Establishment Of A Quick Method For Purification Of Adenoviruses And Constrution Of Recombinant Adenovirus Co-expressing HLYZ And BoIFN-γ

Adenoviral vectors have been widely used as the gene transfer vectors for gene therapy and vaccine development with advantages of wide host range, high efficient transduction, low pathogenicity, and low risk to cause insertional mutagesis. Several methodologies have been developed for concentration and purification of Ad vectors, but these methods are usually laborious and expensive, as well as requirement for specialized equipment.Bovine mastitis is one of the most common dairy cow diseases worldwide causing heavy economical losses to dairy cow industry. The control of this disease relies mainly on antibiotics treatment of lactating cows and internal teat sealing during dry off, which resualts in the widespread of antibiotic-resistant bacterial strains. Therefore, new strategies for more efficient control of the disease are urgently needed. Lysozyme (LYZ) is a well-known muramidase with potent antibacterial activities against a variety of microorganisms. It has been shown that intramammary injection of LYZ-expressing plasmid vector had long-lasting therapeutic effect against bovine mastitis. Interferon-γ (IFN-γ) is a multifunctional cytokine with antiviral, antiproliferative, immunomodulatory and antiangiogenesis effects. The recombinant BoIFN-γ expressed in Escherichia coli (E. coli) has also been used to treat bovine mastitis. Therefore, the recombinant adenovirus co-expressing LYZ and IFN-γ is expected to have better therapeutic effect against bovine mastitis.In this study, we established a single-step method for Ad concentration and purification by combing coxsachievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. Then, we screened a better eukaryotic internal ribosome entry sites (IRES) from 5 IRESs for construction of co-expression adenoviral vector. Finally, we generated a recombinant adenovirus co-expressing hLYZ and BoIFN-γ for treatment bovine mastitis.Expression and purification of recombinant ELP-CAR protein:To construct a CAR-ELP fusion protein expression vector, the coding sequences for ELP, CAR spacer and CAR-D1 domain were inserted into pET-30a vector, and the resultant pET-ELP-CAR vector was transformed into BLR E. coli. The expression of ELP-CAR fusion protein was induced at 20℃ with IPTG. The ELP-CAR fusion protein was purified from the cell extract by ELP-mediated inverse transition cycling (ITC) using optimized conditions. The results showed that the ELP-CAR fusion protein was expressed in a soluble form. After first round of ITC, the purity of ELP-CAR fusion protein was 89.4%, which was increased to 94.5% after an additional round of ITC. Western blotting showed that the purified ELP-CAR fuaion protein was recognizable by anti-CAR serum. These results showed that the ELP-CAR fusion protein was purified successfully in this study.Establishment of a single-step method for concentration and purification of adenoviruses:To confirm the specific binding of ELP-CAR protein to adenoviruses (Ads), the rAd-GFP expressing green fluorescent protein (GFP) gene was incubated with ELP-CAR or His-ELP protein for 1 h at 4℃, and then precipitated by one round of ITC. Both bound (pellets) and unbound fractions (supernatants) were collected for DNA extraction and PCR analysis using GFP-specific primers. An expected 720-bp product was amplified from the bound fraction of ELP-CAR-incubated rAd, but not from that of His-ELP-incubated rAd. To optimize the conditions for rAd concentration and purification, the clarified culture medium or extract of rAd-transduced cells was incubated at different temperatures and pH for different times with different concentrations of ELP-CAR protein. The protein-bound rAd was precipitated by one round of ITC. Viral titration assay showed that the binding of ELP-CAR protein to rAd-GFP was dose-dependent, and slightly pH-, temperature-and time-dependent. To optimize the conditions for rAd elution, the ELP-CAR protein-bound rAd-GFP was incubated at different temperatures, pH for different times, and the released ELP-CAR protein was removed by an additional round of ITC. Viral titration assay showed that the elution of rAd off ELP-CAR protein was pH-, time-dependent and slightly temperature-dependent. The rAd was purified from the culture medium or cell extract of transduced cells by one round of ITC under the optimized conditions with recovery of 76.2% or 73.3%. The rAd was eluted off and the released ELP-CAR protein was removed by one round of ITC with rAd recoveries ranging from 30.6% to 34.5%(viral titration assay). The final rAd recovery of this method was comparable to that for high performance liquid chromatography process and cesium chloride density-gradient centrifugation. Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). Further viral titration assay showed that the ELP-CAR-bound rAd had significant lower transduction efficiencies than the eluted rAd, which were decreased slightly or significantly in the presence of fetal bovine serum. In addition, the rAd was efficiently recovered from the "spiked" PBS and top water with recovery of-74%or-60%. This work demonstrates the usefulness of the ELP-CAR protein-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.Construction of a recombinant Adenovirus co-expressing hLYZ and GFP:The sequences for IRESs from encephalomyocarditis virus, eukaryotic initiation factor 4G, human vascular collagen inducible protein, mouse vascular collagen inducible protein and homeodomain protein Gtx were amplified by PCR or chemically synthesized. Each of the five IRESs was cloned into NotI and Xholl sites of pShuttle-CMV vector, resulting in the co-expression vector pShuttle-IRES. The coding sequence for hLYZ and GFP were amplified from pcDNA-LYZ and PEGFP-Nl vector and then inserted into pShuttle-IRES vectors, resulting in five co-expression vectors pShuttle-LYZ-IRES-GFPs. After confirmation of their correct identity, the five bicistronic constructs were transfected into cell lines PK-15, AAV-293, CHO-K1 and MDBK, and the downstream GFP gene expression was determined by FACS. The results showed that EIF4G IRES had significantly higher cap-independent expression efficiency than other IRESs.Constrution and identification of the recombinant adenovirus co-expressing hLYZ and BoIFN-y:The coding sequence for BoIFN-ywas amplified from pGEX-IFNyvector. The coding sequence for GFP in pShuttle-LYZ-EIRES-GFP vector was replaced with the PCR-amplified BoIFN-ysequence, resulting in LYZ and IFNyco-expression vector pShuttle-LYZ-EIRES-IFNy. The vector was transformed into E. coli and a homologous recombinant plasmid was generated by homologous recombination. After transfection into AAV-293 cells, the recombinant adenovirus rAd-LYZ-EIRES-IFNywas generated. Typical morphology of the rAd was confirmed by electron microscopy. The presence of LYZ and IFNycoding sequences in the viral genome was confirmed by PCR detection. In the rAd-transfected PK-15 cells, expression of hLYZ and BoIFNywas detected by RT-PCR and indirect immunofluorescence.