G418 (800 mg/L) and analyzed by indirect immunofluorescence and SDS PAGE and Western blotting. It was confirmed that a MDBKPK cells expressing PK protein was obtained. It is useful to study the biological function of PrV PK gene in the process of PrV inducing culture cells apoptosis.