Expression Of Chicken Interferonα In Bacillus Subtilis And Its Activity Assay

ABSTRACT: Interferons (IFNs) are ubiquitous inducible cytokines with a powerful immunomodulatory activity on animal immune response in vivo and a broad-spectrum competence in antiviral and antitumor. Nowadays, recombinant IFN was successfully expressed in Escherichia coli, yeast, insect and even mammalian cells. While in prokaryotic expression system, the renaturation of the inclusion bodies within recombinant protein, the exogenous pyrogen of E. coli etc. were problems to be solved for E. coli host strain to express recombinant IFN. Bacillus subtilis, as a beneficial intestinal bacteria, has a huge capacity for secreting proteins directly into the growth medium, which greatly facilitates their downstream processing. In recent years, B. subtilis as a genetically engineered expression system developed rapidly with the development of molecular biology and genetic engineering. Besides, B. subtilis as a new drug or antigen delivery systems also show a promising prospect. In the current study, the nucleotide sequence was cloned to construct E. coli-B. subtilis shuttle expression plasmid pWBAO-ChIFN-a. Then it was was transformed into engineered B. subtilis WB800. Western blotting was used to detect the expression of recombinant ChIFN-a, while microplate cytopathic inhibition assay was employed to exam its antiviral activity.Part1:The construction of E. Coli-B. subtilis shuttle expression plasmid pWBAO-ChIFN-a.The ChIFN-a nucleotide sequence of489bp was correctly cloned using RT-PCR after the spleen RNA of immunized chicken was extracted. The construction of pWBAO-ChIFN-a solved the extremely inefficient problem of the ligation product to transform into B. subtilis. The recombinant shuttle plasmid was constructed based on pWB980. The E. coli origin of replication sequence and ampicillin resistance gene (1800bp) was inserted ahead of sacB signal peptide to make it successfully replicated in E. coli. After that, E. coli-B. subtilis shuttle expression plasmid pWBAO-ChIFN-a was constructed within DH5a.Part2:The study of the methods to transform pWBAO-ChIFN-a into WB800and the optimization of its expression.In the current study, pWBAO-ChIFN-a was transformed intoWB800using chemical transformation and electro transformation respectively. Chemical transformation:according to the nutritional needs of B. subtilis at different growth stages, WB800competent cells was prepared in the case of poor nutrition. Then transform pWBAO-ChIFN-α into the competent cells in a relatively mild way. Later,2-3transformants were obtained with this method. Electrotransformation:The recombinant shuttle vector was transformed into WB800by electric shock when the corresponding competence cells were prepared in a hypertonic solution. It is known that the voltage is a key factor affecting electrotransformation. Hence the electroporation voltage and the electric constant were determined at1.0-1.2kv/mm and4.5-5.5ms respectively after a large number of experimental exploration. We successfully harvested10～12transformants with this method. And the positive rate of them were100%when identified by bacterial PCR. Optimize the expression conditions of B. subtilis so as to determine the optimal expression time.Part3:The detection of rChIFN-α expressed in B. subtilis and its antiviral activity.The tests were divided into the sucrose inducible group and the non-inducible group.2%sucrose solution (final concentration) was added into the former group3hours post-inoculation in order to induce the expression of recombinant ChIFN-α, while no sucrose was added into the latter with the same culture conditions. The protein concentration was detected via CBA Kit. Moreover the molecular weight of rChIFN-α was about20ku when assayed using SDS-PAGE and Western Blot. Later, the virus neutralization assay was performed with the MDCK cells infected by NDV to detect its antiviral activity. In the test, the blank control, the positive control, the negative control and the experiment group were set to make the result more credible. The results showed that the2-6-dilution of the recombinant ChIFN-α extract failed to restrain the cytopathic effect. It suggested that the ChIFN-α do have biological inhibition activity against virus.The investigation results confirmed that intestinal bacteria B. subtilis was capable to express ChIFN-α with biological activity. The successful secretive expression of ChIFN-α also laid a foundation for the intestinal mucosal immune and the study on delivery carrier of drugs and immunogen. Whereas, Some heterologous genes have been expressed successfully in the B. subtilis expression system, but there are still one vital drawback, the low level of protein production, which limit its application potential.