Cloning And Expression Of Canis Interferon α Gene And The Study Of Its Modified Product

As economy developing, raising dogs is increasingly popular; however, the threat of animal pathogens, particularly viruses, is also becoming more serious; it usually causes a fatal disease in dogs and loss in humans both psychologically and economically. Interferon (IFN) is a cytokine family with antiviral activity; among them, interferon a (IFNa) has been used in the treatment of virus infectious diseases. Canis IFNa (CaIFNa) has been proved to be effect in the treatment of many viral diseases, thus it has promising clinical potential. But the traditional production method had low yields and high costs, causing the high price of CaIFNa. Moreover, due to the short activity of CaIFNa, it needs to be injected frequently and has long treatment cycle. As a result, cheap CaIFNa with long activity needs to be developed.Large amounts of protein can be obtained through genetic engineering, but detailed reports about how to get active CaIFNa through genetic engineering were not much. Amony them, escherichia coli (E.coli) was the most widely used system, and the majority of CaIFNa was expressed as inclusion body. Inclusion body needs to be renatued, the yield of which is usually low. Thus researchers usually prefer soluble protein. Here we used E.coli as expression system and three fusion proteins were obtained by fusing CaIFNa with different tags. Fusion CaIFNa was expressed as inclusion body or soluble protein; by comparing them, we aimed to produce large amounts of active CaIFNa.Nano drug carrier, especially those made by amphiphilic polymer, can prolong drugs’ acivity and deliver drugs to target position. Thus the activity of CaIFNa may be prolonged with drug carriers. Polyglutamic acid (PGA), a kind of macromolecule with cheap source and low toxicity, has been proved to be suitable for carring drugs. But PGA is soluble in water and can’t form particles automatically, so it can’t carry CaIFNa directly. Here we hydrophobically modified PGA with phenylalanine ethyl ester (PAE). The PGA-PAE material has hydrophilic backbone and hydrophobic segment, thus it can self-assemble into nanoparticle (NP) in water. Also, it can encapsulate Trx-CaIFNa (NP-CaIFNa) during self-assembling. We prepared NP and NP-CaIFNa, characterized them, optimized the preparion process, and studied their properties.The results can be devided into two parts:1. Cloning, expression, purification and activity assessment of CaIFNaThe sequence of CaIFNa was optimized according to E.coli’s preference codon. We fused CaIFNa to three different protein tags:thioredoxin (Trx), glutathione S-transferase (GST), and NusA (Nus) and got three fusion proteins:Trx-CaIFNα、 GST-CaIFNa and Nus-CaIFNa. Amony them, Trx-CaIFNa’s expression level was the highest (71.9%), while Nus-CaIFNa’s solubility was the highest (100% when the induction temperature was 25℃). Trx-CaIFNa was refolded and purified, while Nus-CaIFNa was purified under native conditions. The purity of Trx-CaIFNa and Nus-CaIFNa was greater than 90%, and their yields were 74.8% and 6.5%, respectively. Both Trx-CaIFNa and Nus-CaIFNa had antiviral activity in vitro. Their anti-viral activity was (2.14±2.79)×1014 U/mol and (1.87±0.92)×1012 U/mol, respectively, on Madin-Darby canine kidney cells. Trx-tag was removed from Trx-CaIFNa using enterokinase. The total yield following the digestion was 38%, and the antivirl activity of CaIFNa was (1.87±0.76)×1015 U/mol.Here we used Nus-tag and lowered temperature to soluble Nus-CalFNa. The soluble protein didn’t need refolding, saving time and cost, and this method can be studied. The expression level of inclusion body Trx-CaIFNa was the highest among other two fusion proteins, and was even higher than CaIFNa in other reports. This study also proved that fusion protein had activity without removing tags.2. The preparion and study of NP-CaIFNaPolyglutamic acid was fermented by Bacillus subtilis DL. PGA with 15-30kD was prepared by precipitation and acid-degradation-method. PAE was attached to PGA via an amide bond with water-soluble EDC as catalyst. The PGA-PAE material was confirmed by NMR. To obtain NP, the PGA-PAE material was mixed with water of same volume. It was monodispersed nanoparticle with uniform size. When the concentration of PGA-PAE was 40mg/mL, the degradation time was 9min and the rate of charge of PAE was 0.68, NP’s diameter was 132nm, its Polydispersity Index (PDI) was 0.114, zeta potential was 40.7mV and its concentration was 1.77x1013 particles/mL. To investigate NP-CaIFNa, we used Trx-CaIFNa as a model. PGA-PAE material was mixed with Trx-CaIFNa solution of same volume. NP-CaIFNa was also monodispersed nanoparticles. Under the optimized preparation condition, NP-CaIFNa’s diameter was 165nm, its PDI was 0.114, zeta potential was 40.7mV, and its concentration was 7.29x1012 particles/mL. The antiviral activity of NP-CaIFNa was (0.44±3.59) x1014U/mol, which meant that it retained about 20% of Trx-CaIFNa activity. The Trx-CaIFNa-release-experiment proved that NP-CaIFNa was stable in PBS, but would release Trx-CaIFNa when incubated with serum:about 30 days was required for 80% protein’s release. Thus NP-CaIFNa may have prolonged activity in vivo.In conclusion, we obtained Nus-CaIFNa and Trx-CaIFNa by genetic engineering. Nus-CaIFNa was soluble, thus the method of soluble CaIFNa may worth studying. Inclusion body Trx-CaIFNa had the highest expression level, being 71.9% when the inducing temperature was 37℃. Anyway, we developed two pathways for producing large amounts of CaIFNa. We prepared NP-CaIFNa with PGA-PAE material. We proved that NP-CaIFNa was monodispersed nanoparticles and had in vitro antiviral activity. Also, it could release Trx-CaIFNa when incubated with serum, suggesting that it may have long-lasting activity in vivo. We used amphiphilic nano drug carrier to carry CaIFNa for the first time, and developed a new way for preparing CaIFNa with prolonged activity.