[Construction of baculovirus vector with Cytomegaloviruse promoter to express eGFP in primary chicken embryo cells].

OBJECTIVE

Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells. The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter, VSV-GED and different functional regulatory elements. The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells, which provides us a new approach to develop engineered poultry vaccines.

METHOD

The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor, VSV-GED, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs). The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells, and the eGFP protein expression levels mediated by different baculoviruses were analyzed.

RESULTS

The expression of eGFP was detected at 12 hours after infection. The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED. The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10 mmol/L butyrate. However, the WPRE elements are nontoxic to cells. Within 72 hours, the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually.

CONCLUSION

The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells. The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.