Eukaryotic Expression Of PTP2 Gene Of Encephalitozoon Cuniculi In The Blue Fox (Alopex Lagopus) And Its Immunogenicity Study

As one of the important economic animals in the international fur market,blue fox has a large number of breeding bases in northern China,especially in Liaoning province,which has the largest fur trading market in Asia.Microsporidiosis in blue foxes is mostly an insidious infection,and there are many infected people with developmental stagnation,rapid spread within the group,and vertical transmission is difficult to control,thus leading to the culling of animals including breeding animals,and there are no strong and effective preventive and therapeutic measures.The Encephalitozoon cuniculi targeted in this study is a microsporidial pathogen that infects blue foxes,specializing in intracellular parasitic organisms of the eukaryotic fungal kingdom.It causes the most pronounced lesions mainly in the brain tissue and kidney of blue foxes,and is also a key concern and potential zoonotic disease.Mature spores are about 1～3μm in length and have three spore walls,which are highly resistant and can germinate and pop out unique infestation structures,the diodes,under certain water environment.Ec PTP1 and Ec PTP2,identified in the genus Encephalitozoon,are two important proteins that constitute the basic structure of the transistor and have good medical research and application value.In order to break the barrier of immunoprophylaxis constructed by Microsporidia,the immunogenicity study of Ec PTP2 gene was conducted as follows.1,E.cuniculi was isolated and purified from the disease material and identified by ITS1 gene PCR and Gram staining,PTP2 gene was amplified and p MD18T-PTP2 vector was constructed,and PTP2 gene bioinformatics analysis was performed.2,Then the positive blue fox serum Ig G was crude extracted by ammonium sulfate precipitation method and stored for spare.No TAA-PTP2 primers were designed and PCR amplified to construct pcDNA3.1-PTP2 and pcDNA3.1-EGFP-PTP2 recombinant vectors,respectively.MDCK cells were transfected with pcDNA3.1-EGFP-PTP2 recombinant vector gradient,and the transfection ratio of recombinant vector to liposome was screened as 2:1,and this system was used to transfect pcDNA3.1-The cells were collected at 24 h and 48 h after changing the transfection solution without antibiotics,and the reverse transcription PCR and Western Bolt test showed that pcDNA3.1-PTP2 was successfully expressed in vitro and the size of PTP2 protein was 35 KDa.3.pcDNA3.1-PTP2 recombinant vector was extracted in large quantities,the concentration was measured;the body weight of the mice was measured,and after calculation,a total of 36 mice in 4 groups(10 blank mice were not injected)were injected intramuscularly into the legs,in which the pcDNA3.1-PTP2 group(P2 group)was immunized three times and the high concentration of pcDNA3.1-PTP2(high immunity P2 group)was immunized twice for a maximum of 21 d.Reverse transcription PCR,Western Bolt of serum and indirect ELISA tests on collected mouse liver,spleen,and both leg muscles showed that pcDNA3.1-PTP2 was successfully expressed in mice with PTP2 protein and stimulated the production of PTP2 antibodies in mice.Spores were used as substrates for an indirect ELISA to monitor serum potency in mice.The within-group control showed that when the serum dilution was increased to 1:6400,14 days out of 5 periods were significantly higher in the pcDNA3.1-PTP2 group than in 7 and 21days(maximum P<0.0001).The high concentration of pcDNA3.1-PTP2 experimental group was significantly higher in 14 days out of 3 periods(maximum P<0.0001).Between-group control showed significant differences between the blank and NC groups only at 1:1600 dilution(P<0.04)and no significant differences between the rest(minimum P>0.2).Therefore,the blank,NC and PC groups were combined into a complete control group.Analysis of the two experimental groups versus the complete control group when the serum dilution was increased to 1:6400.pcDNA3.1-PTP2 group was significantly higher than the complete control group at 8 and 14 days(max P=0.0165).The high concentration pcDNA3.1-PTP2 group was significantly higher than the complete control group at 11 and 14 days(maximum P<0.0001)and significantly higher at 14 days than at 11 days(P=0.0073).There were no significant differences between the pcDNA3.1-PTP2 and high concentration pcDNA3.1-PTP2 groups at 7 and 14 days at all dilutions.However,day 14 in the high concentration pcDNA3.1-PTP2 group(immunized twice)was found to be higher than day 21 in the pcDNA3.1-PTP2 group(immunized three times)at all dilutions,and day 14 was significantly higher than day 21 at a serum dilution of 1:6400(P<0.0001).The results showed that Ec PTP2 can be independently expressed in animals and activate the immune system of animal organism to produce specific PTP2 antibodies.The recombinant vector of pcDNA3.1-PTP2 at a concentration of 0.63 μg/μLand a volume of 100-150 μL/animal was immunized in mice with a potency of 1:6400 at day 14.