Safety Evaluation Of Aspergillus Niger ANSTJ01 Strain And Expression Of EtMIC2 Protein Of Chicken Coccidiosis In Aspergillus Niger

Filamentous fungi have a very strong ability of protein secretion.Filamentous fungi have unique advantages over the prokaryotic expression system and yeast system.Among them,Aspergillus Niger has the advantages of high extracellular efficiency,large expression,natural activity and protein modification mode approaching to higher eukaryotes.Aspergillus niger as a cell factory is widely used in many production areas,such as enzyme preparation,heterologous protein,organic acid,antigen production,and so on.FDA and WHO in the United States identified A.niger Generally regarded as safe microorganism.Through the study Eimeria tenella microneme protein 2 can be used as a candidate antigen for blocking the sporozoite invasion of animal host cells.In order to obtain a large number of high efficient antigens,the high efficient expression of EtMIC2 protein must be realized.In Aspergillus niger expression system,which not only has simple operation,fast growth,fermentation expression,fermentation has the advantages of low cost high,but also has higher eukaryotic expression of various posttranslational modification systems,such as protein phosphorylation and glycosylation function etc..The immunological activity and biological function of the product expressed by system are close to that of natural protein.Therefore,the construction of Aspergillus niger as a carrier provides an important support for the research and development of animal production.Aspergillus niger 2485(cellulase strain)were isolated,after verifying the biological safety of the strain Then.we use the strain to express EtMIC2 protein and display EtMIC2 on the AN.niger cell surface.The specific research is as follows:1.The biosafety test of Aspergillus niger ANSTJ01 strainThe toxicity test of Aspergillus Niger culture was carried out.blood routine examination,histological section and intestinal examination were employed as the basis of judgement.During the test period,the strain could not lead to the death of the experimental chicks,no abnormal blood routine.No hypha was found in the tissue sections,and the intestinal tract did not cause lesions.2.Construct express system of Aspergillus niger and optimize the transformationThe isolated strains of Aspergillus niger were screened by antibiotics,and the suitable resistance marker genes were selected according to different concentrations and different antibiotics.Screening suitable genetic marker antibiotics.In YTD solid medium,30 degree static training,the experimental results show that 200 mg/ml HYB has the most obvious effect.The internationally recognized Aspergillus niger strong promoter PglaA(amplified Aspergillus niger isolated from laboratory)has been studied in the promoter region.The exocrine GlaA singal,and Hyg gene fragment of Aspergillus strains were integrated with PglaA,and the target plasmid was successfully constructed and named PPSHD.The Et MIC2 gene was successfully cloned and successfully incorporated into the PPSHD-GFP target plasmid constructed successfully.At the same time,the plasmid PPSHD-CBM-EtMIC2 was constructed by adding CBM anchored protein gene fragment before the Et MIC2 gene fragment.By changing the carbon source,nitrogen source,increasing solid attachment and changing the fermentation temperature,the fermentation conditions of Aspergillus niger were optimized,and the expression level of target gene was increased.In 48 h,mycelium fluorescence of mycelium fluorescence intensity of maltose fermentation liquid > fluorescence of glucose fermentation liquid > fiber two sugar fermentation liquid > microcrystalline cellulose fermentation liquid.In the xylose fermentation broth,the weak fluorescence was detected.The fermentation condition of maltose concentration was the best.The growth medium and growth conditions of Aspergillus niger were changed,the lysase system of protoplast was optimized and the yield of Aspergillus niger protoplast was increased.The protoplast transformation technique was used to transfect the transient plasmids and homologous recombinant linearized fragments to Aspergillus niger strains.The results showed that the positive rate of transient plasmid transformation reached 83%,and the homologous recombination efficiency reached 48%,which was significantly lower than that of transient plasmid transformation.However,the positive plasmid was inserted into the genome randomly,while the homologous recombination fragment was inserted into the designated location.In this study,the EtMIC2 gene wassuccessfull cloned and insected into PJET1.2 plasmid and successfully joined it into the PPSHD-GFP expression plasmid and PPSHD-CBM plasmid successfully constructed in our laboratory.The positive strains were screened by the enzyme digestion and the sequencing results were not correct.The target plasmid was linearized by AvrII restriction enzyme,and the target gene fragment was successfully transfected into Aspergillus niger by the original plasmid technology.Homologous recombination technology was used to reconstitute the target gene to the fixed site in the genome.The positive strains were screened by PCR technology and the target gene was successfully expressed by fluorescence microscopy.The surface protein of the surface was detected by IFA.In the later period,the expression of the protein was increased by changing the fermentation conditions,and the expression of the target protein was successfully verified by Western-blot technology.the production of the aim protein was improved.