| PurposeAutoimmune hepatitis(AIH)is a chronic inflammatory disease of the liver whose cause is unclear.The majority of female patients are characterized by elevated serum transaminases and immunoglobulins,and positive serum autoantibodies.Since each type of autoimmune liver disease has different characteristics and different autoantibodies,the detection of serum autoantibodies in AIH patients is very important.The purpose of this project is to express six kinds of protein antigens of AIH,make them into membrane strips,and combine with quantum dot-labeled secondary antibodies to detect AIH autoantibodies.At present,there are many systems for expressing proteins,including prokaryotic expression systems,eukaryotic expression systems,cell-free expression systems,and other expression systems.Among them,prokaryotic and eukaryotic expression systems are more commonly used.When foreign proteins are expressed recombinantly in a prokaryotic host In the absence of post-transcriptional modifications such as glycosylation and phosphorylation,large molecular weight proteins are difficult to successfully express with bacterial expression systems,and inclusion bodies are likely to form during protein expression,and eukaryotic expression systems can avoid these deficiencies.Therefore,this study uses the insect baculovirus expression system commonly used in eukaryotic expression systems to express AIH-related protein antigens AMA,GP210,SP100,LC-1,LKM-1,and SLA / LP.At present,the commonly used methods for detecting AIH autoantibodies include indirect immunofluorescence,immunoblotting,enzyme-linked immunosorbent assay and chemiluminescence.However,these methods have certain deficiencies: the indirect immunofluorescence method is more complicated to operate manually,has more influencing factors and is more subjective;the detection antibody of the immunoblotting method and the enzyme-linked immunosorbent assay is an enzyme label,and the product color is unstable,It is impossible to observe for a long time;the detection antibody of chemiluminescence method is usually labeled with the chemiluminescent agent acridinium ester(AE),the product is unstable in luminescence,and is easily affected by environmental factors.Quantum dots are a kind of emerging fluorescent markers.The luminescence is relatively stable and can be imaged repeatedly.The operation steps are simple and the detection is fast.The results can be observed under ultraviolet light.Therefore,in this experiment,successfully expressed protein antigens were made into membrane strips.After optimizing the detection system,the clinical sera of 40 AIH-positive patients and 40 healthy persons were tested,and the quantum dot-labeled goat anti-human IgG was added.Observe the results under the light,and compare indirect immunofluorescence and immunoblotting to compare the coincidence rates of quantum dot-labeled immunoblotting with indirect immunofluorescence and immunoblotting.Methods1.Gene cloning: PCR,agarose gel electrophoresis,gel recovery and DNA product purification of the purchased plasmids containing the target genes AMA,GP210,SP100,LC-1,LKM-1,SLA / LP,verified by enzyme digestion After the target gene was successfully connected to the insect cell expression vector p Fast HTA,it was expanded and cultured with competent cell DH5α and then transformed into DH10 Bac Escherichia coli to extract the bacmid.After PCR,agarose gel electrophoresis was performed to verify the successful bacmid extraction.2.Protein expression: culture SF9 insect cells,transfect the extracted bacmid into SF9 insect cells,obtain the first generation virus(P1),obtain the second generation virus(P2)and the third generation virus(P3)by repeated infection),Calculate the virus titer of P3 by Q-PCR method,add an appropriate amount of high virus titer P3 to suspension cultured SF9 insect cells,culture for 72 hours,the target protein AMA,GP210 in the cells by ultrasonic disruption,SP100,LC-1,LKM-1,SLA / LP are released.3.Protein purification: The mixed protein containing the target antigen protein AMA,GP210,SP100,LC-1,LKM-1,SLA / LP is purified by nickel column(Ni column)and imidazole of different concentrations,and then by dialysis Replace the imidazole in the purified protein,and use the BCA method to detect the concentration of the target protein4.Detection of positive specimens: The purified target protein was made into a membrane strip by Yahuilong Biotechnology Co.,Ltd.After the detection system was optimized,the clinical sera of 40 AIH positive patients and 40 healthy persons were tested respectively.Quantum dot-labeled goat anti-human IgG was added,and the results were observed under ultraviolet light.Indirect immunofluorescence and immunoblotting were used as controls to compare their coincidence rates.Results1.Successfully amplified the gene fragments of AMA,GP210,SP100,LC-1,LKM-1,SLA / LP,which have been successfully ligated into pFastHTA vector after enzyme digestion;verified by PCR and successfully extracted to 6 purposes The bacmid corresponding to the gene.2.SF9 insect cells successfully expressed AMA,GP210,SP100,LC-1,LKM-1,SLA/ LP six antigen proteins,SDS-PAGE,Western Blotting results showed that the recombinant target protein was well purified by affinity chromatography,And the size of the expected protein molecule is consistent,with good antigenicity.3.After optimization of a series of reaction conditions,the concentration of quantum dot-labeled secondary antibody was determined to be diluted 1: 100,positive mixed serum was diluted 1: 100,and a better detection level can be reached when the reaction time is 15 minutes;quantum dot-labeled immunity Compared with indirect immunofluorescence and immunoblotting methods,the detection results of AIH autoantibodies by blotting method were 90% positive,92.5% negative,and 91.25%overall.ConclusionQuantum dot-labeled immuno-imprinting method for detecting AIH-related autoantibodies has better compliance than indirect immunofluorescence and immunoblotting methods.The method is simple in operation,rapid in detection,time-consuming,stable and easy to judge. |
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