| Objective: An improved protein purification method was used for the purification of acetylcholine receptor protein(Ach R)from the electrical organs of Naraine timlei;the purified Ach R protein was used to replicate the experimental myasthenia gravis(EAMG)rat model by a secondary immunization method.Based on the successful replication of the EAMG rat model,dynamic observation the levels of three cytostatic inhibitory cytokines interleukin 35(IL-35),interleukin 10(IL-10),and transforming growth factor β1(TGF-β1)in serum,and explored the possible mechanism of three cytokines involved in the pathogenesis of EAMG.Methods:1.Ach R protein was purified from the electrical organs of Naraine timlei using a modified affinity chromatography.Affinity chromatography columns were prepared by Chinese cobra venom neurotoxin and Bungarus multicinctus venom neurotoxin.The Ach R protein was identified by SDS-PAGE and the amount of protein that can be purified was detected by BCA.2.Twenty female Lewis rats at 6-8 weeks were divided into control group and experimental group.In the experimental group we used purified Ach R +Freund’s adjuvant,the Lewis rats were immunized with a primary immunization at day 0 and a secondary immunization after 4 weeks.The adjuvant group used the same dose of PBS + Freund’s adjuvant.The following tests are performed after the primary immunization:(1)Weakness scores,weight changes,and neostigmine tests were used to observe behavioral changes.(2)Tail vein blood was collected after the onset of weakness,and serum was extracted to detect the level of acetylcholine receptor antibody(Ach R-ab)to confirm the immunological changes in rats.(3)Blood was collected form tail vein weekly,and serum were extracted and the serum levels of IL-35,IL-10 and TGF-β1 were tested by ELISA.Results:(1)The Ach R protein was successfully purified by using modified method.Four bands were seen around 35-66.2KD by SDS-PAGE.(2)Using the Chinese cobra venom neurotoxin chromatography column,an average of 1.568 ± 0.5235 mg of Ach R protein can be purified from about 30 g of electrical organs each time.Under the same conditions 0.590 ± 0.2847 mg of Ach R protein can be purified by Bungarus multicinctus venom neurotoxin.The Chinese cobra venom neurotoxin chromatography column was more effective in purifying Ach R protein(P <0.01).(3)Behavioral changes: No abnormal muscle strength was found in the control group rats.Of the ten rats in the experimental group,two died during the experiment.The remaining eight rats showed muscle weakness after the fourth week and the symptoms of weakness were gradually increased after the second immunization.The muscle weakness score of EAMG rats was higher than the control group(P <0.01).Compared with adjuvant rats,EAMG rats lost weight after the onset of disease(P <0.01);EAMG rats were positive for the neostigmine test.(4)The levels of acetylcholine receptor antibody(Ach R-ab)in EAMG rats were higher than that in the control group at the eighth week(P <0.01).(5)There was no significant difference in IL-35 levels between EAMG rats and control rats at first five weeks after immunization(P> 0.05);IL-35 levels in EAMG rats showed an increasing trend at sixth week.At the sixth,seventh and eighth weeks,the levels of IL-35 in EAMG rats were significantly higher than those in the first five weeks of EAMG rats and control group rats at the same period(P <0.01);No significant change was seen in each week in control group(P> 0.05).(6)There was no significant difference in IL-10 and TGF-β1 levels between EAMG rats and control rats at first four weeks(P> 0.05);The levels of IL-10 and TGF-β1 in EAMG rats began to decrease after the fourth week.And the levels of IL-10 and TGF-β1 in EAMG rats at week fifth,sixth,seventh,andeighth were lower than those of EAMG rats in the first four weeks and the same time of the control group(P <0.01);IL-10 and TGF-β1 in the control group had no significant changes in each week(P> 0.05).(7)The symptoms of EAMG rats were negatively correlated with IL-10 and TGF-β1 levels,and positively correlated with IL-35 levels(P <0.01).Conclusion:(1)Adequate Ach R protein can be purified from electrical organs of Naraine timlei by improved method.The use of Chinese cobra snake venom neurotoxin was more effective in purifying Ach R protein.(2)Ach R protein purification using an improved method can successfully replicate the Lewis rat EAMG animal model.(3)After the second immunization,the levels of IL-10 and TGF-β1gradually decreased in EAMF rats;The levels of IL-35 in EMAMG rats gradually increase after the onset of disease.IL-35,IL-10 and TGF-β1 are closely related to the severity of EAMG rats。... |
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