Cloning,Expression,Purification And Antigenicity Identification Of ?1-antitrypsin

?1-antitrypsin??1-antitrypsin,?1-AT?is an important protease inhibitor in human body.It mainly is a glycoprotein synthesized by hepatocytes,and also exists in extrahepatic tissues such as intestinal epithelial renal parenchyma and certain tumor cells.It inhibits a variety of proteases and serine endopeptidases.In electrophoresis,the trans-location site is in the?1 protein band,therefore also referred to as the?1protease inhibitor.The trypsin-antitrypsin system plays a key role in a variety of pulmonary inflammatory diseases.Some of the diagnosis and treatment of?1-antitrypsin relevant diseases,especially?1-antitrypsin and liver diseases,need further study.There is a large amount of?1-antitrypsin in gastric juice of gastric cancer patients,the specific reason remains unclear,and further research is needed.Objective:To clone human?1-antitrypsin gene and express it in cells for antigenic identification.Methods:?1?Construction of expression plasmid:the human?1-AT cDNA sequence?1200 bp?was obtained by RT-PCR using the mRNA of human embryonic kidney cell line 293T as the template,cloned into vector and identified by enzyme digestion for sequence analysis.?2?Purification and expression of the fusion protein:the recombinant plasmid with the correct sequence was extracted and transferred into E.coli M15 for expression,and the supernatant was purified by GST affinity purification method?Column Chromatography?,the protein was washed with the reduced glutamic acid solution,,the target protein was washed with PBS?pH 7.4?buffer,and 10?l of the supernatant before the column chromatography,the supernatant after the column chromatography,and the obtained target protein were subjected to SDS-PAGE analysis.?3?Antigenicity identification of recombinant protein:Western blot was used to verify whether the target protein conforms to the antigenicity of?1-AT.Results:The human?1-AT gene cDNA was obtained by gene cloning method.The obtained human?1-AT sequence was identical to the nucleotide sequence reported in literature in Gene bank.Western blot analysis confirmed that the target protein met the immunological properties of?1-AT.Conclusion:?1?Using the RNA of human embryonic kidney cell line 293T as template,the?1-AT gene cDNA is obtained by RT-PCR,and the human?1-AT expression vector PQE₃₀-?1-AT is constructed.Highly expressed?1-AT protein can be obtained in E.coli M₁₅.?2?The human?1-AT protein obtained and purified by genetic engineering has the same antigenicity as the native?1-AT.