| Interferon gamma (IFN-γ) is also known as a kind of immune interferon, mainly produced byactivated T cells, NK cells, which plays a role in inhibiting viral replication, antitumor, antipar-asitic activity and immune regulation. With the development of canine industry, species quantity ofdogs is increasing, especially viral disease harm to health and survival of dogs. So as an importantimmunopotentiator, canine interferon gamma (CaIFN-γ) has a good application prospect in preven-tion, immunotherapy and vaccine adjuvants for viral disease.For obtaining the CaIFN-γ antiserum from rabbit, the CaIFN-γ gene was digested by EcoR I,Sal I and then inserted into plasmid pET30a to construct recombinant plasmid pET30a-CaIFN-γ,final-ly transformed into Escherichia coli. The expression results of CaIFN-γ protein by westernblot and SDS-PAGE shows that, the recombinant protein was about23Ku and mainly existed inthe form of inclusion body. The rabbit was injected intramuscularly with purified recombinantprotein to obtaining CaIFN-γ antiserum. The serum titer is1:12800.Based on the Codon bias of amino acid of lactic acid bacteria, CaIFN-γ gene is optimized andsynthesized. Secreted plasmid pAMJ399induced by Lactococcus lactis pH is used as expressionvector, to construct the recombinant plasmid pAMJ399-CaIFN-γ, and then pAMJ399-CaIFN-γ istransformed to Lactococcus lactis PSM565by electricity facing, to construct recombinant Lactoco-ccus lactis pAMJ399-CaIFN-γ/PSM565, which induced by the acid production. SDS-PAGE resultsshow that there is about20ku protein in cells; western blot results show that there is about20kuprotein in the culture supernatant and the cells, which have specific reaction with CaIFN-γantiserum from rabbit. So CaIFN-γ protein is expressed in recombinant Lactococcus lactis, whichcan exist in medium in the form of secretion and in cells.In order to improve the expression level of recombinant Lactococcus lactis, the growth curveand the effect of pH on growth curve to determine the fermentation conditions. The results showedthat32℃, stirring speed50rpm, and adding50%glucose with0.5s/min rate after6h can increasethe rate of live bacteria of recombinant lactic acid bacteria, in which pH was maintained at5.5and5.75, expression level of protein in the supernatant were more than which in pH6and6.25.For the detection of antiviral activity of CaIFN-γ expressed in recombinant Lactococcus lactis, supernatant of recombinant strains is filtered and incubated with MDCK cells for24h, Cytopathiceffect can be observed by the microscope when cells is inoculated vesicular stomatitis virus (VSV)for30h. The results showed that cells treated with CaIFN-γ are good, cells of control withoutCaIFN-γ are wrinkled, agminated, round, deciduous. The statistical analysis results by MTTshowed that5times concentrated supernatant group with CaIFN-γ compared with virus group havesignificant difference extremely (P<0.01), CaIFN-γ of supernatant of recombinant Lactococcuslactis have antiviral activity; virus load of MDCK cells treated with interferon is detected by SYBRGreen real-time quantitative PCR methods. The results showed that pAMJ399-CaIFN-γ/PSM565group have significantly difference extremely (P <0.01) compared with pAMJ399/PSM565groupand virus control group, Virus load of MDCK cells treated with supernatant of recombinant strainspAMJ399-CaIFN-γ/PSM565is lower than which in virus control group.It means that CaIFN-γexpressed in recombinant strains have obvious antiviral effect.The above results show that CaIFN-γexpressed in recombinant Lactococcus lactis providing a reliable basis for antiviral clinicalapplication and research of cytokines as adjuvants. |
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