An Efficient Method For The Construction Of Recombinant Pseudorabies Virus Vector And Its Delivery To Mammalian Cells

Pseduorabies virus (PRV) is a swine herpesvirus that belongs to Alphaherpesvirinae subfamily. It causes Pseudorabies disease of swine and results in enormous economic losses worldwide every year. PRV can infect a wide range of mammalian and avian hosts. However, it is non-pathogenic to human and other high-order species of primates. Swine is the natural host of PRV. PRV has been of interest to those who concerned about disease control in swine agriculture. On the other hand, PRV has been served as an excellent model to study the gene function and molecular processes of herpesviruses in general. The virus has also been used as a "live" tracer of neuronal pathways, making use of its remarkable propensity to infect synaptically connected neurons.PRV has a large double-strand DNA genome (about 150 kb) with some region that can be replaced with foreign fragments. PRV is highly neurotropic and may be latent in the neural tissues. Therefore, PRV can also be used as a vector for gene therapy or vaccine manufacture, which has been used to delivery and express target genes in mammalian cells or product polyvalent vaccines. The classic technique for inserting foreign genes to PRV genome is based on homologous recombination between a transfer plasmid and the virus genome in mammalian cells. This method usually requires laborious screening or selection. PRV genome had been cloned as a BAC vector which enables manipulation the PRV genome DNA in E. coli.In this study, a simple clone approach——mating-assisted genetically integrated clone (MAGIC) for the rapid construction of recombinant PRV vectors was developed based on PRV-BAC plasmid pBecker2, and a efficient method for transferring PRV-BAC vectors DNA into mammalian cells through bactofection was described here.1. The construction of Escherichia coli DH10B-IS2 expressingⅠ-SceⅠ.The araC gene was amplified with pBAD-TOPO as template and cloned into the vector pML294 to generate the plasmid pML294C. pML294C was digested with EcoRI/KpnI, and the fragment containing umuC-araC-ParaBAD-Ⅰ-SceⅠ-FRT-npt-FRT-umuC was transformed into E.coli DH10B, followed by insertion into the umuC locus by homologous recombination. This strain is referred as E.coli DH10B-ISK. The npt was later removed in vivo through Flp-mediated excision using plasmid pCP20 containing the Flp gene to generate strain DH10B-IS2.2. Construction of the recipient plasmid pBecker2-KHThe kanr gene cassette (H1-Ⅰ-SceⅠ-kanr-Ⅰ-SceⅠ-H2) was amplified with pShuttleK as a template and electroporated into E.coli JPBecker2 in which theλrecombinase was induced during the period of the preparation of the electroporation-competent cells. The recipient plasmid pBecker2-KH was generated by insertion of the kanr gene cassette into TK gene of pBecker2 via homologous recombination. Sequence analysis demonstrated the kanr gene cassette was inserted precisely in the target site and the sequence of the homologous regions (H1 and H2) used to target recombination was correct. Restriction map analysis and Southern blot analysis indicated the vector backbone had no obvious insertion or deletion and the recombination occurred only at the target site. pBecer2-KH was transfected into PK-15 cells. Plaques were observed 2-3 days postinfection, indicating pBecker2-KH was able to produce infectious virus particles (vBec2-KH).3. Construction of recombinant PRV plasmids through MAGIC methodThe donor strain DH10βcontaining pRTRA and the recepient strain DH10B-IS2 bearing the plasmid pML300 and recipient plasmid pBecker2-KH were incubated and mixed. The mixture was diluted and spread on the LB plates supplemented with Cam (12.5μg/ml), Amp (100μg/ml) and Ara (0.2% w/v). The antibiotic-resistant colonies were further verified by colony PCR. The result demonstrated all Ampr resistant colonies containing the expected recombinant plasmid pBecker2-red. Sequence analysis confirmed no point mutation or frame shift present at the insertion site. Restriction map analysis and Southern blot analysis indicated the vector backbone had no obvious insertion or deletion and the recombination occurred only at the target site. pBecer2-red was transfected into PK-15 cells. Plaques emitted red fluorescence under fluorescence microscope were observed 2-3 days postinfection. The virus vBecker2-red was harvested from the cells. Single-step growth curves of vBecker2-red and vBecker2-KH were indistinguishable from the parental vBecker2.The recombinat vector pBecker2-O5 which cloned ORF5 of PRRSV was constructed by MAGIC method, and transformed into PK-15 cells. The recombinant virus vBecker2-O5 was acquired. The ORF5-6×His infusion protein expressed in PK-15 cells was detected by Western blot through anti-His antibody.4. The clone efficiency of MAGIC methodPlasmid pRThGA which contains gfp expression cassette between H1 and H2 was constructed and used as another donor vector. After mating, the mixture was incubated on the LB plates supplemented with Cam and Ara. The colonies were picked randomly and identified by colony PCR. Forty-three of 48 randomly selected colonies containing the expected recombinant vector pBecker2-gfp. The efficiency of correct recombination catalyzed byλ-Red recombinase was high as 89.4±8.5%.pBecker2-gfp was transfected into PK-15 cells. Plaques emitted obvious green fluorescence under fluorescence microscope were observed 2-3 days postinfection. The virus vBecker2-gfp was harvested from the cells. The GFP-6×His infusion protein expressed in PK-15 cells was detected by Western blot through anti-His antibody.5. Invasive E.coli delivers BAC DNA into mammalian cellsThe plasmid pGBQinv-hly contains Yersinia pseudotuberculosis invasin gene and Listeria monocutogenes listeriolysin O (LLO) gene. pGBΩinv-hly was transformed into E.coli DH10B (pBecker2), DH10B(pBecker-KH) or DH10B(pBecker2-red) to generate invasive E.coli DH10B-pBGihl, DH10B-pBGih2 and DH10B-pBGih3, respectively. Each of the invasive E.coli DH10B stains was used to bactofect PK-15 cells. Cytopathic effects were observed, and productive viruses were harvested from the cells. Plaques from the sample infected by DH10B-pBGih3 were observed under fluorescence microscope, which emitted red fluorescence.pBecker2 DNA can be directly transferred from E.coli DH10B-pBGihl to PK-15, IBRS-2 and Hela cells. However, it can't be deliveried to Vero cells.6. Viral titers varied with multiplicity of infection and time of incubationTo determine the optimal multiplicity of infection (MOI) and time of incubation in the process of E.coli-mediated gene delivery, E.coli DH10B-pBGihl was incubated with IBRS-2 and Hela cells with at different MOIs ranging from 0 to 3000 or different time ranging from 10 min to 180 min. High-titer PRV stocks were generated from IBRS-2 cell culture at MOIs of 300 to 3000, or from Hela cell culture at MOIs of 600 to 3000. The high level PRV stocks was generated at a time rang of incubation of 60-150min. 7. Invasive E.coli can not deliver BAC DNA into miceDH10B-pBGihl (109 CFU) was injected intraperitoneally or intramuscular into each mouse. Main organs of the mouse, such as livers, spleens and brains etc were collected, and organ homogenates were tested by plaque assay on PK-15 for the presence of infectious PRV. No infectious PRV was detected from the samples above. The mice immunited with DH10B-pBGihl can not surviv the challenge with lethal doses of PRV.In conclusion, we introduced a simple method to construct PRV-BAC vectors through MAGIC strategy and an efficient method to deliver BAC DNA to mammalian cells through invasion of E.coli. The simple and low-cost methods described here may not only greatly extend the potential of PRV as an expression vector but also be adapted to construct other kinds of herpesvirus BAC vectors and delivery corresponding vectors into mammalian cells.