Assembly of pseudorabies virus genome-based transfer vehicle carrying major antigen sites of S gene of transmissible gastroenteritis virus: potential perspective for developing live vector vaccines

Abstract

Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.

Fig. 1

Construction of transfer vector pUGS-LacZ. After construction of a recombinant pUG via inserting Ga and Gb of PRV into vector pUC119, another continuous fragment containing S1 of TGEV, MCS of vector pPROEX HTc as well as SV 40 polyA sequences was introduced into gG promoter downstream of pUG. Finally, a reporter gene LacZ controlled by the immediate early promoter of CMV was located at the PstI site of pUGS-LacZ. For details, see Section 2.

Fig. 2

Identification of recombinant pUGS-LacZ. (A) Identification of S1 fragment of TGEV by PCR: lane 1, the expected PCR product, about 2210 bp; lane 2, negative control. (B) Identification SV40 polyA fragment by PCR: lanes 1–2, negative controls; lane 3, the expected PCR product, about 548 bp. (C) Identification of pUGS-LacZ with restriction enzyme digestion: lane 1, pUGS-LacZ digested with PstI produced two bands, about 7675 bp (recombinant pUGS) and 4300 bp (LacZ expression cassette) respectively; lane 2, pUGS-LacZ digested with NspV, about 11,975 bp. (D) Validation of the MCS in pUGS-LacZ: lanes 1–3, pUGS-LacZ digested with StuI, SstI, SpeI respectively produce the linear pUGS-LacZ, about 11,975 bp as calculated. The DNA marker used here was designated M.

Fig. 3

Recovery of recombinant virus rBartha/S1. (A) S1 region (containing S1 fragment, SV40 PolyA, LacZ and MCS sequences) was integrated into parental virus genome. (B) The recombinant virus carrying S1 region. Therefore, gG gene is not intact due to the insertion.

Fig. 4

Blue plaques on Vero cells in presence of X-gal (×150). (A) Uninfected Vero cell monolayer. (B) Blue plaques appeared after adding X-gal on Infected Vero cell monolayer with rBartha/S1, several of which have been indicated with black arrows.

Fig. 5

PCR analysis of recombinant virus rBartha/S1. Lane 1, the PCR product from genome of rBatha/S1, about 1344 bp as expected. Lane 2, the PCR result from genome of BarthaK-61. Lane 3, The PCR result from pUGS-LacZ. Lane 4, negative control for PCR reaction.

Fig. 6

Multistep growth curve of Bartha K-61 and rBartha/S1. Vero cells monolayer in 35-mm-diameter dishes was infected with virus at a MOI of 0.001 and incubated at 37 °C in DMEM containing 5% fetal bovine serum. Supernatants were harvested at 6-h interval for virus titration. In this panel, A, B and C are the values of triplicate experiments, respectively.